In mammalian cells genes that are in close proximity can be transcriptionally coupled: silencing or activating one gene can affect its neighbors. Understanding these dynamics is important for natural processes, such as heterochromatin spreading during development and aging, and when designing synthetic gene regulation circuits. Here, we systematically dissect this process in single cells by recruiting and releasing repressive chromatin regulators at dual-gene synthetic reporters, and measuring how fast gene silencing and reactivation spread as a function of intergenic distance and configuration of insulator elements. We find that silencing by KRAB, associated with histone methylation, spreads between two genes within hours, with a time delay that increases with distance. This fast KRAB-mediated spreading is not blocked by the classical cHS4 insulators. Silencing by histone deacetylase HDAC4 of the upstream gene can also facilitate background silencing of the downstream gene by PRC2, but with a days-long delay that does not change with distance. This slower silencing can sometimes be stopped by insulators. Gene reactivation of neighboring genes is also coupled, with strong promoters and insulators determining the order of reactivation. Our data can be described by a model of multi-gene regulation that builds upon previous knowledge of heterochromatin spreading, where both gene silencing and gene reactivation can act at a distance, allowing for coordinated dynamics via chromatin regulator recruitment. Drosophila and was originally coined position effect variegation (Eukaryotic gene expression is regulated by chromatin regulators (CRs) and the histone and DNA modifications they read, write, and remove . Chromatiegation . The meciegation , and hasiegation . HoweverTo understand the effects of intergenic distance and insulators on spreading, we studied the dynamics of gene silencing and reactivation after recruitment and release, respectively, of different types of CRs to a dual-gene reporter . This reThe KRAB repressive domain from zinc finger 10 is commonly used in synthetic biology applications , is one We wanted to compare the effects of KRAB with those of another fast silencer that is not associated with histone methylation positive feedback, and instead is only associated with removal of acetylation, such as HDAC4 We have previously shown that HDAC4 recruitment leads to fast gene silencing, comparable to that of KRAB . TherefoWe investigated how silencing by these two rapid-acting chromatin regulators with and without positive feedback mechanisms, KRAB and HDAC4, respectively, affects gene expression of neighboring genes when separated by either distance or the cHS4 insulator, as well as how the dynamics of reactivation are affected after removal of each chromatin regulator. We used single-cell time-lapse microscopy and flow cytometry to monitor gene expression during and after KRAB or HDAC4 recruitment to the upstream gene. We found that transcriptional silencing of the upstream gene by recruitment of either KRAB or HDAC4 affects the downstream gene even when separated by up to 5 kb of distance or with the cHS4 insulator and its core region. For KRAB, the time required for spreading to occur is short and largely distance-dependent. For HDAC4, however, the delay time between silencing of the two genes is longer, does not increase with intergenic distance, and can be influenced by insulators. Reactivation also spreads between the two genes with a delay that is distance-dependent, and is affected by promoters and insulators. We can summarize our findings with a simple kinetic model that describes the dynamics of silencing and reactivation as a competition between: (1) the silencing rates associated with each repressive CR, and (2) the activation rates associated with strong promoters and insulators, where both of these rates decrease with genomic distance. Our results show that targeted transcriptional silencing affects neighboring genes with different dynamics and provide insight for designing novel synthetic systems in mammalian cells.To study the spreading of transcriptional silencing and chromatin modifications, we site-specifically integrated reporters consisting of two neighboring fluorescent reporter genes (mCitrine and mCherry) with each gene driven by a constitutive promoter . Both flTo investigate the spreading of transcriptional silencing, we first recruited KRAB upstream of the mCitrine gene for 5 days in CHO-K1, K562, and HEK293T cell lines with the addition of dox . Using fSurprisingly, HDAC4 recruitment also resulted in silencing of both genes , albeit Additionally, we noticed that in CHO-K1 cells in the absence of dox, mCherry expression is lower and has a wider distribution in the 5 kb reporter compared to NS . This obTo see if the changes in gene expression were accompanied by the expected changes in chromatin modifications, we used CUT&RUN  to map aRecruitment of HDAC4 for 5 days in CHO-K1 cells to the 5 kb and NS reporters showed a decrease in acetylation levels, and no change in H3K9me3 , as expeWe measured the spreading dynamics of transcriptional silencers at finer temporal resolution by first taking time-lapse movies of CHO-K1 cells with the NS and 5 kb spreading reporters . Cells wS, at the gene rather than protein level for all spreading reporters in CHO-K1 and K562 with different CRs recruited ), suggesting the distance-dependent silencing rates induced by KRAB dominate over the activation rates associated with the promoters and insulators .We developed a kinetic model that summarizes our observations of gene dynamics and chromatin modifications as a competition between the distance-dependent silencing rates associated with each tethered chromatin regulator and the reactivation rates driven by our promoters and insulators. In this model, each gene can be either active or silent, leading to four possible states in our two-gene reporters . Each gebs2k ) of pRSV by endogenous polycomb complexes as indicated by the appearance of H3K27me3  at a rate that does not change as a function of the distance between the two genes. We hypothesize that pRSV-mCherry silencing is not due to the direct action of the HDACs recruited upstream of the pEF, but rather due to background silencing , kbs2  oH3K27me3 , loss ofH3K27me3 , and theH3K27me3 .a1(dk)). Once the pEF-mCitrine gene is silenced by HDAC4, it can no longer bolster the overall rate of reactivation at the pRSV-mCherry gene (a1(dk) is missing in Because we see much less pRSV-mCherry background silencing when the upstream pEF- mCitrine is active (in the absence of dox), we conclude that when pEF is active, it can increase activity of the downstream pRSV and prevent it from background silencing , increases with the distance between the genes, and is not affected by insulators. HDAC4 spreading, associated with histone deacetylation and H3K27me3, is slower (days) and does not depend on the distance between the genes. HDAC4-mediated silencing can also spread past insulators, though insulators seem to have a stronger effect on it, even blocking it in the DC configuration.KRAB was previously shown to lead to spreading of both gene silencing and histone methylation when recruited near a gene as a fusion to either TetR  or to prThe fact that KRAB-mediated silencing can spread to neighboring genes quickly raises two concerns for synthetic gene control and epigenetic editing. First, in mammalian genetic circuits where multiple genes need to be integrated together in a cell, genes controlled by KRAB need to either be placed far away from other genes to avoid unwanted interference and feedback, or other mechanisms of escaping silencing potentially through recruitment of H3K9me demethylases such as PHF8 , as may In contrast to KRAB, silencing by HDAC4  is not traditionally associated with spreading of heterochromatin in mammalian cells, so we were initially surprised to observe that silencing mediated by HDAC4 can affect neighboring genes. In general, spreading of heterochromatin-mediated silencing is commonly associated with reader-writer feedback, which has been shown to contribute to heterochromatin spreading in theoretical models  and syntWe were surprised to discover that the cHS4-based insulators were inefficient at blocking the spreading of chromatin-mediated targeted silencing, since they are traditionally thought to prevent spreading of heterochromatin and are commonly used in synthetic biology constructs to protect them from heterochromatin encroachment. None of the insulator variants derived from the cHS4 region of the beta-globin locus prevented spreading of silencing after KRAB recruitment . HoweverPrevious models have proposed the idea that insulators, similar to enhancers and transcription factors, are anti-silencers which oppose repressive forces , and traBy monitoring gene reactivation dynamics after release of the CRs, we conclude that activation can also spread to nearby genes in a distance-dependent manner. However, which gene reactivates first depends not only on the distance between the genes, but also on their promoter size and strength and overall reactivation propensity of the locus. These results led us to modeling promoters and insulators as regions with increased rates of reactivation. In this model, acetylation from strong promoters, such as pEF, can spread via looping and positive feedback to the downstream weaker pRSV promoter, as seen in CHO-K1 cells. This model can be used in other contexts in which spreading of active modifications is relevant, such as enhancer-promoter interactions, activation of nearby endogenous genes after epigenetic editing and perhaps in understanding aging where loss of heterochromatin can lead to changes in gene expression  or in diTogether, our experimental results and the model based on them have broad implications for understanding chromatin mediated gene regulation and building mammalian synthetic biology applications. They suggest that genes in close proximity (< 5 – 10 kb) in a synthetic gene system can respond to changes in chromatin regulators recruited at the promoter of one gene in a coordinated manner, during both silencing and activation, similar to endogenous gene clusters where neighboring genes tend to be co-regulated, including cell cycle, circadian rhythm, and housekeeping genes . In our The CHO-K1 PhiC31 reporters  were assThe K562 and HEK293T AAVS1 reporter constructs  were assThe Piggybac plasmids containing the rTetR-CR were assembled into the PB CMV-MCS- EF1α-Puro PiggyBac vector backbone (System Biosciences #PB510B-1), which was modified via Gibson Assembly to add H2B-rTetR-Zeo from pEx1-pEF-H2B-mCherry-T2A-rTetR-KRAB- Zeo (Addgene #78352), mIFP from pSLQ2837-1, and for K562 plasmids the pGK promoter from pSLQ2818 . The CRs used were: rat KRAB from pEx1-pEF-H2B-mCherry-T2A-rTetR-KRAB (Addgene #78348) for CHO-K1, human KRAB ZNF10 from pSLQ2815 CAG-Puro-WPRE_PGK-KRAB-tagBFP-dCas9  for K562, and human HDAC4 from pEx1-pEF-H2B-mCherry- T2A-rTetR-HDAC4 (Addgene #78349) for both CHO-K1 and K562.All plasmids used in this study have been deposited to Addgene (see Key Resources Table).The CHO-K1 cell line with the human artificial chromosome  was a giReporter lines in CHO-K1 cells were created by integrating the reporter plasmids at the phiC31 integration site on the MI-HAC  by co-transfecting 750 ng of reporter plasmid with 250 ng of the phiC31 integrase using Lipofectamine 2000 . Cells were plated 24 hr before transfection and media change was performed 12 hr after transfection. Selection was started 48–72 hr after transfection with 600 ng/mL geneticin  for 1–2 weeks. CRs were integrated randomly with the Piggybac system  by co-transfecting 750 ng of CR plasmid and 250 ng PiggyBac transposase with Lipofectamine 2000. Cells were selected with 400 ng/mL Zeocin starting 24 hr or later after transfection for 1–2 weeks. Single clones were isolated for each lambda reporter, and correct integration into the MI-HAC was verified by PCR of genomic DNA. Note that rat KRAB was used for CHO-K1cells and human KRAB ZFN10 was used for K562; both are driven by CMV promoter and have mIFP as a fluorescent marker followed by T2A before the rTetR-CR fusion.TGTCCCCTCCACCCCACA-3′ and AAVS1-TALEN-R (Addgene #35432) targeting 5′-TTTCTGTCACCAATCCTG-3′ (Reporters were integrated in K562 cells at the AAVS1 safe harbor site using TALENs: AAVS1-TALEN-L (Addgene #35431) targeting 5′-TCCTG-3′ . RoughlyBoth CHO-K1 and K562 reporter cell lines with CRs were sorted (Sony SH800) for triple positive fluorescence: mCitrine and mCherry of the dual gene reporter and mIFP transcribed along with the CR.2. CHO-K1 cells were grown in Alpha MEM Earle’s Salts media with 10% Tet Approved FBS  and 1 X Penicillin/Streptomycin/L-glutamine (Gibco 10378016). CHO-K1 cells were passaged by rinsing with Dulbecco’s Phosphate-Buffered Saline , and incubating at room temperature with 0.25% Trypsin . K562 cells were grown in RPMI 1640 medium  with 10% Tet Approved FBS  and 1 X Penicillin/Streptomycin/L-glutamine (Gibco 10378016). For long-term storage, cells were frozen in growth media with 10% DMSO  and placed at – 80°C (for up to a month), and then transferred to liquid nitrogen for long term storage.Cells were cultured at 37°C in a humidified incubator (Panasonic MCO-230AICUVL) with 5% COhttps://antebilab.github.io/easyflow/) and a Python-based package, Cytoflow (https://cytoflow.github.io/). All cells were gated for mIFP positive cells based on fitting the background fluorescence of wildtype cells to a sum of Gaussians, and setting the threshold 2 standard deviations away from the mean of the main peak. The percentages of cells with mCitrine and mCherry active  was added to the media to a final concentration of 1000 ng/mL unless otherwise stated. Fresh dox diluted from frozen aliquots was added to the media before each cell passage during recruitment, every 2 – 3 days. Flow cytometry data was collected on two different flow cytometers due to machine access: CytoFLEX S (Beckman Coulter) and ZE5 Cell Analyzer (BioRad). Cells were filtered through 40 µm strainers to remove clumps before flow, and 10,000 – 30,000 cells were collected for each time point. For CHO-K1 NS and 5 kb clonal lines were used for replicates; for the remaining multiclonal constructs, data for each time course was collected from experiments started on different days for use as biological replicates. For EZH2 inhibitor experiments, Tazemetostat-6438  was dissolved in DMSO  at 1 mM and was added to media at final concentration of 5 µM. Data was analyzed using a Matlab program called EasyFlow . CUT&RUN for the CHO-K1 HDAC4 cell lines was performed using the EpiCypher CUTANA ChIC/CUT&RUN Kit (Cat. No.: 14 – 1048) according to the User Manual Version 2.0 with the following conditions: an input of 500,000 CHO-K1 cells per antibody condition; a final digitonin concentration of 0.01%; an overnight antibody incubation at 4°C with 1:100 dilutions of the anti-H3Kac and anti-H3K9me3 antibodies described above along with a 1:50 dilution of rabbit anti-H3K27me3 ; and DNA purification with the provided spin columns. Dual-indexed sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina  with 12 – 14 cycles of PCR, and size selection was performed with either Agencourt AMPure XP  or SPRIselect  magnetic beads. Library concentrations were quantified with the Qubit 1 X dsDNA HS Assay Kit , and library fragment sizes were assessed with the High Sensitivity D1000 ScreenTape  and High Sensitivity D1000 Reagent  on an Agilent 4,200 TapeStation System. Libraries were pooled for genome-wide sequencing.CUT&RUN for the K562 KRAB and CHO-K1 KRAB cell lines was performed according to the third version of the published protocol on protocols.io . For eacGenome-wide libraries were paired-end sequenced by the Stanford Center for Genomics and Personalized Medicine on a HiSeq 4000 with 2 × 101 cycles. A custom genome with our reporter sequence appended to the end of the hg19 human genome assembly was constructed with bowtie2-build. Paired-end alignment was performed with the following bowtie2 command: bowtie2 --local --very-sensitive-local --no-unal --no-mixed --no-discordant --phred33 -I 10 X 700 x {reference genome} -p 8 – 1 {first mate of pair} – 2 {second mate of pair} -S {output SAM file name}. Fragments that mapped completely within non-unique reporter elements  were ambiguous and thus removed to avoid confounding. Samtools was used to convert SAM files to BAM files and to subsequently sort and index BAM files. Picard was used to mark and remove duplicates with the following command: java -jar {picard tool} MarkDuplicates -I {input sorted BAM file} -O {output deduplicated BAM file} -M {output metrics file} --REMOVE_DUPLICATES true. Reads were normalized to counts per million (CPM) and bedgraph files were generated with the following bamCoverage command: bamCoverage --bam {input deduplicated BAM file} -o {output bedgraph file} --outFileFormat bedgraph --extendReads --centerReads --binSize 10 --normalizeUsing CPM. CUT&RUN data were visualized via the Broad Institute and UC San Diego Integrative Genomics Viewer. Additional data analyses and visualization were performed with custom scripts in Python.2 fold-change in ChromaBlocks analysis was performed in R using the Repitools package. Normalized bedgraph files were read in as dataframes and converted to GRangesList objects with the annoDF2GR and GRangesList methods. GRangesList objects for H3K9me3, H3Kac, or H3K4me3 samples were specified for the rs.ip argument within the ChromaBlocks method, and the respective dox-treated or untreated IgG sample was specified as the rs.input argument. ChromaBlocks was called with the ‘large’ preset for H3K9me3 and H3Kac and with the ‘small’ preset for H3K4me3 to identify regions of enrichment. Start coordinates and widths of enriched regions were used to construct bed files for visualization in IGV and for signal integration to compute the logEach cell line was treated with dox (1 ug/mL) for the indicated number of days before harvesting. RNA was harvested using RNeasy Mini Kit  using the on-column RNase-Free DNase . cDNA was synthesized using the iScript Reverse Transcription Supermix  for qPCR experiments and qScript cDNA SuperMix  for PCR experiments. qPCR on cDNA was performed using SsoFast EvaGreen Supermix on a CFX Connect Real-Time PCR System (both from Bio-Rad Laboratories). For qPCR primer sequences, see 2 (Praxair Cat. BI NICD5O6B-K), and with humidity control. CHO-K1 cells were seeded 24 hr prior to the start of movie acquisition on optically clear 96-well µ-Plates (Ibidi Cat. 89626). Border wells were filled with PBS (Thermo Fisher Scientific Cat. 10010023) and plates were sealed with Breath-Easy gas-permeable membranes (Diversified Biotech Cat. BEM-1) to reduce evaporation and maintain humidity during image acquisition. Cells were imaged in alpha MEM media with no phenol red  with 10% Tet-Free FBS , and 1% Penicillin/Streptomycin/Glutamine (Fisher Scientific Cat. 10378 – 016) to reduce background fluorescence. Images were acquired every 20 min in three different fluorescent channels: YFP, RFP and CY5, corresponding to the mCitrine, mCherry and mIFP fluorophores, respectively. The light source was a Sola Light Engine, and a Lumencor control pod was used to attenuate the light to < 1 mW range. The FIM was set to 10% and Lumencore was set to 5%. Exposure times were 150 ms, 200 ms, and 100 ms for the CY5, RFP and YFP channels, respectively . One to two time-lapse movies were analyzed for each cell line, where each movie consisted of cells in dozens of different wells, and in each well 3 – 5 non-overlapping sites were imaged. Cell culture media was changed every 24 hr to replenish doxycycline and remove toxic reactive oxygen species which may have formed due to prolonged imaging. LasX software was used to control the microscope in the Mark and Find module to image up to 200 positions in total and AFC on demand mode adjusted focus for every cycle and position of imaging.Fluorescent time-lapse imaging was conducted using a DMi8 inverted epifluorescence microscope  with a 20 X Plan Apo 0.8 NA objective and a Leica DFC9000 GT CMOS Camera using 2 × 2 binning. The microscope was enclosed in a cage incubator  held at 37 °C, and samples were placed in a stage top chamber (Okolab H301-K-FRAME) also held at 37°C, 5 % COhttps://github.com/brookstaylorjr/MACKtrack). Nuclei were segmented and tracked using H2B-mIFP signal or H2B-mCherry signal (on a case-by-case basis based on expression level) and single-cell intensities for integrated nuclear intensity of mCitrine, mCherry, and mIFP channels were measured at each frame. Tracked single-cell output traces were then filtered in a semi-automated fashion to remove incomplete or mistracked cells which were defined as traces exhibiting numerous aberrant drops in fluorescence intensity inconsistent with cell divisions. Additionally, traces consisting of background silenced cells with low mCitrine expression prior to the 24-hr time point were omitted from analysis. In order to call transcriptional silencing events, the gradient of the cumulative intensity traces for mCherry and mCitrine were used. To generate cumulative traces, mitotic events were first computationally identified based on periodic dilution of the stable H2B-fluorophore signal by approximately 50% upon cell division. Using these points as reference, raw cumulative traces for mCherry and mCitrine were generated by computationally re-adding the lost fluorescence due to cell division events and adding these values to all subsequent time points after a division event to ‘stitch together’ intensity values across divisions  unless otherwise stated. Fluorescent imaging data were obtained by automated image segmentation and single-cell tracking using the MACKtrack package  between chromatin-regulator spreading rates between NS and 5 kb reporters.mCitrine gene, followed by the downstream mCherry gene. The deterministic equations are then used in the probabilistic component to derive a probability density function that describes flow cytometry data. By deriving this model and fitting it to our data, we estimate the silencing parameters of our system such as the time delay between reporter silencing and the magnitude of transcriptional interference.We have developed a model that describes the time dependent evolution of the fluorescence distributions of our two fluorescent reporters after the recruitment of CRs. This model has two components: a deterministic component describing the decay of fluorescence due to mRNA and protein degradation and dilution, and a probabilistic component taking into account that cells can stochastically transition between the active and silent states at different times. In the deterministic component, we will derive equations that describe the fluorescence of cells in either the active or silent state, beginning with the simpler upstream mCitrine gene. We begin at the level of transcription where t=0 with the addition of doxycycline, but transcriptional silencing of mCitrine does not occur until some time later, tS,C. By assuming a constant mRNA production rate, αm, and a constant coefficient of mRNA degradation and dilution, βm , we can write a piecewise differential equation for mRNA concentration:In this component of the model, we derive deterministic equations for the fluorescence of cells in the active and silent state. First, we will derive these equations for the simpler upstream mCitrine gene. We assume a steady-state initial condition and continuity among the pieces to arrive at the following solution for mCitrine mRNA as a function of time:This differential equation describes transcription of the upstream Next, we move on to the level of translation, where mRNA is translated into protein. The protein concentration is represented by m(t) into the equation above and combining Substituting our solution for Again, we assume a steady-state initial condition and continuity among the pieces to arrive at the following solution for mCitrine concentration:herex in our model. We assume that fluorescence increases proportionally to concentration after the addition of some background fluorescence xB. The proportionality constant is absorbed into the production parameter, α. Therefore, fluorescence can simply be expressed as protein concentration with the addition of the background fluorescence in Arbitrary Fluorescence Units (AFU):Now that we have derived equations for the concentration of protein over time, we need to convert from concentration to fluorescence, represented by Using this relationship and the solution to mCitrine gene, but they are too simple to describe silencing at the downstream mCherry gene, as we often observe an increase in mCherry after the recruitment of the CR. We hypothesize that this upwards spike in mCherry production is due to the loss of transcriptional interference upon silencing of the upstream mCitrine gene. Therefore, we modified the differential equation for mRNA with the addition of a new term, γ, which represents the factor by which mRNA production is reduced by transcriptional interference. This factor is removed after the average silencing time of the mCitrine gene, tS,C, causing a delayed spike in the mRNA concentration of mCherry before it silences and begins to degrade at its own tS,M:These two equations describe the fluorescence for the upstream mCherry mRNA over time is:To solve this differential equation, we again assume a steady-state initial condition and continuity among the pieces. The solution for wherem(t) into the general translation equation and again combining α’s into an integrated production parameter α, we arrive at a piecewise differential equation for the concentration of mCherry over time:Substituting this solution for mCherry concentration over time:Again, we assume a steady-state initial condition and continuity among the pieces to arrive at the following solution for wherep(t) assumes the more likely scenario that the upstream mCitrine gene silences before the downstream mCherry gene mCherry may silence before mCitrinemCherry promoter silences without the loss of transcriptional interference concomitant with mCitrine silencing. Therefore, the following solution for mCherry concentration over time when mCherry silences first is the same as mCitrine, only divided by γ:While the previous equation for Now that we have defined two equations of mCherry concentration, we again combine these equations with the fluorescence equation and split them such that there is one equation for the active state where These two equations describe the downstream mCherry fluorescence at any given point in time and with any given ● ● ● ● ● In the probabilistic component of the model, we include a stochastic transition between active and silent states in the derivation of a probability density function of fluorescence en cells  , we incTo consider all cells and for tS. We add stochasticity to this transition by allowing cells to transition at different times and defining random variable TS. We choose a gamma distribution for To determine the fraction of active cells and silent cells at any point in time, we consider that a cell transitions from an active state to a silent state after a time of Using this definition, we will attain solutions for This cumulative density function represents the fraction of active cells that have transitioned into the silent state until To get the weight of active cells, we simply subtract the cumulative density function from 1 to find the fraction of active cells that have not silenced until With analytical expressions for each weight, we now consider each independent probability density function of fluorescence for each of the three cell states. First, the probability density function for the fluorescence of background silent cells does not have a time dependence and is log-normal with a median of the background fluorescence, Next, the probability density function for the fluorescence of active cells is also log-normal, but the median of this distribution is given by Finally, the probability density function for the fluorescence of silent cells is a convolution of Again, we used the same shape parameter,, so that when the fluorescence decays to background levels, the distribution for fluorescence of background silent cells matches the distribution for silent cells:We have now defined the weights and probability density functions for each of the three possible states in terms of some new parameters and the equations derived in the deterministic component, which concludes the probabilistic component of the derivation. The additional constant parameters of the model are:Fitting our probabilistic model to the flow cytometry data allows us to estimate the relevant silencing parameters. Of particular interest are the interference parameter, γ and the mean silencing times for mCitrine and mCherry, μS, C and μS, M as well as the difference between the two, or ΔμS. Our model fitting was conducted in Python 3 Jupyter notebooks.γ,First, we defined functions F_o and F_s to calculate mentally . Then weWith functions defined to calculate the model solution given a set of parameters, we next defined an error function to calculate error between the data and the model. To calculate this error, the fluorescence values are binned into 100 equally spaced bins for each timepoint in the data. The probability densities of each bin are calculated. Then, we evaluate the model with the given parameters at the midpoint of each bin. The error is calculated by subtracting the bin densities from the solution to the model’s probability density function. The error arrays from each timepoint are appended to generate a single array of errors for the whole time course.The mCitrine and mCherry reporters were each fit separately and had differences in the parameters used. Therefore, we used the general error function described above to define two separate error functions for mCitrine and mCherry. Since we never saw a spike in mCitrine expression, the interference parameter, γ, was intentionally set to one and the spike delay, The mCherry fit was conducted after the mCitrine fit as some of the parameters are carried from mCitrine to mCherry. One of these parameters is the protein dilution There is one additional consideration for the mCherry fits. Due to fast mCherry silencing by KRAB, it was difficult estimate both the interference factor, γ, and the mean silencing delay, One final adjustment to the model was made to fit non-saturating dox concentrations (100 ng/mL and 200 ng/mL). When dox concentration was low, we did not observe silencing of mCitrine in all cells as was the case for 1000 ng/ml, and accordingly did not observe subsequent spreading of silencing to mCherry in those cells. To account for only a fraction of cells being silenced, an additional parameter was added to the model to represent cells that are always active, To estimate the unconstrained parameters for each reporter, we fit the model to the data using a least-squares approach, scipy.optimize.least_squares. This function solves for the parameters that minimize the sum of the squares of the error array that we defined. Initial guesses for each parameter were reasonably estimated by manual inspection of the data where possible. Bounds for each parameter were set generously as to avoid bounding of the solution where possible. The fits were performed independently for each replicate or single clone and took several hours for each cell line. However, the resulting fits were satisfactory by visual inspection, and the parameters were in accordance with those found in time course movie analysis.The 90% confidence interval (CI) for the parameters extracted from fitting daily flow cytometry data to the model, was estimated using a t-distribution with t-score 2.92 and sample standard deviation from all replicates. This study describes a novel approach to investigate how the transcriptional repressors KRAB and HDAC4 repress gene expression, how repression spreads and the role of insulator elements in blocking the spread of repression and in reactivation of repressed genes. Despite some inherent limitations resulting from the use of artificial reporters compared to previous genomic studies, it addresses the question of repression in a rather novel manner adding a dimension of time and single cell resolution. The results allow modeling of the coordinated repression or activation of closely linked genes and should be of wide interest to researchers interested in chromatin, gene expression and synthetic biology. The authors have made a thorough revision of the paper and have addressed all of the major issues raised by the referees. They have better discussed their results in the context of previous studies and they have added new data with EZH2 inhibitors that support a role for PRC2 in HDAC4-mediated repression. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Irwin Davidson as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Kevin Struhl as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Didier Trono (Reviewer #2).Thank you for submitting your article "Dynamic spreading of chromatin-mediated gene silencing and reactivation between neighboring genes in single cells" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.While the referees found the paper reported interesting observations, a major issue raised by all three referees regards the generalities drawn from a very limited set of observations . In addition, previously published data was not discussed in the context of this current study. For example, Amabile et al. 2015 and Groner et al. 2010 (both referenced but not much commented on) were much broader and already made many of the points stated in the present work.A major revision of the manuscript would considerably gain from placing its results in the context of the sum of previous studies, discussing specifically what the current study adds to the field.Additional data.The authors show that HDAC4 repression is associated with gain of H3K27me3. To investigate the role of PRC2 in this process and the requirement for the K27me3 mark in HDAC4 repression, the authors should test silencing of the locus in the presence of EZH2 inhibitor. In addition, there may be published data / databases where HDAC inhibition triggers silencing likely via H3K27me3 deposition may be seen in broader contexts.Particular attention should be paid to the following issues.1. The authors should mention in the text of the paper what is known about the differences in half-life/stability of the mCitrine and mCherry reporter proteins. This is important to assess the kinetics of repression. While imaging and flow cytometry allow assessment of their expression at the single cell level.2. The authors do not show kinetics and overall expression of the Dox-induced KRAB and HDAC proteins and how these levels compare with the endogenous cellular levels. If as may be expected, the levels are much higher than endogenous, how does this affect the interpretation of the results in terms of kinetics and spreading of repression?3. The authors should better explain why they chose KRAB and HDAC4 for their experiments. They should also comment on the histone deacetylase activity recruited by the KRAB-binding KAP1. KRAB is not a protein, but a domain, which often but not always recruits KAP1, and some KRAB-containing proteins also harbour other domains and some are not repressors. This should be explained as well to help the non-specialists situate the relevance of the work in a broader context.4. Line 74: "a set of mammalian cell lines" could be advantageously replaced by "two mammalian cell lines".5. A statistical test should be performed to qualify the yellow and red subpopulations.6. A spacing of 0 or 5kb hardly qualifies as "various spacer lengths" as stated on line 79.7. Lines 112-114: spreading from the HDAC4 docking site takes 20 days, is observed in only a fraction of cells, and the interpretation of the results is complicated by promoter interference intrinsic to the vector design (spreading is more efficient in the 5kb- than in the 0kb-spacing configuration). The data thus do not warrant the statement "these results show for the first time that silencing mediated by a histone deacetylase can also extend to nearby genes".8. The models presented in 2F and 2G are not clear: The way the picture is drawn, it seems that loss of H3Kac is a temporal prerequisite of H3K9me3 and H3K27me3 deposition in both models. This might be true for HDAC4 , but not for KRAB.9. Line 299: reference to the figure should be corrected .10. Lines 331-333: "These results suggest that the insulators can help the downstream gene remain active in conditions where its silencing is already slow, such as after indirect silencing by HDAC4 recruitment in K562." Again, this hypothesis relies on so few observations  that it should be formulated with much caution.11. Lines 510-515: one cannot say that the referenced previous studies yielded "disparate" results, notably on the extent of KRAB-induced heterochromatin spreading. Compared to the present work, they covered far greater variety of settings relative to the strengths of the targeted promoters, their distance from the KRAB docking site and their immediate or broader chromatin environment, so that a broad range of phenotypes was expected.12. The authors should integrate in their thinking results reported ten years ago . This work reported an extensive analysis of chromatin features conducive or not of KRAB-induced long-range silencing, making interesting conclusions on the "repression-primed" state of the most sensitive promoters and their paradoxical location in a surrounding of highly expressed genes, and on the failure of CTCF recruitment to exert any barrier effect on H3K9me3 spreading.13. The authors should also comment on KRAB zinc finger protein genes, many of which recruit KAP1 at their 3' end and are grouped in tightly packed clusters, yet can escape self- or mutually inflicted silencing. And on the interesting observation by K. Helin's group that the H3K9me1/2 demethylase PHF8 is recruited to the promoter of many of these genes, perhaps explaining this phenomenon (Mol Cell 2010 38: 165).14. The potential roles of histone acetylation functioning as insulator sequences require more rigorous testing and validation. Perhaps the author should mine some published genomic data to find potential correlations between various histone acetylation marks and heterochromatin vs euchromatin compartments in development or disease conditions.Reviewer #1 (Recommendations for the authors):The authors should mention in the text of the paper what is known about the differences in half-life/stability of the mCitrine and mCherry reporter proteins. This is important to assess the kinetics of repression. While imaging and flow cytometry allow assessment of their expression at the single cell level, additional RT-qPCR experiments for mRNA levels for each gene would strengthen the analyses.The authors do not show kinetics and overall expression of the Dox-induced KRAB and HDAC proteins and how these levels compare with the endogenous cellular levels. If as may be expected, the levels are much higher than endogenous, how does this affect the interpretation of the results in terms of kinetics and spreading of repression?The authors have investigated the role of the cSH4 insulator element, can they better justify why this one was chosen. As the properties of insulator elements are not as well described as promoters or enhancers, experiments with other insulators would be required before general statements can be made on their ability to block repression or contribute to reactivation of gene expression.It is now well accepted that regulation of gene expression is mainly driven by enhancer elements. The reporter plasmid used in this study comprises only proximal promoter elements. Do the authors know whether their promoter activity is driven/influenced by distal enhancers located around the integration sites? More specifically it would be informative to generate a reporter with a strong enhancer element and assess how this impacts repression. Insertion of an enhancer between the two genes may reveal whether it can block the spreading of repression to the mCHERRY gene.Reviewer #2 (Recommendations for the authors):Introduction:1. The authors should better explain why they chose KRAB and HDAC4 for their experiments. They should also comment on the histone deacetylase activity recruited by the KRAB-binding KAP1. KRAB is not a protein but a domain, which often but not always recruits KAP1, and some KRAB-containing proteins also harbour other domains and some are not repressors. This should be explained as well to help the non-specialists situate the relevance of the work in a broader context.Regarding Figure 11. Line 74: "a set of mammalian cell lines" could be advantageously replaced by "two mammalian cell lines".2. A statistical test should be performed to qualify the yellow and red subpopulations.3. A spacing of O or 5kb hardly qualifies as "various spacer lengths" as stated on line 79.4. Lines 111-112: "Moreover, in K562 cells, HDAC4-mediated silencing of mCherry was much slower, taking up to 20 days, compared to 5 days in CHO-K1 cells . Any lead to explain the difference? Shouldn't other chromosomal loci be tested before attributing the difference to the cellular background?5. Lines 112-114: spreading from the HDAC4 docking site takes 20 days, is observed in only a fraction of cells, and the interpretation of the results is complicated by promoter interference intrinsic to the vector design (spreading is more efficient in the 5kb- than in the 0kb-spacing configuration). The data thus do not warrant the statement "these results show for the first time that silencing mediated by a histone deacetylase can also extend to nearby genes". Rare are the pairs of genes, the promoters of which are at such short distances.Regarding Figure 21. The models presented in 2F and 2G are not clear: The way the picture is drawn, it seems that loss of H3Kac is a temporal prerequisite of H3K9me3 and H3K27me3 deposition in both models. This might be true for HDAC4 , but not for KRAB.2. Lines 181-183: "… that can lead to spreading of these chromatin modifications . This observation suggests that silencing of the RSV promoter is indirectly due to PRC2 action, but it can only take place once HDAC4 removes acetylation at the locus." Artificial recruitment of PRC2 at this locus in the absence of HDAC4 could be used to validate this hypothesis.Regarding Figure 3The authors used time-lapse movies to analyse the spreading of epigenetic marks with high temporal resolution. They conclude that KRAB-mediated spreading of transcriptional silencing is distance-dependent in CHO-K1 cells while HDAC4-mediated spreading is distance-independent at these length scales.1. A limitation of time-lapse movies is the low output. However, the authors developed a mathematical model to optimize the information recovered from "high output" data (flow cytometry combined with gene expression). The output of this method is nicely consistent with time-lapse movies opening perspectives to use this system on a larger scale. A useful contribution to the field!2. There should be published data / databases allowing to verify that in broader contexts HDAC inhibition triggers silencing likely via H3K27me3 deposition (Line 176), rather than it being a peculiarity of the dual system used here.3. Further down (lines 178-180) it is stated that "This repressive modification, which is associated with polycomb repressive complex 2 (PRC2), has not been reported in association with HDAC4, and was not observed after HDAC4 recruitment at a pEF-mCitrine reporter flanked by insulators in the same locus of CHO-K1 cells". To consolidate their hypothesis, the authors could test silencing of the locus in the presence of EZH2 inhibitor to see in which extent it depends on PRC2, and measure PRC2 enrichment by CUTandTAG or CUTandRUN.4. Lines 223-226: "The observation that the delays of pRSV-mCherry silencing do not increase with distance  suggests that its silencing initiates at the RSV promoter, most likely by the action of PRC2 .". If the silencing starts from the RSV promotor, why is it different without the 5Kb spacer? Can the dynamics of H3K27me3 deposition at the RSV promotor be assessed on both constructs? What would be the mechanism leading to the recruitment of PRC2 at the RSV instead of the pEF since HDAC4 is recruited at pEF? What is the effect of inverting the two promoters?Regarding Figure 4In this figure, the authors document the lack of effect of cHS4 insulators on KRAB/HDAC4-induced silencing in CHO cells, but observe some impact on the latter in K562 cells.1. Line 299: reference to the figure should be corrected .2. Lines 331-333: "These results suggest that the insulators can help the downstream gene remain active in conditions where its silencing is already slow, such as after indirect silencing by HDAC4 recruitment in K562." Again, this hypothesis relies on so few observations  that it should be formulated with much caution.Regarding Figure 5Here the authors investigate the re-activation of the genes in their system. Again, they formulate overly general conclusions based on a very limited set of parameters. Lines 395-398: "These results show that the extent of epigenetic memory depends strongly not only on the chromatin regulator recruited, but also on the configurations of promoters and insulators at the target locus, with stronger promoters closely surrounded by core insulators diminishing memory".Regarding Figure 6 (Model)The model is fine as long as it is clearly restricted to the parameters tested here, and not proposed as a paradigm.Discussion1. Lines 510-515: one cannot say that the referenced previous studies yielded "disparate" results, notably on the extent of KRAB-induced heterochromatin spreading. Compared to the present work, they covered far greater variety of settings relative to the strengths of the targeted promoters, their distance from the KRAB docking site and their immediate or broader chromatin environment, so that a broad range of phenotypes was expected, without this heterogeneity being shocking (which is what the term "disparate" suggests).2. Lines 560-561: "By monitoring gene reactivation dynamics after release of the CRs, we conclude that activation can also spread to nearby genes in a distance-dependent manner." Only one distance (5Kb) was tested, which pales with the thousands of distances evaluated in some previously published studies.3. Lines 571-572: "They suggest that genes in close proximity (<5-10kb) can respond to signals in a coordinated manner, during both silencing and activation". Right, although confirmatory of a large series of observations previously made on co-regulated gene clusters.4. Lines 579- 582: "Finally, this experimental and theoretical framework can serve as a starting point for measuring and modeling the effects of targeting various epigenetic editors at endogenous loci in order to guide the time necessary for efficient on-target effects without unwanted off-target spreading". The authors fail to demonstrate convincingly that their system is really suited for the kind of high throughput analyses that would be necessary to consolidate, give a general value or even validate some of their conclusions.5. The authors should integrate in their thinking results reported ten years ago . This work proceeded to an extensive analysis of chromatin features conducive or not of KRAB-induced long-range silencing, making interesting conclusions on the "repression-primed" state of the most sensitive promoters and their paradoxical location in a surrounding of highly expressed genes, and on the failure of CTCF recruitment to exert any barrier effect on H3K9me3 spreading.6. The authors should also comment on KRAB zinc finger protein genes, many of which recruit KAP1 at their 3' end and are grouped in tightly packed clusters, yet can escape self- or mutually inflicted silencing. And on the interesting observation by K. Helin's group that the H3K9me1/2 demethylase PHF8 is recruited to the promoter of many of these genes, perhaps explaining this phenomenon (Mol Cell 2010 38: 165).Reviewer #3 (Recommendations for the authors):The authors should include a more critical discussion of the differences of this model with prior studies where KRAB domains were recruited to natural genomic sequences.The potential roles of histone acetylation functioning as insulator sequences require more rigorous testing and validation. Perhaps the author should mine some published genomic data to find potential correlations between various histone acetylation marks and heterochromatin vs euchromatin compartments in development or disease conditions. While the referees found the paper reported interesting observations, a major issue raised by all three referees regards the generalities drawn from a very limited set of observations . In addition, previously published data was not discussed in the context of this current study. For example, Amabile et al. 2015 and Groner et al. 2010 (both referenced but not much commented on) were much broader and already made many of the points stated in the present work.A major revision of the manuscript would considerably gain from placing its results in the context of the sum of previous studies, discussing specifically what the current study adds to the field.We agree with these comments, and have made major edits throughout the entire text of the manuscript to decrease the generalities of our claims and restrict them to our system, describe in more detail what has been shown in previous studies, motivate our experimental design choices better, emphasize the novel results from our work, and place our results in the context of previous work. Please see point-by-point responses to each reviewer.Additional data.The authors show that HDAC4 repression is associated with gain of H3K27me3. To investigate the role of PRC2 in this process and the requirement for the K27me3 mark in HDAC4 repression, the authors should test silencing of the locus in the presence of EZH2 inhibitor. In addition, there may be published data / databases where HDAC inhibition triggers silencing likely via H3K27me3 deposition may be seen in broader contexts.We added the new required data: To further test this hypothesis of PRC2-mediated silencing of the downstream pRSV-mCherry gene, we measured HDAC4-mediated silencing in the presence of EZH2 inhibitor Tazemetostat (Taz). We found that Taz blocked pRSV-mCherry silencing, while not affecting the pEF-mCitrine silencing. These results support the idea that pRSV-mCherry silencing we observe after HDAC4-mediated silencing of the pEF-mCitrine results from PRC2 mediated silencing of the pRSV-mCherry. We have added these data to Figure 2F-H as well as new supplemental Figure 2 —figure supplement 3. Additions to the main text include:– Line 180, added text: “To test whether the silencing of mCherry was mediated by PRC2, we recruited HDAC4 in the presence of Tazemetostat (Taz), a small molecule inhibitor of EZH2, which is the H3K27 methyltransferase in PRC2 . First, we tested Taz in the NS reporter line with recruitment of EED, the H3K27 reader in PRC2, and showed that inhibition reduced silencing of mCitrine and mCherry . We found that in the presence of dox and Taz while the mCitrine gene was still silenced by HDAC4, the mCherry gene was not silenced and even slightly increased in expression in the 5kb reporter  and the NS reporter .– Line 419, add to sentence: “loss of pRSV-mCherry silencing upon PRC2 inhibition, and the fact that the silencing delay is distance-independent.”– Line 536, add to sentence: “which was confirmed by the loss of mCherry silencing when PRC2 was inhibited .“– Line 21, edit sentence in abstract to reflect the role of PRC2: “Silencing by histone deacetylase HDAC4 of the upstream gene can also facilitate background silencing of the downstream gene by PRC2, but with a days-long delay that does not change with distance.”– Edits to legend of Figure 2:– (F) Addition of Tazemetostat (Taz) inhibits the EZH2 methyltransferase from PRC2. (G) Fluorescence distributions of mCitrine (left) and mCherry (right) measured by flow cytometry either without CR recruitment  or after 7 days of HDAC4 recruitment  and with Taz at the 5kb reporter in CHO-K1. (H) Percentages of cells OFF normalized by the -dox+DMSO condition, based on threshold shown in panel G (dotted line).– (I) In the absence of CR recruitment, both genes have H3Ac across the reporter (top). Upon recruitment of KRAB (bottom left), we see a loss of H3Kac and gain of H3K9me3 across the dual-gene reporter through both DNA looping from rTetR-KRAB as well as positive feedback loops for spread of methylation, resulting in a distance-dependent delay of transcriptional silencing between two genes. Upon recruitment of HDAC4 (bottom right), we see a loss of H3Kac as well as a gain of H3K27me3 across the reporter through positive feedback loops for spread of methylation by PRC2, resulting in distance-independent delay of transcriptional silencing between two genes.New figure legend:Figure 2 —figure supplement 3. EZH2 inhibitor Tazemetostat affects silencing after recruitment of EED and HDAC4.(A) Addition of Tazemetostat (Taz) inhibits the EZH2 methyltransferase from PRC2. (B-C) Fluorescence distributions of mCitrine (top left) and mCherry (bottom left) measured by flow cytometry either without CR recruitment  or after 7 days of (B) EED or (C) HDAC4 recruitment  and with Taz at the NS reporter in CHO-K1. Percentages of cells OFF normalized by the -dox+DMSO condition (right), based on threshold (dotted line).With respect to the connection between HDAC inhibition, loss of acetylation, and H3K27me3 deposition in broader contexts, we have modified the manuscript to describe published results which support our hypothesis that acetylation removal can lead to PRC2associate chromatin modifications:– Added two sentences at line 183: “ In accordance with our hypothesis, previous studies have shown that by co-ChIP of primary cells H3K27me3 and H3Ac are negatively correlated , that lack of transcription and histone deacetylation both drive an increase in H3K27me3 deposition steadily over time , and that recruitment of HDAC1 leads to repressed chromatin associated with a strong increase in H3K27me3 . Additionally, knockdown of p300, a histone acetyltransferase, resulted in a global decrease of H3K27Ac in tandem with an increase in H3K27me3 .”– In Discussion section, line 536 added: “In accordance with our observations, previous studies have shown that H3K27me3 and H3Ac are negatively correlated on individual nucleosomes , and specifically that deacetylation by either lack of transcription or recruitment of HDAC1 leads to increases in H3K27me3 . Additionally, studies where cells are treated with HDAC inhibitor show in an increase in histone acetylation which is correlated with reduced recruitment of EZH2, EED and Suz12 proteins from the PRC2 as well as a decrease in H3K27me3 , showing the bidirectionality of the inverse correlation between H3K27me and H3K27Ac . This supports our observation that low levels of acetylation allow PRC2 to work more effectively.”Particular attention should be paid to the following issues.1. The authors should mention in the text of the paper what is known about the differences in half-life/stability of the mCitrine and mCherry reporter proteins. This is important to assess the kinetics of repression. While imaging and flow cytometry allow assessment of their expression at the single cell level.We agree with the reviewer that the half-life/stability of the reporter proteins are important parameters for the kinetics of repression. Our reporter genes are fusions between H2B and a fluorescent protein: H2B-mCitrine and H2B-mCherry. Fusions between H2B and fluorescent proteins (H2B-FPs) are used for studying chromatin dynamics due to their high signal to noise ratio from nuclear localization and incorporation into nucleosomes without inhibiting cell cycle progression . H2B-FPs are very stable, with very long protein halflives on the order of weeks; therefore degradation is mostly driven by cell division . At the mRNA level, we have previously shown that the half-life of H2B-mCitrine mRNA transcript is about 3.9 hours as measured by inhibiting transcription with actinomycin D and quantifying mRNA overtime . Although we have not measured the lifetime of the H2B-mCherry mRNA our video data and our model from daily flow cytometry data shows that the delay between mCitrine and mCherry silencing (δ T) is close to zero in the NS reporters , suggesting that mCitrine and mCherry lifetimes are very close together. Small differences in mRNA stability could change silencing times of each gene but ultimately would shift the δ T delay equally for all reporter constructs, and therefore not change our conclusion that the delay increases with distance, which is also corroborated by our video results which measure silencing at the protein level.We have added this information into the main text as follows:– Line 73-76, first sentence edited:– Original: “ To study the spreading of transcriptional silencing and chromatin modifications, we engineered a set of mammalian cell lines containing two neighboring fluorescent reporter genes and a CR, with each gene driven by constitutive promoters .”-New: “To study the spreading of transcriptional silencing and chromatin modifications, we site-specifically integrated reporters consisting of two neighboring fluorescent reporter genes (mCitrine and mCherry) with each gene driven by a constitutive promoters . Both fluorescent proteins are fused to H2B; this approach increases the signal to noise ratio due to nuclear localization and is commonly used for studying chromatin dynamics . In each reporter cell line, we also stably integrated a recruitable rTetR fused to either KRAB or HDAC4.”Line 195, added sentence: “Since H2B fusions to fluorescent proteins have long halflives on the order of weeks, their loss is mostly driven by cell division , which allows the silencing of the gene to be detected based on the change in fluorescent protein accumulation. Stitching together single-cell traces across cell divisions results in cumulative traces of fluorescent protein levels over time for specific lineages . The slope of these traces is then proportional to promoter activity, and its decrease can be used to determine silencing of a gene.”Line 218, add to sentence:“… especially if we consider the additional time necessary for mRNA degradation of about ~4 hours included in the flow fits , as determined previously by inhibiting transcription with actinomycin D and quantifying the decay of H2B-mCitrine mRNA over time. Since we observed a delay time between mCherry and mCitrine silencing of less than 4 hrs after KRAB recruitment , we assumed that the H2B-mCherry mRNA degrades at a similar rate as H2B-mCitrine.”2. The authors do not show kinetics and overall expression of the Dox-induced KRAB and HDAC proteins and how these levels compare with the endogenous cellular levels. If as may be expected, the levels are much higher than endogenous, how does this affect the interpretation of the results in terms of kinetics and spreading of repression?Indeed, we would expect the expression levels of our chromatin regulators to be higher than endogenous levels, and we also expect the amount of rTetR-CR recruited at the reporter to affect the dynamics of silencing.. In fact, with our dox inducible system we are able to tune the number of rTetR-KRAB or rTetR-HDAC4 proteins that can be recruited to the reporter locus by altering dox concentration. As shown in Figure 4 – supplement 4, at lower dox concentrations, in the insulator reporters silencing of mCherry still occurs in cells where mCitrine is silenced. We find that although the fraction of the population that silences mCitrine is lower, in the cells with mCitrine silent, silencing spreads to the downstream mcherry within 5 days for both HDAC4 and KRAB. We have also collected additional flow cytometry data with KRAB recruitment to NS and 5kb reporters at lower dox concentrations. As seen with the insulator reporters, we find that lower dox concentration reduces the fraction of cells that silence but that in cells where mCitrine is silenced, mCherry silencing follows in both NS and 5kb reporters. Although the lower dox concentration slows down the rate of spreading, it does not alter coordinated dynamics of the two genes. In the NS reporter, both genes silence simultaneously at 4 ng/mL . In the 5kb reporter after 5 days at lower dox concentrations, mCitrine still silences first with slower spreading to mCherry, this progression can be seen in the 4 ng/mL and 10 ng/mL concentrations respectively .We have now added this additional data as Figure 1 —figure supplement 3, and it is referenced in the main text as follows:– Line 107, added sentence: “Tuning the probability of of KRAB recruitment at the reporter by varying dox concentration, we found that spreading of silencing still occurs in both the NS and the 5kb reporters, and the percentage of cells with both genes silenced increases with dox concentration .”New figure legend:Figure 1 —figure supplement 3. Silencing by KRAB at lower dox concentrations.(A-B) 2D scatter plots of mCitrine and mCherry fluorescence measured by flow cytometry in CHO-K1 cells with KRAB recruitment for 5 days at 4 ng/mL, 10 ng/mL and 1000 ng/mL in the (A) NS reporter or (B) 5kb reporter. Percent of cells in each quadrant is shown with standard error of mean (n=2).3. The authors should better explain why they chose KRAB and HDAC4 for their experiments. They should also comment on the histone deacetylase activity recruited by the KRAB-binding KAP1. KRAB is not a protein, but a domain, which often but not always recruits KAP1, and some KRAB-containing proteins also harbour other domains and some are not repressors. This should be explained as well to help the non-specialists situate the relevance of the work in a broader context.Indeed, we have modified the paragraphs introducing KRAB and HDAC4 to better reflect why we chose them, as well as the known biology of KRAB as follows (additions highlighted):New KRAB paragraph (lines 46-52): “The Kruppel associated box (KRAB) repressive domain from zinc finger 10 is commonly used in synthetic biology applications , is one of the strongest repressor domains in human cells , and is associated with spreading of heterochromatin and epigenetic memory through positive feedback mechanisms 8,15–22. KRAB-mediated gene silencing operates through recruitment of cofactors, including KAP1, HP1, and SETDB1, that read and write the repressive histone modification, histone 3 lysine 9 trimethylation (H3K9me3), creating a positive feedback loop that establishes stable gene silencing . Through this type of feedback mediated by the ability of KAP1 to recruit HP1, spreading of epigenetic effects beyond the target locus can affect nearby genes in a distance-dependent manner .Targeted recruitment of the KRAB domain from ZNF10 has been shown to repress gene expression, and lead to loss in histone 3 acetylation and gain of H3K9me3 across several tens to hundreds of kilobases around the target gene depending on the method and duration of recruitment . However, the spatial-temporal dynamics of these effects and the capacity of the commonly used cHS4 insulator to influence KRAB-mediated spreading of silencing have not been systematically characterized. In addition, the dynamics of reactivation at neighboring genes after removal of KRAB has also not been well characterized in a synthetic system with variable distance or insulators. Understanding the drivers of reactivation could also be important for diseases, where developmentally silenced genes reactivate(Das and Chadwick 2021).”New HDAC4 paragraph (previously at line 108): We wanted to compare the effects of KRAB with those of another fast silencer that is not associated with histone methylation positive feedback, and instead is only associated with removal of acetylation. We have previously shown that HDAC4 recruitment leads to fast gene silencing, comparable to that of KRAB. Therefore, we expected silencing of the targeted gene to occur at a similar rate as with KRAB, while differences in the downstream gene silencing would be due to the lack of positive feedback with HDAC4 recruitment.4. Line 74: "a set of mammalian cell lines" could be advantageously replaced by "two mammalian cell lines".We agree, it is better to specify exactly how many cell types we have tested. We have added additional data with another human cell line, HEK293T with the reporters integrated at the AAVS1 locus, as a new supplemental figure to Figure 1. As a result, we now have three cell lines, and, as suggested, we modified the text to remove “a set of mammalian cell lines” from the first sentence, and replace it with: “we site-specifically integrated our reporters in three cell types”, and also added “and HEK293T cells” to line 84.5. A statistical test should be performed to qualify the yellow and red subpopulations.We have now included a statistical test between the fraction of cells off in +dox/-dox for mCitrine and mCherry cell population replicates. The results from the statistical tests is now included in Appendix 1- table 1, which includes percent of cells off in each condition with the standard error of mean (SEM) and p-value from a Welch’s unequal variances T-test comparing the percent of cells off in -dox versus +dox for each gene. All comparisons were shown to be statistically significant (p<0.05), indicating that silencing of both genes is reproducible at the indicated timepoints.This is referenced in the main text  and the legend for Figure 1: “ A Welch’s unequal variances T-test comparing the percent of cells off in -dox versus +dox for each gene, showed that the percent of cells silenced were statistically significant and reproducible for NS and 5kb reporters in all cell types tested (Appendix 1 - table 1).”Note, that we also added a third replicate to the 20 day recruitment of HDAC4, which in our original visualization only had 2 replicates .New table legend:Appendix 1 - table 1. Statistics of reporters after silencing. Percent of cells off with standard error of mean (SEM) for each gene in the -dox and +dox condition after the number of days indicated, with threshold drawn in Figure 1 (dotted line). Welch’s unequal variances T-test was run on the percent cells off from each replicate (n=3) and p-value is shown for comparison of -dox versus +dox for each gene.6. A spacing of 0 or 5kb hardly qualifies as "various spacer lengths" as stated on line 79.We deleted the “various spacer lengths”. Instead, the text reads: “we separated the two fluorescent genes by either no spacer (NS)  or 5kb λ phage DNA (5kb) .”7. Lines 112-114: spreading from the HDAC4 docking site takes 20 days, is observed in only a fraction of cells, and the interpretation of the results is complicated by promoter interference intrinsic to the vector design (spreading is more efficient in the 5kb- than in the 0kb-spacing configuration). The data thus do not warrant the statement "these results show for the first time that silencing mediated by a histone deacetylase can also extend to nearby genes".It is true that most protein coding genes in mammalian cells are generally spaced farther apart. However these distances are common, and in fact desired, for synthetic circuits, including expression of antibiotic markers. We have modified the text to reflect this emphasis:“These results show that in a synthetic reporter system, where genes are separated by short distances up to 5kb, silencing mediated by a histone deacetylase can also extend to a nearby gene.”In addition, we have tested the effects of HDAC4 in a different human cell line (HEK293T) with the 5kb reporter integrated at the AAVS1 locus, and the silencing of mCherry occurs in 7 days, much faster than in K562 and more similar to CHO-K1. We added these data as a Figure 1 —figure supplement 2, as mentioned above.8. The models presented in 2F and 2G are not clear: The way the picture is drawn, it seems that loss of H3Kac is a temporal prerequisite of H3K9me3 and H3K27me3 deposition in both models. This might be true for HDAC4 , but not for KRAB.Indeed, good point, the removal of histone acetylation need not happen first, we have changed this cartoon to remove the implication that acetylation is a temporal prerequisite to methylation .9. Line 299: reference to the figure should be corrected .Thank you, we have corrected this error.10. Lines 331-333: "These results suggest that the insulators can help the downstream gene remain active in conditions where its silencing is already slow, such as after indirect silencing by HDAC4 recruitment in K562." Again, this hypothesis relies on so few observations  that it should be formulated with much caution.We have rephrased this sentence as follows:“These results suggest that in K562, where silencing by HDAC4 recruitment has slower dynamics compared to CHO, the cHS4 insulators can help the downstream gene mCherry remain active.”11. Lines 510-515: one cannot say that the referenced previous studies yielded "disparate" results, notably on the extent of KRAB-induced heterochromatin spreading. Compared to the present work, they covered far greater variety of settings relative to the strengths of the targeted promoters, their distance from the KRAB docking site and their immediate or broader chromatin environment, so that a broad range of phenotypes was expected.We have rephrased this sentence as follows:“Our results help frame the extent of spreading of KRAB-mediated silencing as a dynamic process that depends on the time and strength of recruitment as well as the level of activation at the target locus, which can be influenced by neighboring promoters and insulators.”12. The authors should integrate in their thinking results reported ten years ago . This work reported an extensive analysis of chromatin features conducive or not of KRAB-induced long-range silencing, making interesting conclusions on the "repression-primed" state of the most sensitive promoters and their paradoxical location in a surrounding of highly expressed genes, and on the failure of CTCF recruitment to exert any barrier effect on H3K9me3 spreading.We have integrated the findings from this work into our discussion:– Line 520, added text: “Additionally, in our system, we see that silencing is slower and occasionally inhibited by the presence of the cHS4 insulator, which contains high levels of acetylation. In our model, we propose that genes in active regions can also cooperate with neighboring genes in addition to insulators to oppose silencing by chromatin regulators. However, there are other factors at play in targeting of endogenous loci which affect gene silencing. In previous studies, targeting dCas9KRAB to hundreds of repeated sgRNA sites forms a large domain of H3K9me3 heterochromatin on the order of megabases in a few days, but does not result in widespread gene silencing, rather the silencing of genes is controlled by the loss of H3K27Ac and H3K4me3 . Additionally previous studies have also shown that while genes that are more susceptible to KRAB-mediated silencing with KAP1 have higher levels of repressive histone marks at the promoter and gene body, though surprisingly they have higher levels of active histone marks surrounding the gene , indicating that the genomic locus affects silencing by KRAB. Therefore, more work needs to be done to understand how the genomic and epigenetic environment surrounding a group of genes can affect their repressibility.13. The authors should also comment on KRAB zinc finger protein genes, many of which recruit KAP1 at their 3' end and are grouped in tightly packed clusters, yet can escape self- or mutually inflicted silencing. And on the interesting observation by K. Helin's group that the H3K9me1/2 demethylase PHF8 is recruited to the promoter of many of these genes, perhaps explaining this phenomenon (Mol Cell 2010 38: 165).We have added this to the discussion by editing sentence on line 521-523:Original: “First, in mammalian genetic circuits where multiple genes need to be integrated in the same cell, genes controlled by KRAB need to be placed far away from other genes to avoid unwanted interference and feedback.”New: “First, in mammalian genetic circuits where multiple genes need to be integrated together in a cell, genes controlled by KRAB need to either be placed far away from other genes to avoid unwanted interference and feedback, or other mechanisms of escaping silencing potentially through recruitment of H3K9me demethylases such as PHF8 should be considered .”14. The potential roles of histone acetylation functioning as insulator sequences require more rigorous testing and validation. Perhaps the author should mine some published genomic data to find potential correlations between various histone acetylation marks and heterochromatin vs euchromatin compartments in development or disease conditions.We agree that using genomic data to connect histone modifications with insulators which help protect euchromatin from heterochromatin is a good approach. A recent paper has done this and used ENCODE ChIP-seq data to identify insulators, and found candidates which were then screened for their ability to prevent transgene silencing. They additionally show that when tested in the presence of an HDAC inhibitor, the expression of the insulated transgene increased. We have added this in our discussion along with more references to previous studies showing that the HS4 insulator’s capabilities are associated with acetylation and previous models that have characterized the role of insulators as activating forces which combat repressive forces:– Line 547-549, add to sentence: “We can explain the function of insulators on spreading and background silencing by thinking of them as areas in an active chromatin state, that can for example recruit additional writers of acetylation(Zhao and Dean 2004), and other properties similar to promoters(Raab and Kamakaka 2010).”– Line 549-550, add: “It has been shown that the core region of the HS4 insulator has high levels of acetylation , and it has been previously proposed that the HS4 insulator protects from spreading of H3K9me3 through maintenance of high levels of acetylation . A recent study (Rudina and Smolke 2019) used ChIPseq ENCODE data of histone modifications, including the presence of active acetylation marks and absence of repressive methylation marks, to identify candidate insulator sequences which were then screened for their ability to prevent background transgene silencing. Additionally, when these insulators were tested in the presence of an HDAC inhibitor, the expression of the insulated transgene increased (Rudina and Smolke 2019).– Line 550, new paragraph: “Previous models have proposed the idea that insulators, similar to enhancers and transcription factors, are anti-silencers which oppose repressive forces , and transcriptional activators have been shown to insulate against heterochromatin and even drive reactivation . Our findings are in line with these models, and we further propose that active regions, such as insulators and promoters, cooperate with each other to inhibit gene silencing and drive gene reactivation.”We have also added text to reference previous work that has shown strong correlations between acetylation and euchromatin, line 127:– “Previous studies looking at histone modifications across the genome in different cell types have shown that active promoters and strong enhancers in euchromatin have high levels of acetylation , which is in agreement with the general histone code by which acetylation confers open and active regions of euchromatin, while methylation results in silenced and compact heterochromatin (Jenuwein and Allis 2001).”We have added information about facioscapulohumeral dystrophy (FSHD), which is a disease that is potentially caused by loss of spreading of heterochromatin causing aberrant gene activation:– Line 568, add to sentence: “and perhaps in understanding aging where loss of heterochromatin can lead to changes in gene expression (Villeponteau 1997) or in diseases such as facioscapulohumeral dystrophy (FSHD). FSHD is a neuromuscular disorder that is potentially caused by loss of spreading of heterochromatin causing aberrant gene activation.”Reviewer #1 (Recommendations for the authors):The authors should mention in the text of the paper what is known about the differences in half-life/stability of the mCitrine and mCherry reporter proteins. This is important to assess the kinetics of repression. While imaging and flow cytometry allow assessment of their expression at the single cell level, additional RT-qPCR experiments for mRNA levels for each gene would strengthen the analyses.We agree with the reviewer that the half-life/stability of the reporter proteins are important parameters for the kinetics of repression. Our reporter genes are fusions between H2B and a fluorescent protein: H2B-mCitrine and H2B-mCherry. Fusions between H2B and fluorescent proteins (H2B-FPs) are used for studying chromatin dynamics due to their high signal to noise ratio from nuclear localization and incorporation into nucleosomes without inhibiting cell cycle progression . H2B-FPs are very stable, with very long protein halflives on the order of weeks; therefore degradation is mostly driven by cell division . At the mRNA level, we have previously shown that the half-life of H2B-mCitrine mRNA transcript is about 3.9 hours as measured by inhibiting transcription with actinomycin D and quantifying mRNA overtime . Although we have not measured the lifetime of the H2B-mCherry mRNA our video data and our model from daily flow cytometry data shows that the delay between mCitrine and mCherry silencing (δ T) is close to zero in the NS reporters , suggesting that mCitrine and mCherry lifetimes are very close together. Small differences in mRNA stability could change silencing times of each gene but ultimately would shift the δ T delay equally for all reporter constructs, and therefore not change our conclusion that the delay increases with distance, which is also corroborated by our video results which measure silencing at the protein level.We have added this information into the main text as follows:– Line 73-76, first sentence edited:– Original: “ To study the spreading of transcriptional silencing and chromatin modifications, we engineered a set of mammalian cell lines containing two neighboring fluorescent reporter genes and a CR, with each gene driven by constitutive promoters .”– New: “To study the spreading of transcriptional silencing and chromatin modifications, we site-specifically integrated reporters consisting of two neighboring fluorescent reporter genes (mCitrine and mCherry) with each gene driven by a constitutive promoters . Both fluorescent proteins are fused to H2B; this approach increases the signal to noise ratio due to nuclear localization and is commonly used for studying chromatin dynamics . In each reporter cell line, we also stably integrated a recruitable rTetR fused to either KRAB or HDAC4.”– Line 195, added sentence: “Since H2B fusions to fluorescent proteins have long halflives on the order of weeks, their loss is mostly driven by cell division , which allows the silencing of the gene to be detected based on the change in fluorescent protein accumulation. Stitching together single-cell traces across cell divisions results in cumulative traces of fluorescent protein levels over time for specific lineages . The slope of these traces is then proportional to promoter activity, and its decrease can be used to determine silencing of a gene.”– Line 218, add to sentence:“… especially if we consider the additional time necessary for mRNA degradation of about ~4 hours included in the flow fits , as determined previously by inhibiting transcription with actinomycin D and quantifying the decay of H2B-mCitrine mRNA over time. Since we observed a delay time between mCherry and mCitrine silencing of less than 4 hrs after KRAB recruitment , we assumed that the H2B-mCherry mRNA degrades at a similar rate as H2BmCitrine.”The authors do not show kinetics and overall expression of the Dox-induced KRAB and HDAC proteins and how these levels compare with the endogenous cellular levels. If as may be expected, the levels are much higher than endogenous, how does this affect the interpretation of the results in terms of kinetics and spreading of repression?Indeed, we would expect the expression levels of our chromatin regulators to be higher than endogenous levels, and we also expect the amount of rTetR-CR recruited at the reporter to affect the dynamics of silencing.. In fact, with our dox inducible system we are able to tune the number of rTetR-KRAB or rTetR-HDAC4 proteins that can be recruited to the reporter locus by altering dox concentration. As shown in Figure 4 – supplement 4, at lower dox concentrations, in the insulator reporters silencing of mCherry still occurs in cells where mCitrine is silenced. We find that although the fraction of the population that silences mCitrine is lower, in the cells with mCitrine silent, silencing spreads to the downstream mcherry within 5 days for both HDAC4 and KRAB. We have also collected additional flow cytometry data with KRAB recruitment to NS and 5kb reporters at lower dox concentrations. As seen with the insulator reporters, we find that lower dox concentration reduces the fraction of cells that silence but that in cells where mCitrine is silenced, mCherry silencing follows in both NS and 5kb reporters. Although the lower dox concentration slows down the rate of spreading, it does not alter coordinated dynamics of the two genes. In the NS reporter, both genes silence simultaneously at 4 ng/mL . In the 5kb reporter after 5 days at lower dox concentrations, mCitrine still silences first with slower spreading to mCherry, this progression can be seen in the 4 ng/mL and 10 ng/mL concentrations respectively .We have now added this additional data as Figure 1 —figure supplement 3, and it is referenced in the main text as follows:– Line 107, added sentence: “Tuning the probability of of KRAB recruitment at the reporter by varying dox concentration, we found that spreading of silencing still occurs in both the NS and the 5kb reporters, and the percentage of cells with both genes silenced increases with dox concentration .”New figure legend:Figure 1 —figure supplement 3. Silencing by KRAB at lower dox concentrations.(A-B) 2D scatter plots of mCitrine and mCherry fluorescence measured by flow cytometry in CHO-K1 cells with KRAB recruitment for 5 days at 4 ng/mL, 10 ng/mL and 1000 ng/mL in the (A) NS reporter or (B) 5kb reporter. Percent of cells in each quadrant is shown with standard error of mean (n=2).The authors have investigated the role of the cSH4 insulator element, can they better justify why this one was chosen. As the properties of insulator elements are not as well described as promoters or enhancers, experiments with other insulators would be required before general statements can be made on their ability to block repression or contribute to reactivation of gene expression.We agree with this comment and we have:1) modified the text to better explain why we chose the cHS4 element , and 2) edited the conclusions we draw about the ability to block repression to refer only to the cHS4 insulator and its derivatives we have tested as indicated below.We have added more reasoning as to why we chose to use the cHS4 insulator:– Line 44, add sentence: “The cHS4 insulator is a commonly used insulator in synthetic biology in mammalian cells , as it has been shown act as a barrier against heterochromatin , thus preventing transgene silencing in many cell lines including CHO and K562 .”– Line 276, edit first part of sentence to : “The cHS4 insulator is a commonly used insulator for protecting from transgene silencing in many cell lines; …”We have clarified the conclusions are restricted to the cHS4 insulator:– Line 62: “different insulator arrangements” changed to “the cHS4 insulator and its core region”– Lines 285, 289, 291, 298, 302, 318, 320, 322, 324, 326, 334, 337, 346, 354, 372, 385,389, 395, 504 specify: “cHS4 insulator(s)”/”insulator configurations” in place of “insulator(s)”Line 473: “Similar to promoters, the core region of the cHS4 insulator”– Modified Figure 6B-D and 6HandI to replace “insulator core” with “cHS4 insulator core”.In Figure 6 legend, we changed “Insulators” to “The core element of the cHS4insulator”Line 555, edit sentence:– Original: “Screening different insulators in a similar recruitment assay in the future could help go beyond the binary classification of sequences as insulators or non-insulators and actually quantify how well they perform against different mechanisms of silencing.”– New: “Screening different sequences with insulating function, such as Matrix Attachment Regions (MARs) and ubiquitous chromatin opening elements(UCOEs)(Rudina and Smolke 2019), along with other insulators such as H19Igf2 and SNS , in a similar recruitment assay in the future could help go beyond the binary classification of sequences as insulators or non-insulators and help specify and quantify how well they perform against different mechanisms of silencing.”It is now well accepted that regulation of gene expression is mainly driven by enhancer elements. The reporter plasmid used in this study comprises only proximal promoter elements. Do the authors know whether their promoter activity is driven/influenced by distal enhancers located around the integration sites? More specifically it would be informative to generate a reporter with a strong enhancer element and assess how this impacts repression. Insertion of an enhancer between the two genes may reveal whether it can block the spreading of repression to the mCHERRY gene.We agree this would be an interesting idea to test, potentially in a high-throughput screen that could look at different enhancers as well as different insulators. We have added this idea to the discussion:– Line 559, add sentence: “Additionally, it would also be interesting to test enhancers between the two reporter genes to study whether they can also block spreading of silencing to the downstream gene.”We’ve also highlighted previous work on the effect of recruiting KRAB to an enhancer:– Line 516: “For example, when KRAB is recruited for a short period at an enhancer in the hemoglobin locus in K562, the extent of spreading of methylation is very small (<1 kb).”Reviewer #2 (Recommendations for the authors):Introduction:1. The authors should better explain why they chose KRAB and HDAC4 for their experiments. They should also comment on the histone deacetylase activity recruited by the KRAB-binding KAP1. KRAB is not a protein but a domain, which often but not always recruits KAP1, and some KRAB-containing proteins also harbour other domains and some are not repressors. This should be explained as well to help the non-specialists situate the relevance of the work in a broader context.Indeed, we have modified the paragraphs introducing KRAB and HDAC4 to better reflect why we chose them, as well as the known biology of KRAB as follows:New KRAB paragraph (lines 46-52): “The Kruppel associated box (KRAB) repressive domain from zinc finger 10 is commonly used in synthetic biology applications , is one of the strongest repressor domains in human cells , and is associated with spreading of heterochromatin and epigenetic memory through positive feedback mechanisms 8,15–22. KRAB-mediated gene silencing operates through recruitment of cofactors, including KAP1, HP1, and SETDB1, that read and write the repressive histone modification, histone 3 lysine 9 trimethylation (H3K9me3), creating a positive feedback loop that establishes stable gene silencing . Through this type of feedback mediated by the ability of KAP1 to recruit HP1, spreading of epigenetic effects beyond the target locus can affect nearby genes in a distance-dependent manner .Targeted recruitment of the KRAB domain from ZNF10 has been shown to repress gene expression, and lead to loss in histone 3 acetylation and gain of H3K9me3 across several tens to hundreds of kilobases around the target gene depending on the method and duration of recruitment . However, the spatial-temporal dynamics of these effects and the capacity of the commonly used cHS4 insulator to influence KRAB-mediated spreading of silencing have not been systematically characterized. In addition, the dynamics of reactivation at neighboring genes after removal of KRAB has also not been well characterized in a synthetic system with variable distance or insulators.Understanding the drivers of reactivation could also be important for diseases, where developmentally silenced genes reactivate(Das and Chadwick 2021).”New HDAC4 paragraph (previously at line 108) : We wanted to compare the effects of KRAB with those of another fast silencer that is not associated with histone methylation positive feedback, and instead is only associated with removal of acetylation. We have previously shown that HDAC4 recruitment leads to fast gene silencing, comparable to that of KRAB . Therefore, we expected silencing of the targeted gene to occur at a similar rate as with KRAB, while differences in the downstream gene silencing would be due to the lack of positive feedback with HDAC4 recruitment.Regarding Figure 11. Line 74: "a set of mammalian cell lines" could be advantageously replaced by "two mammalian cell lines".We agree, it is better to specify exactly how many cell types we have tested. We have added additional data with another human cell line, HEK293T with the reporters integrated at the AAVS1 locus, as a new supplemental figure to Figure 1. As a result, we now have three cell lines, and, as suggested, we modified the text to remove “ a set of mammalian cell lines” from the first sentence, and replace it with: “we site-specifically integrated our reporters in three cell types”, and also added “and HEK293T cells” to line 84.2. A statistical test should be performed to qualify the yellow and red subpopulations.We have now included a statistical test between the fraction of cells off in +dox/-dox for mCitrine and mCherry cell population replicates. The results from the statistical tests is now included as a Appendix 1 - table 1, which includes percent of cells off in each condition with the standard error of mean (SEM) and p-value from a Welch’s unequal variances T-test comparing the percent of cells off in -dox versus +dox for each gene. All comparisons were shown to be statistically significant (p<0.05), indicating that silencing of both genes is reproducible at the indicated timepoints.This is referenced in the main text  and the legend for Figure 1: “A Welch’s unequal variances T-test comparing the percent of cells off in -dox versus +dox for each gene, showed that the percent of cells silenced were statistically significant and reproducible for NS and 5kb reporters in all cell types tested (Appendix 1 - table 1).”Note, that we also added a third replicate to the 20 day recruitment of HDAC4, which in our original visualization only had 2 replicates .New table legend:Appendix 1 - table 1. Statistics of reporters after silencing. Percent of cells off with standard error of mean (SEM) for each gene in the -dox and +dox condition after the number of days indicated, with threshold drawn in Figure 1 (dotted line). Welch’s unequal variances T-test was run on the percent cells off from each replicate (n=3) and p-value is shown for comparison of -dox versus +dox for each gene.3. A spacing of O or 5kb hardly qualifies as "various spacer lengths" as stated on line 79.We deleted the “various spacer lengths”. Instead, the text reads: “we separated the two fluorescent genes by either no spacer (NS)  or 5kb λ phage DNA (5kb) .”4. Lines 111-112: "Moreover, in K562 cells, HDAC4-mediated silencing of mCherry was much slower, taking up to 20 days, compared to 5 days in CHO-K1 cells . Any lead to explain the difference? Shouldn't other chromosomal loci be tested before attributing the difference to the cellular background?We agree that in general we expect both genomic locus and cell type to be able to affect the speed of spreading, and we have emphasized in our discussion the interesting results at different genomic loci reported in , please see response to Discussion point 5 below.In the case of HDAC4 recruitment, we believe that the silencing of pRSV-mCherry is due to background silencing of the pRSV promoter by PRC2 in the absence of protective activation by pEF. pRSV promoter is stronger in K562 cells, as indicated by higher mCherry expression compared to CHO-K1 cells , therefore it likely can resist this background silencing better. This is consistent with the fact that in K562 we also see faster reactivation of the pRSV-mcherry . In support of this point, we also added text pointing to previous work comparing the two promoters: “In line with our hypothesis, previous studies have shown that the pRSV promoter is more prone to transgene background silencing than the pEF promoter when integrated into mammalian genomes .”Moreover, we were curious what happens if we change the cellular background while still having the reporter integrated at the AAVS1 locus: so we created HEK293T lines with the 5kb reporter at the AAVS1 locus, and recruited either KRAB and HDAC4 . We find that KRAB-mediated silencing can consistently spread over 5kb, consistent with the CHO-HAC and K562 AAVS1 results. With HDAC4-mediated recruitment at the 5kb dual reporter in HEK293T at the AAVS1 locus, the majority of cells that silence mCitrine also silence the downstream mCherry within 7 days, similar to the CHO-HAC system, and much faster than in K562. These results suggest that cellular background can play a strong role in the speed of silencing and spreading, as all other factors in the systems are held constant such as reporter set up and genomic locus.We have edited the paragraph from line 86-114 to reflect the addition of these new data:Lines 86-90: “To investigate the spreading of transcriptional silencing, we first recruited KRAB upstream of the mCitrine gene for 5 days in CHO-K1, K562 and HEK293T cell lines with the addition of dox . Using flow cytometry to measure fluorescence intensity, we observed silencing of both the upstream mCitrine gene and the downstream mCherry gene in the NS  and 5kb  reporter lines for the cell types tested.–Line 112, add sentences: “ With HDAC4-mediated recruitment at the 5kb dual reporter in HEK293T at the AAVS1 locus, the majority of cells that silence mCitrine also silence the downstream mCherry within 7 days , similar to the CHO-HAC system, and much faster than in K562. These results suggest that cellular background can play a strong role in the speed of silencing and spreading, as all other factors in the systems are held constant such as reporter set up and genomic locus.”New figure legend:Figure 1 —figure supplement 2. Recruitment of KRAB and HDAC4 to 5kb reporter in HEK293T.(A-B) Fluorescence distributions of mCitrine and mCherry measured by flow cytometry either without CR recruitment (-dox) or after 5 or 7 days of recruitment (+dox) of either (A) KRAB or (B) HDAC4 at the 5kb reporters in HEK293T. Biological replicates of multiclonal populations are shown as overlaid semi-transparent distributions (n=3).5. Lines 112-114: spreading from the HDAC4 docking site takes 20 days, is observed in only a fraction of cells, and the interpretation of the results is complicated by promoter interference intrinsic to the vector design (spreading is more efficient in the 5kb- than in the 0kb-spacing configuration). The data thus do not warrant the statement "these results show for the first time that silencing mediated by a histone deacetylase can also extend to nearby genes". Rare are the pairs of genes, the promoters of which are at such short distances.It is true that most protein coding genes in mammalian cells are generally spaced farther apart. However these distances are common, and in fact desired, for synthetic circuits, including expression of antibiotic markers. We have modified the text to reflect this emphasis:“These results show that in a synthetic reporter system, where genes are separated by short distances up to 5kb, silencing mediated by a histone deacetylase can also extend to a nearby gene.”In addition, we have tested the effects of HDAC4 in a different human cell line (HEK293T) with the 5kb reporter integrated at the AAVS1 locus, and the silencing of mCherry occurs in 7 days, much faster than in K562 and more similar to CHO-K1. We added these data as a Figure 1 —figure supplement 2, as mentioned above.Regarding Figure 21. The models presented in 2F and 2G are not clear: The way the picture is drawn, it seems that loss of H3Kac is a temporal prerequisite of H3K9me3 and H3K27me3 deposition in both models. This might be true for HDAC4 , but not for KRAB.Indeed, good point, the removal of histone acetylation need not happen first, we have changed this cartoon to remove the implication that acetylation is a temporal prerequisite to methylation .2. Lines 181-183: "… that can lead to spreading of these chromatin modifications . This observation suggests that silencing of the RSV promoter is indirectly due to PRC2 action, but it can only take place once HDAC4 removes acetylation at the locus." Artificial recruitment of PRC2 at this locus in the absence of HDAC4 could be used to validate this hypothesis.We agree that recruitment of PRC2 to our reporter would help support our hypothesis. We generated a CHO-K1 cell line where EED, from PRC2, is recruited upon addition of dox. We see that upon EED recruitment to the NS reporter that both genes are silenced . We go on further to test the effects of PRC2 inhibition on HDAC4mediated silencing (see comments to point 3 regarding figure 3).Regarding Figure 3The authors used time-lapse movies to analyse the spreading of epigenetic marks with high temporal resolution. They conclude that KRAB-mediated spreading of transcriptional silencing is distance-dependent in CHO-K1 cells while HDAC4-mediated spreading is distance-independent at these length scales.1. A limitation of time-lapse movies is the low output. However, the authors developed a mathematical model to optimize the information recovered from "high output" data (flow cytometry combined with gene expression). The output of this method is nicely consistent with time-lapse movies opening perspectives to use this system on a larger scale. A useful contribution to the field!Thank you!2. There should be published data / databases allowing to verify that in broader contexts HDAC inhibition triggers silencing likely via H3K27me3 deposition (Line 176), rather than it being a peculiarity of the dual system used here.We’ve modified the text to describe published results which support our hypothesis that acetylation removal can lead to PRC2-associate chromatin modifications: both here (line 183) and in our Discussion section:– Added two sentences at line 183: “ In accordance with our hypothesis, previous studies have shown that by co-ChIP of primary cells H3K27me3 and H3Ac are negatively correlated , that lack of transcription and histone deacetylation both drive an increase in H3K27me3 deposition steadily over time , and that recruitment of HDAC1 leads to repressed chromatin associated with a strong increase in H3K27me3 . Additionally, knockdown of p300, a histone acetyltransferase, resulted in a global decrease of H3K27Ac in tandem with an increase in H3K27me3 .”– In Discussion section, line 536 added: “In accordance with our observations, previous studies have shown that H3K27me3 and H3Ac are negatively correlated on individual nucleosomes , and specifically that deacetylation by either lack of transcription or recruitment of HDAC1 leads to increases in H3K27me3 . Additionally, studies where cells are treated with HDAC inhibitor show in an increase in histone acetylation which is correlated with reduced recruitment of EZH2, EED and Suz12 proteins from the PRC2 as well as a decrease in H3K27me3 , showing the bidirectionality of the inverse correlation between H3K27me and H3K27Ac . This supports our observation that low levels of acetylation allow PRC2 to work more effectively.”3. Further down (lines 178-180) it is stated that "This repressive modification, which is associated with polycomb repressive complex 2 (PRC2), has not been reported in association with HDAC4, and was not observed after HDAC4 recruitment at a pEF-mCitrine reporter flanked by insulators in the same locus of CHO-K1 cells". To consolidate their hypothesis, the authors could test silencing of the locus in the presence of EZH2 inhibitor to see in which extent it depends on PRC2, and measure PRC2 enrichment by CUTandTAG or CUTandRUN.Thank you for this suggestion! To further test this hypothesis of PRC2-mediated silencing of the downstream pRSV-mCherry gene, we measured HDAC4-mediated silencing in the presence of EZH2 inhibitor Tazemetostat (Taz). We found that Taz blocked pRSV-mCherry silencing, while not affecting the pEF-mCitrine silencing. These results support the idea that pRSV-mCherry silencing we observe after HDAC4-mediated silencing of the pEF-mCitrine results from PRC2 mediated silencing of the pRSV-mCherry. We have added these data to Figure 2F-H as well as new supplemental Figure 2 —figure supplement 3. Additions to the main text include:– Line 180, added text: “To test whether the silencing of mCherry was mediated by PRC2, we recruited HDAC4 in the presence of Tazemetostat (Taz), a small molecule inhibitor of EZH2, which is the H3K27 methyltransferase in PRC2 . First, we tested Taz in the NS reporter line with recruitment of EED, the H3K27 reader in PRC2, and showed that inhibition reduced silencing of mCitrine and mCherry . We found that in the presence of dox and Taz while the mCitrine gene was still silenced by HDAC4, the mCherry gene was not silenced and even slightly increased in expression in the 5kb reporter  and the NS reporter .– Line 419, add to sentence: “loss of pRSV-mCherry silencing upon PRC2 inhibition, and the fact that the silencing delay is distance-independent.”– Line 536, add to sentence: “which was confirmed by the loss of mCherry silencing when PRC2 was inhibited .“– Line 21, edit sentence in abstract to reflect the role of PRC2: “Silencing by histone deacetylase HDAC4 of the upstream gene can also facilitate background silencing of the downstream gene by PRC2, but with a days-long delay that does not change with distance.”– Edits to legend of Figure 2:(F) Addition of Tazemetostat (Taz) inhibits the EZH2 methyltransferase from PRC2. (G) Fluorescence distributions of mCitrine (left) and mCherry (right) measured by flow cytometry either without CR recruitment  or after 7 days of HDAC4 recruitment  and with Taz at the 5kb reporter in CHO-K1. (H) Percentages of cells OFF normalized by the -dox+DMSO condition, based on threshold shown in panel G (dotted line).(I) In the absence of CR recruitment, both genes have H3Ac across the reporter (top). Upon recruitment of KRAB (bottom left), we see a loss of H3Kac and gain of H3K9me3 across the dual-gene reporter through both DNA looping from rTetR-KRAB as well as positive feedback loops for spread of methylation, resulting in a distance-dependent delay of transcriptional silencing between two genes. Upon recruitment of HDAC4 (bottom right), we see a loss of H3Kac as well as a gain of H3K27me3 across the reporter through positive feedback loops for spread of methylation by PRC2, resulting in distance-independent delay of transcriptional silencing between two genes.New figure legend:Figure 2 —figure supplement 3. EZH2 inhibitor Tazemetostat affects silencing after recruitment of EED and HDAC4.(A) Addition of Tazemetostat (Taz) inhibits the EZH2 methyltransferase from PRC2. (B-C) Fluorescence distributions of mCitrine (top left) and mCherry (bottom left) measured by flow cytometry either without CR recruitment  or after 7 days of (B) EED or (C) HDAC4 recruitment  and with Taz at the NS reporter in CHO-K1. Percentages of cells OFF normalized by the -dox+DMSO condition (right), based on threshold (dotted line).4. Lines 223-226: "The observation that the delays of pRSV-mCherry silencing do not increase with distance  suggests that its silencing initiates at the RSV promoter, most likely by the action of PRC2 .". If the silencing starts from the RSV promotor, why is it different without the 5Kb spacer? Can the dynamics of H3K27me3 deposition at the RSV promotor be assessed on both constructs? What would be the mechanism leading to the recruitment of PRC2 at the RSV instead of the pEF since HDAC4 is recruited at pEF? What is the effect of inverting the two promoters?We show that although the silencing dynamics of KRAB silencing are dependent on distance between the two genes, with HDAC4 recruitment the silencing of the pRSVmCherry gene is independent of distance. If PRC2 were brought by HDAC4 at the pEF, we would expect the pRSV-mCherry silencing to take longer in the 5kb construct than in the NS, just as we see with KRAB (as spreading of the modifications depends on distance).Therefore, our hypothesis is that PRC2 gets recruited at the pRSV promoter independently of HDAC4, at all times. We have illustrated this point more clearly in Figure 6 (by adding PRC2 above the background repressive arrow targeting the pRSV-mCherry). The pEF promoter when active helps prevent background silencing of the pRSV-mCherry gene by PRC2 . Therefore, when HDAC4 silences the pEF-mCitrine gene, the pEF can no longer prevent the background silencing of the downstream pRSV-mCherry gene by PRC2 . This hypothesis is supported by the additional PRC2 inhibitor data we collected . If PRC2 were recruited directly by HDAC4 near the pEF promoter, we would expect the silencing delay to increase with distance, as we see with KRAB; however, we see that the delay with HDAC4 recruitment is distance-independent. Therefore, the observation that the delays of pRSV-mCherry silencing do not increase with distance upon HDAC4mediated silencing , together with the loss of mCherry silencing but not mCitrine silencing upon HDAC4 recruitment in the presence of Taz , suggests that the silencing of mCherry is initiated at the pRSV promoter, by the action of PRC2 .”– We have also clarified an earlier sentence on line 177-183 that was inadvertently misleading the reader to think we propose HDAC4 directly recruits PRC2:Original: “This repressive modification, which is associated with polycomb repressive complex 2 (PRC2), has not been reported in association with HDAC4, and was not observed after HDAC4 recruitment at a pEF-mCitrine reporter flanked by insulators in the same locus of CHO-K1 cells. Like H3K9me3 , H3K27me3 has reader-writer positive feedback that can lead to spreading of these chromatin modifications . This observation suggests that silencing of the RSV promoter is indirectly due to PRC2 action, but it can only take place once HDAC4 removes acetylation at the locus.”New version: “This repressive modification is associated with polycomb repressive complex 2 (PRC2), and not with HDAC4. We have previously observed at a pEF-mCitrine reporter flanked by insulators in the same locus of CHO-K1 cells that HDAC4 recruitment does not lead to deposition of H3K27me3 . Therefore, we hypothesized that the pRSV promoter gets silenced not by HDAC4, but by PRC2 after the pEF-mCitrine gene is silenced. Once the H3K27me3 modification is present, like H3K9me3 , H3K27me3 also has reader-writer positive feedback that can lead to spreading of these chromatin modifications .”Measuring the dynamics of H3K27me3 deposition by CUTandRUN are difficult to capture and to quantify as a function of time for such a heterogeneous population where some cells silence pRSV at 2 days and some at 3-5 days. We see these silencing times in movies where we can measure the fluorescent protein production rate in single cells; however, we cannot sort on these rates, since it takes time for the H2B-FP to dilute after silencing. In a bulk measurement of histone modifications, cells that have silenced and likely have histone modifications are mixed with cells that are not yet silenced and do not have modifications present. So the signal would likely be an average measurement of a heterogeneous population and not reflective of the true dynamics of the spread of the histone modifications.We have also added evidence from previous works showing that the pRSV promoter is more prone to transgene silencing than the pEF promoter:– Line 419, add sentence: “In line with our hypothesis, previous studies have shown that the pRSV promoter is more prone to transgene background silencing than the pEF promoter when integrated into mammalian genomes .”Additionally we agree that changing the promoters would be an interesting experiment to further test and expand our hypotheses summarized in Figure 6, and have added this point to our discussion.– Line 539, add sentence: “Testing reporters with inverted promoters as well as testing other promoter combinations and configurations would be informative for building synthetic constructs where genes are in close proximity.”Regarding Figure 4In this figure, the authors document the lack of effect of cHS4 insulators on KRAB/HDAC4-induced silencing in CHO cells, but observe some impact on the latter in K562 cells.1. Line 299: reference to the figure should be corrected .Thank you, we have corrected this error.2. Lines 331-333: "These results suggest that the insulators can help the downstream gene remain active in conditions where its silencing is already slow, such as after indirect silencing by HDAC4 recruitment in K562." Again, this hypothesis relies on so few observations  that it should be formulated with much caution.We have rephrased this sentence as follows: “These results suggest that in K562, where silencing by HDAC4 recruitment has slower dynamics compared to CHO, the cHS4 insulators can help the downstream gene mCherry remain active.”Regarding Figure 5Here the authors investigate the re-activation of the genes in their system. Again, they formulate overly general conclusions based on a very limited set of parameters. Lines 395-398: "These results show that the extent of epigenetic memory depends strongly not only on the chromatin regulator recruited, but also on the configurations of promoters and insulators at the target locus, with stronger promoters closely surrounded by core insulators diminishing memory".We have rephrased this sentence as follows: “These results show that the extent of epigenetic memory in our system depends on the chromatin regulator recruited and the configurations of promoters and insulators at the target locus, with the stronger pEF promoter closely surrounded by cHS4 insulators diminishing memory.”Regarding Figure 6 (Model)The model is fine as long as it is clearly restricted to the parameters tested here, and not proposed as a paradigm.We agree that the original text introducing the model in Figure 6 was phrased in terms that were too general. We have now clarified that this model is a way of summarizing and making sense of all our results, and removed all generalizations throughout this section of the results and Figure 6, as follows:– Original, Lines 401-404: “We used our gene dynamics and chromatin modifications data to develop a generalizable kinetic model that captures distance-dependent silencing associated with a tethered chromatin regulator and distills the roles of promoters and insulators as elements associated with high reactivation rates.” New version: “We developed a kinetic model that summarizes our observations of gene dynamics and chromatin modifications as a competition between the distancedependent silencing rates associated with each tethered chromatin regulator and the reactivation rates driven by our promoters and insulators.”– Lines 428: “In summary, in our system both silencing and reactivation rates are affected by the distance between two genes.”– Line 463: “In our system, promoters can be thought of as regions that are associated with high rates of gene activation and can drive reactivation after silencing.– Line 473: “Similar to promoters, the core region of the cHS4 insulator can be modeled as a DNA element that increases the rate of reactivation of nearby genes in our setup in a distance-dependent manner . This action, combined with our promoter reactivation rates,”– Line 483: “Thinking of the promoters and core insulators in our synthetic constructs as elements associated with higher reactivation rates”– Modified Figure 6B-D and 6HandI to replace “insulator core” with “cHS4 insulator core”.– In Figure 6 legend, we changed “Insulators” to “The core element of the cHS4 insulator”This phrasing now better matches the overall text in this section and the rest of the figure, where we refer to the specific elements used in our experiments: pEF, pRSV, cHS4 insulator core, KRAB, HDAC4, rather than general promoters, insulators or chromatin regulator.Along the same line, we modified our abstract to replace “We propose a new model” with “Our data can be described by a model”. In addition, we have modified the discussion heavily to clearly delineate how this model that summarizes our data connects with previous experimental results and previous models proposed in the literature .Discussion1. Lines 510-515: one cannot say that the referenced previous studies yielded "disparate" results, notably on the extent of KRAB-induced heterochromatin spreading. Compared to the present work, they covered far greater variety of settings relative to the strengths of the targeted promoters, their distance from the KRAB docking site and their immediate or broader chromatin environment, so that a broad range of phenotypes was expected, without this heterogeneity being shocking (which is what the term "disparate" suggests).We have rephrased this sentence as follows: “ Our results help frame the extent of spreading of KRAB-mediated silencing as a dynamic process that depends on the time and strength of recruitment as well as the level of activation at the target locus, which can be influenced by neighboring promoters and insulators.”2. Lines 560-561: "By monitoring gene reactivation dynamics after release of the CRs, we conclude that activation can also spread to nearby genes in a distance-dependent manner." Only one distance (5Kb) was tested, which pales with the thousands of distances evaluated in some previously published studies.Increasing the distance between a target gene and the KRAB recruitment site has been shown to decrease the susceptibility of a gene to be silenced , which we have added in the silencing portion (see section in introduction and discussion). However, here we are specifically referring to reactivation of genes after release of the silencing CR . To our knowledge there have not been studies looking at the effect of distance and the cHS4 insulator on reactivation.We have also emphasized this aspect of reactivation in the introduction, line 40-42, edit sentence:“ To understand the effects of intergenic distance and insulators on spreading, we studied the dynamics of spreading of gene silencing and reactivation after recruitment and release, respectively, of different types of CRs to a dual-gene reporter .”3. Lines 571-572: "They suggest that genes in close proximity (<5-10kb) can respond to signals in a coordinated manner, during both silencing and activation". Right, although confirmatory of a large series of observations previously made on co-regulated gene clusters.We have rephrased this sentence to emphasize that this coordinated response, which has been studied on co-regulated gene clusters endogenously is also seen in synthetic dualgene constructs:“They suggest that genes in close proximity (<5-10kb) in a synthetic system can respond changes in chromatin regulators recruited at the promoter of one gene in a coordinated manner, during both silencing and activation, similar to endogenous gene clusters where neighboring genes tend to be co-regulated,including cell cycle, circadian rhythm and housekeeping genes .”We have also edited the first sentence of our abstract to:“In mammalian cells genes that are in close proximity can be transcriptionally coupled: silencing or activating one gene can affect its neighbors.”We have also added previous observations about co-regulated ZNF-KRAB clusters, line 524:“… as may be the case in regulation of zinc finger gene clusters which are auto- and coregulated by KAP1 recruitment ”4. Lines 579- 582: "Finally, this experimental and theoretical framework can serve as a starting point for measuring and modeling the effects of targeting various epigenetic editors at endogenous loci in order to guide the time necessary for efficient on-target effects without unwanted off-target spreading". The authors fail to demonstrate convincingly that their system is really suited for the kind of high throughput analyses that would be necessary to consolidate, give a general value or even validate some of their conclusions.We agree that future work on systematically studying dynamics at endogenous loci would require a different experimental system, such as dCas9. We have edited this sentence accordingly: “Finally, our experimental results and theoretical framework highlight the need for further measuring and modeling the dynamics of gene expression after targeting various epigenetic editors at endogenous loci systematically,  in order to determine the time necessary for efficient non-target effects without unwanted off-target spreading.”5. The authors should integrate in their thinking results reported ten years ago . This work proceeded to an extensive analysis of chromatin features conducive or not of KRAB-induced long-range silencing, making interesting conclusions on the "repression-primed" state of the most sensitive promoters and their paradoxical location in a surrounding of highly expressed genes, and on the failure of CTCF recruitment to exert any barrier effect on H3K9me3 spreading.We have integrated the findings from this work into our discussion:– Line 520, added text: “Additionally, in our system, we see that silencing is slower and occasionally inhibited by the presence of the cHS4 insulator, which contains high levels of acetylation. In our model, we propose that genes in active regions can also cooperate with neighboring genes in addition to insulators to oppose silencing by chromatin regulators. However, there are other factors at play in targeting of endogenous loci which affect gene silencing. In previous studies, targeting dCas9-KRAB to hundreds of repeated sgRNA sites forms a large domain of H3K9me3 heterochromatin on the order of megabases in a few days, but does not result in widespread gene silencing, rather the silencing of genes is controlled by the loss of H3K27Ac and H3K4me3 . Additionally previous studies have also shown that while genes that are more susceptible to KRAB-mediated silencing with KAP1 have higher levels of repressive histone marks at the promoter and gene body, though surprisingly they have higher levels of active histone marks surrounding the gene , indicating that the genomic locus affects silencing by KRAB. Therefore, more work needs to be done to understand how the genomic and epigenetic environment surrounding a group of genes can affect their repressibility.6. The authors should also comment on KRAB zinc finger protein genes, many of which recruit KAP1 at their 3' end and are grouped in tightly packed clusters, yet can escape self- or mutually inflicted silencing. And on the interesting observation by K. Helin's group that the H3K9me1/2 demethylase PHF8 is recruited to the promoter of many of these genes, perhaps explaining this phenomenon (Mol Cell 2010 38: 165).We have added this to the discussion by editing sentence on line 521-523:Original: “First, in mammalian genetic circuits where multiple genes need to be integrated in the same cell, genes controlled by KRAB need to be placed far away from other genes to avoid unwanted interference and feedback.”New: “First, in mammalian genetic circuits where multiple genes need to be integrated together in a cell, genes controlled by KRAB need to either be placed far away from other genes to avoid unwanted interference and feedback, or other mechanisms of escaping silencing potentially through recruitment of H3K9me demethylases such as PHF8 should be considered .”Reviewer #3 (Recommendations for the authors):As noted in the public comments, the authors should include a more critical discussion of the differences of this model with prior studies where KRAB domains were recruited to natural genomic sequences.We agree with the suggestion to discuss in more detail KRAB recruitment to natural genomic sequences. We have added in the main text (line 526):– Previous work has shown that dCas9-KRAB targeting of promoters or enhancers results in silencing of the associated gene along with enrichment of H3K9me3 at the targeted locus . Targeting of promoters of endogenous genes appears to result in spreading of H3K9me3 across the gene body , while targeting enhancers can result in either H3K9me3 only at the enhancer  or only at the target gene . However, we are not aware of ChIP-seq data after dCas-KRAB recruitment to an endogenous promoter to determine the extent of spreading of H3K9me3 beyond the gene body.– We have also added more details regarding how our model differs from previous KRAB studiesLine 571-572, original: “They suggest that genes in close proximity (<5-10kb) can respond to signals in a coordinated manner, during both silencing and activation...”New: “They suggest that genes in close proximity (<5-10kb) in a synthetic gene system can respond to changes in chromatin regulators recruited at the promoter of one gene in a coordinated manner, during both silencing and activation…”The potential roles of histone acetylation functioning as insulator sequences require more rigorous testing and validation. Perhaps the author should mine some published genomic data to find potential correlations between various histone acetylation marks and heterochromatin vs euchromatin compartments in development or disease conditions.We agree that using genomic data to connect histone modifications with insulators which help protect euchromatin from heterochromatin is a good approach. A recent paper has done this and used ENCODE ChIP-seq data to identify insulators, and found candidates which were then screened for their ability to prevent transgene silencing. They additionally show that when tested in the presence of an HDAC inhibitor, the expression of the insulated transgene increased. We have added this in our discussion along with more references to previous studies showing that the HS4 insulator’s capabilities are associated with acetylation and previous models that have characterized the role of insulators as activating forces which combat repressive forces:– Line 547-549, add to sentence: “We can explain the function of insulators on spreading and background silencing by thinking of them as areas in an active chromatin state, that can for example recruit additional writers of acetylation (Zhao and Dean 2004), and other properties similar to promoters(Raab and Kamakaka 2010).”– Line 549-550, add: “It has been shown that the core region of the HS4 insulator has high levels of acetylation , and it has been previously proposed that the HS4 insulator protects from spreading of H3K9me3 through maintenance of high levels of acetylation . A recent study (Rudina and Smolke 2019) used ChIP-seq ENCODE data of histone modifications, including the presence of active acetylation marks and absence of repressive methylation marks, to identify candidate insulator sequences which were then screened for their ability to prevent background transgene silencing. Additionally, when these insulators were tested in the presence of an HDAC inhibitor, the expression of the insulated transgene increased (Rudina and Smolke 2019).– Line 550, new paragraph: “Previous models have proposed the idea that insulators, similar to enhancers and transcription factors, are anti-silencers which oppose repressive forces , and transcriptional activators have been shown to insulate against heterochromatin and even drive reactivation . Our findings are in line with these models, and we further propose that active regions, such as insulators and promoters, cooperate with each other to inhibit gene silencing and drive gene reactivation.”We have also added text to reference previous work that has shown strong correlations between acetylation and euchromatin, line 127:– “Previous studies looking at histone modifications across the genome in different cell types have shown that active promoters and strong enhancers in euchromatin have high levels of acetylation , which is in agreement with the general histone code by which acetylation confers open and active regions of euchromatin, while methylation results in silenced and compact heterochromatin (Jenuwein and Allis 2001).”We have added information about facioscapulohumeral dystrophy (FSHD), which is a disease that is potentially caused by loss of spreading of heterochromatin causing aberrant gene activation:– Line 568, add to sentence: “and perhaps in understanding aging where loss of heterochromatin can lead to changes in gene expression (Villeponteau 1997) or in diseases such as facioscapulohumeral dystrophy (FSHD). FSHD is a neuromuscular disorder that is potentially caused by loss of spreading of heterochromatin causing aberrant gene activation .”