Chromatin state and dynamics is modulated by chromatin interacting proteins, in particular histones and their post-translational modifications. We investigate the chromatin-bound proteome in aging to understand how DNA readout is misregulated in senescent cells. We focus on domains of reactivated heterochromatin, i.e. chromatin domains undergoing anomalous decondensation and decorated by histones co-modified with markers of condensed heterochromatin and active transcription. We optimize our mass spectrometry methods 3D cell models, and then apply them on B-cells retrieved from Ashkenazi Jews donors. In this cohort, we particularly focus on offsprings of exceptional longevity, i.e. individuals in their 70s with projected longer lifespan due to their centenarians ancestors. Preliminary data on 60 individuals revealed the enrichment of unusual histone modifications in the OPEL group, the group we utilize as indicator of projected longer lifespan. We have then applied biochemistry techniques to identify the role of these unusual modifications on chromatin readout.


Epigenetic alteration is a pivotal factor in tumor metastasis. PHD finger protein 13 (PHF13) is a recently identified epigenetic reader of H3K4me2/3 that functions as a transcriptional co-regulator. In this study, we demonstrate that PHF13 is required for pancreatic-cancer-cell growth and metastasis. Integrative analysis of transcriptome and epigenetic profiles provide further mechanistic insights into the epigenetic regulation of genes associated with cell metastasis during the epithelial-to-mesenchymal transition (EMT) induced by transforming growth factor β (TGFβ). Our data suggest PHF13 depletion impairs activation of TGFβ stimulated genes and correlates with a loss of active epigenetic marks (H3K4me3 and H3K27ac) at these genomic regions. These observations argue for a dependency of TGFβ target activation on PHF13. Furthermore, PHF13-dependent chromatin regions are enriched in broad H3K4me3 domains and super-enhancers, which control genes critical to cancer-cell migration and invasion, such as SNAI1 and SOX9. Overall, our data indicate a functional and mechanistic correlation between PHF13 and EMT. Epithelial-to-mesenchymal transition (EMT) is a cellular process that plays a central role in embryonic development, wound healing, and malignant tumor progression . AbnormaThe TGFβ pathway is one of the most well-characterized pathways that is known to induce EMT. In response, mesenchymal genes  and EMT-induced transcription factors  are activated. These EMT-TFs, in turn, repress epithelial genes and elevate the expression of mesenchymal genes. These widespread transcriptional changes are accompanied by complex epigenetic regulatory mechanisms, notably alterations in the post-transcriptional modification of histones. Interestingly, TGFβ treatment induces widespread alteration of histone modifications via increasing chromatin accessibility . FurtherThe broad H3K4me3 domains regulate embryonic development and human disease –12. For Enhancers, signed by active histone markers, including H3K27ac and H3K4me1, interact with promoters to form chromatin loops, resulting in transcriptional activation. The epigenetic landscape of transcription activators has identified a novel epigenetic signature termed “super-enhancers” (SEs), which strongly activate the expression of genes that control cell identity –18. OncoPHF13 is a plant homeodomain (PHD) finger-containing protein that plays an essential role in modulating the cell cycle , DNA damIn this study, we demonstrate that PHF13-depleted pancreas ductal adenocarcinoma cells Panc-1 show a significantly decreased cell proliferation in vitro and in vivo. Moreover, cell culture-based assays reveal that PHF13 is required for TGFβ-induced EMT. Using the integrated transcriptomic and epigenetic analysis, we provide the first evidence that a subset of TGFβ-activated genes requires PHF13 to increase the activity of poised regions, broad H3K4me3 domains, and super-enhancers. Together, these findings suggest a novel epigenetic mechanism of PHF13 in promoting cancer-cell migration and identify PHF13 as a potential therapeutic target for human pancreatic cancer.PHF13 in pancreatic adenocarcinoma  and downregulated mesenchymal markers (CDH2 and MMP9)  Fig. . In contP9) Fig. . These rTo determine the epigenetic function of PHF13 in gene expression, we performed ChIP-seq analysis of PHF13. We further overlapped the accessible sites (ATAC-seq) with the PHF13-bound sites to preferentially select the PHF13-occupied regions  via the genomic browser and ChIP-qPCR analysis  were increased by TGFβ treatment in Panc-1 cells  were reduced upon PHF13 depletion  in cancer metastasis, we next sought to examine the role of PHF13 in controlling SEs during TGFβ-induced EMT. Specifically, 594 SEs were distinguished from typical enhancers via ROSE analysis of H3K27ac density  Supplem. We furtOX9 Fig. . This efOX9 Fig.  and provWhole-genome sequencing of primary and metastatic tumors in the same patients has revealed limited heterogeneity of known driver mutations among all subclones . TherefoEZH2, a subunit of PRC2, shows context-dependent oncogenic and tumor suppressor functions –55. For MMP2 and MMP9. Thus, a novel epigenetic regulator pathway has been hypothesized to explain PHF13-mediated cancer-cell invasion, in which the H3K4me3 reader PHF13 stabilizes the TGFβ-activated broad H3K4me3 domain to maintain the highly transcribed level of SNAI1, thereby triggers a transcriptional program leading to EMT.Broad H3K4me3 domains are tightly linked to genes controlling cell identity , 11, 44 In conclusion, we have provided mechanistic insights to explain the role of PHF13 in cancer metastasis by employing genome-wide analysis to understand its effects on chromatin activity and gene expression during TGFβ-induced EMT. Furthermore, our data indicate that PHF13 is required for TGFβ to endorse EMT-related oncogene expression by stabilizing the broad H3K4me3 domains and SEs. Interestingly, PHF13 may have diverse molecular functions in gene transcription. In addition to cooperating with H3K4me2/3 to increase chromatin activity , PHF13 i2. For plasmids transfection, cells were transfected with pcDNA3.1(+)-PHF13 overexpressed vector using FuGENE®HD reagent  according to the manufacturer’s instructions. pcDNA3.1(+) empty vector was set as a negative control. siRNA transfections were performed using RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s instructions. Targeted human PHF13 siRNAs  containing the following sequences: 5′-CCCUUAUCCGAAGGAGGAATT-3′, 5′-UUCCUCCUUCGGAUAAGGGTT-3′, 5′-CCUGAUCAGGUCAAAGAAATT-3′, 5′-UUUCUUUGACCUGAUCAGGTT-3′. Non-targeting siRNA was used as a negative control . Afterward, Panc-1 cells were exposed to 10 ng/ml of TGFβ  supplemented growth medium for 48 h to induce epithelial-mesenchymal transition.Panc-1 cells were kindly provided by Stem Cell Bank, Chinese Academy of Sciences. Panc-1 cells were authenticated by DNA profiling using twenty different and highly polymorphic short tandem repeat (STR) loci. Mycoplasma was also measured by PCR. Panc-1 cells were cultured in high-glucose DMEM/F12  supplemented with 10% FBS  and 1% penicillin-streptomycin  at 37 °C, 5% COPHF13 knockout Panc-1 cells were generated using the CRISPR/Cas9 system as described before . Nuclei were pelleted and suspended in sonication buffer . The chromosome was fragmented in the Bioruptor® Pico via sonicating 5 cycles. The fragmented chromosome samples were diluted in two volumes of dilution buffer . The unspecific binding sites were blocked and pulled down by incubating with 100 µl Sepharose 50% slurry for 1 h. The supernatants were incubated with each primary antibody  overnight at 4 °C. The DNA-protein complex was pulled down by incubating with protein A agarose beads for 1.5 h at 4 °C. The immunoprecipitated beads were washed twice with each ice-cold buffer: IP buffer , LiCl buffer , IP buffer again, TE buffer [10 mM Tris-HCl (pH 8) and 1 mM EDTA (pH 8.0)]. RNAs were degraded in 10 mM Tris-HCl (pH 8.0) buffer supplemented with 0.2 µg/µl RNase A. immunoprecipitated DNA was eluted in high SDS buffer  supplemented with 0.2 µg/µl proteinase-K overnight at 65 °C. DNA was purified using phenol: chloroform: Isoamyl alcohol (25:24:1) and quantitated using Qubit 3.0 according to the manufacturer’s protocol (Life technologies). The experiment was performed in duplicate.ChIP of histone modifications was performed as described previously . 48 h afChIP of EZH2 and PHF13 was performed as previous descriptions , 67. BriThe sequencing library was prepared using the VAHTS Universal DNA Library Prep Kit according to the manufacturer’s protocol  and sequenced on the HiSeq X (Illumina) platforms.For ChIP-qPCR, Input DNA (10% of the amount utilized for immunoprecipitation) was prepared from chromatin extracts and used for the normalization of ChIP samples. The occupancy of histone modifications was determined by RT-qPCR using the indicated primers .Differential binding analysis of ChIP-seq data peaks called by MACS2 was performed using the R package “DiffBind” (Version 2.16.2 ). SignifThe bamCoverage tool was used to generate a coverage track normalized by counts per million (CPM) (Version 3.5.0 ). FurtheThe normalized H3K27ac ChIP-seq signal was used to call super-enhancer with Rank Ordering of Super-Enhancers (ROSE) as described previously . The GREPHF13-bound enhancers were performed in the motif enrichment analysis. Then, the DNA sequences at the selected regions were further loaded to find motifs using HOMER (version 4.8.0) . SimilarReproducibility ChecklistSupplementary Figures and Figure legendsoriginal data of western blotDataset 1Dataset 2Dataset 3Dataset 4Dataset 5


Rbbp4 mutant alleles in mouse embryonic stem cell lines by CRISPR-Cas9. The mutations disrupted Polycomb repressive complex 2 assembly and H3K27me3 establishment on target chromatin and altered histone H3 lysine 27 acetylation genome wide. Moreover, Rbbp4 mutant cells underwent dramatic changes in transcriptional profiles closely tied to the deregulation of H3K27ac. The alteration of H3K27ac due to RBBP4 dysfunction occurred on numerous cis-regulatory elements, especially putative enhancers. These data suggest that RBBP4 plays a central role in regulating histone H3 lysine 27 methylation and acetylation to modulate gene expression.RBBP4 is a subunit of the chromatin remodeling complexes known as Polycomb repressive complex 2 and histone deacetylase 1/2-containing complexes. These complexes are responsible for histone H3 lysine 27 methylation and deacetylation, respectively. How RBBP4 modulates the functions of these complexes remains largely unknown. We generated viable  Histone modifications are critical epigenetic regulators that specify the transcriptome during lineage determination and tightly control transcriptional states to preserve cellular identity over cell generations .PRC2, the methyltransferase with specific activity toward H3K27, is responsible for the mono-, di-, and tri-methylation modifications of this residue (H3K27me1/me2/me3)  complexes essential for normal development, including NuRD, Sin3, and CoREST . RBBP4/7Rbbp4 and Rbbp7 in mouse embryonic stem cells (ESCs), we generated viable ESC colonies with mutated Rbbp4/7. We found that RBBP4 mutations remarkably altered transcriptional profiles and the landscape of H3K27 methylation and acetylation. Our results suggest that RBBP4 is involved in acetylation and deacetylation of H3K27, especially at enhancers, to fine-tune gene activity.Since RBBP4/7 are essential for cell viability, it is difficult to test their roles experimentally. By utilizing CRISPR-Cas9 to target l-glutamine, 0.1 mM minimal essential medium-nonessential amino acids, 0.1 mM β-mercaptoethanol, and leukemia inhibitory factor.Mouse E14 ESCs were cultured in Glasgow Minimum Essential Medium supplemented with 15% fetal bovine serum, 1.0 mM Rbbp4 and Rbbp7 were cloned into eSpCas9(1.1)  using a Golden Gate assembly cloning strategy  using the FuGENE HD reagent (Promega) according to the manufacturer’s instructions. The cells were treated with 2 μg/ml puromycin for 2 days and recovered in a standard culture medium until ESC colonies grew. Targeted colonies were genotyped by PCR and verified by DNA sequencing and Western-blot analyses.sgRNAs targeting ChIP was performed as described . MACSv2 P-values ≤0.01 , and total RNA was isolated using the Direct-zol RNA kit (Zymo). Sequencing libraries were prepared using a Kapa mRNA HyperPrep kit per the manufacturer’s instructions and then sequenced on an Illumina NovaSeq 6000 system (paired-end×50 bp). Sequence reads were aligned to the mm10 genomic sequence with STAR (v2.7.3)  and adjusted to 300 mM NaCl by adding dilution buffer   , p. 12. Rbbp4 and Rbbp7. For Rbbp4, we obtained 2 ESC clones. The first mutant clone, Ins/InsRbbp4, contains a 3-nucleotide insertion on both alleles, resulting in a homozygous missense mutation  and a Cys insertion at the 85th amino acid   .Ins migrated slower relative to controls  or narrowly around the TSS   . In cont3, Wnk2) . TogetheSince the reduction of H3K27me3 on PRC2 loci did not affect global levels of H3K27me3 in mutant cells  and 2a, Since RBBP4 exists in chromatin-modifying complexes responsible for methylation and deacetylation of H3K27, we clustered binding loci into 3 groups based on the enrichment patterns of H3K27me3, H3K27ac, HDAC1, and p300 . ClusterRbbp4 mutant ESCs did reveal numerous loci  with increased H3K27ac. However, to our surprise, there were approximately 2-fold more  genomic regions with decreased H3K27ac levels , H3K4me2 (GSM881353), H3K4me3 (GSM723017), p300 (GSM2360934), and ATAC-seq (GSE120393) in mouse ESCs from GEO. We examined the distribution and enrichment of these features around H3K27ac regions. H3K27ac combined with H3K4 methylation, p300, and open chromatin indicated by ATAC are used to predict cell type-specific enhancers . The colIns/InsRbbp4, 3,637 genes showed upregulated expression (P < 0.01), while 3,642 showed reduced expression relative to controls. Similar genes showed misregulation in Ins/DelRbbp4, with 4,022 upregulated and 3,954 downregulated. For those differentially expressed genes with more than a 2-fold change in transcription and a mean read count of more than 20, 762, and 919 genes showed upregulation, and 936 and 1,066 showed decreased expression Ins/InsRbbp4 and Ins/DelRbbp4, respectively  . As defiMyrf intron is associated with an enhancer  and GSE183292 (ChIP-seq).G3 online.jkac082_Supplementary_TableS1Click here for additional data file.jkac082_Supplementary_TableS2Click here for additional data file.


Drosophila gastrulation bring about an increase in activity-levels of a Notch-responsive enhancer. Mutations that disrupt the timing of gastrulation resulted in concomitant delays in transcription up-regulation that correlated with the start of mesoderm invagination. As a similar gastrulation-induced effect was detected when transcription was elicited by the intracellular domain NICD, it cannot be attributed to forces exerted on Notch receptor activation. A Notch-independent vnd enhancer also exhibited a modest gastrulation-induced activity increase in the same stripe of cells. Together, these observations argue that gastrulation-associated forces act on the nucleus to modulate transcription levels. This regulation was uncoupled when the complex linking the nucleoskeleton and cytoskeleton (LINC) was disrupted, indicating a likely conduit. We propose that the coupling between tissue-level mechanics, arising from gastrulation, and enhancer activity represents a general mechanism for ensuring correct tissue specification during development and that Notch-dependent enhancers are highly sensitive to this regulation.Cells sense and integrate external information from diverse sources that include mechanical cues. Shaping of tissues during development may thus require coordination between mechanical forces from morphogenesis and cell-cell signalling to confer appropriate changes in gene expression. By live-imaging Notch-induced transcription in real time, we have discovered that morphogenetic movements during  Cells continuously sense and respond to their environment. This occurs via signaling pathways that detect and respond to external stimuli, such as morphogen gradients or direct cell-surface ligands. During development, information routed through these pathways feeds into networks of transcription factors that coordinate cell fates to pattern and regulate differentiation. At the same time, cells are exposed to mechanical forces from changes in tissue movements, deformations, and stiffness. In order for tissues to develop and function correctly, there must be mechanisms that couple cell signaling with the mechanical environment. Indeed, there is evidence that changes in cell morphology can impact on gene expression , but thiNotch is a key developmental signaling pathway that transmits information between cells in contact. Prior to ligand binding, the ADAM10 cleavage site in Notch is hidden by the negative regulatory region (NRR). To reveal this site, ligand binding exerts a force on the receptor, leading to a displacement of the NRR . A seconDrosophila embryo using the MS2-MCP system to monitor the transcriptional response live. Notch activity initiates in a stripe of cells flanking the mesoderm, the mesectoderm (MSE), in the last cycle prior to gastrulation , and remains active throughout gastrulation. These cells divide after gastrulation, change their polarity and form a boundary that separates the flanking neuroectoderm  cells constricts and cells shorten along their apico-basal axis . This ocWe set out to investigate whether there is a causal relationship between morphological events occurring at gastrulation and the change in levels of Notch-dependent transcription, using live imaging. Strikingly, we found a strong correlation between the start of mesoderm invagination and the time at which transcription levels increased. This change in transcription was delayed or absent when gastrulation was perturbed using different genetic mutations and manipulations. A Notch-independent enhancer exhibited similar albeit more modest effect, suggesting that the mechanism involves a more general feature. Furthermore, the Notch intracellular fragment, NICD, was also subject to similar regulation. The results indicate therefore that the mechanical context has a significant impact on the transcriptional outcome of Notch signaling but argue that this operates downstream of receptor activation and also affects other developmental enhancer(s). This type of coordination between tissue forces, developmental signaling and nuclear activity is thus likely to be of general importance for shaping tissues and organs as they form.Drosophila embryogenesis, Notch is active in the mesectoderm (MSE), a stripe of cells that border the mesoderm m8-HLH gene that both showed decreased levels also during cellularization , which encodes the ligand for the GPCR in the cascade regulating gastrulation  gene. Unlike many of the other embryonic enhancers studied to date, the vnd early embryonic enhancer (vndEEE) is reported to be active throughout nc14 and to drive expression in a band of cells that overlap the MSE   .m5/m8 enhancer are more marked than those on vnd, Notch-dependent processes may be more sensitive. Furthermore, the consequences are strongest in MSE cells, suggesting that gastrulation exerts differential impacts across the ectoderm, which might be related to the gradients of forces and cell deformation that have been measured , which encodes a KASH protein, was successful, producing embryos with strongly reduced klar mRNA  complex . In sevelar mRNA  . Depletcontrols . Consistg by 11%  .m5/m8[II] transcription. The early phase of transcription was slightly elevated and there was no subsequent transition or reduction in the late phase , as well as several tagged variants of GFP, with and without a nuclear localization signal (nls). None of these exhibited any change in nuclear levels at the time of gastrulation, , includim5/m8[II] does not prevent the increase , inserted in the landing site attP40-25C in the second chromosome  was generated by replacing m5/m8 for the vndEEE enhancer, as defined by GGGAAGCTTGGGTAAGCACAAGGATTCC and GGGACCGGTCGAATAAGCTGCAAGGAGATC with HindIII and AgeI sites. The resulting plasmid was inserted in the same attP landing site (attP40-25C) as m5/m8 by ΦC31 mediated recombination . Control embryos were obtained by crossing His2av-RFP, nos-MCP-GFP females with m5/m8 or vnd males. Hemizygous fog mutant embryos were obtained by crossing fog[S4] / FM6;; His2av-RFP, nos-MCP-GFP with m5/m8 or vnd males and they were recognized by the presence of ectopic folds after gastrulation and embryonic lethality. Other embryos obtained from the same cross were grouped together and labeled fog+/+/-. To combine the fog background with ectopic NICD produced by eve2-NICD, fog[S4] was combined with eve2-FRT-STOP-FRT-NICD and crossed with ovo-FLP;; His2av-RFP, nos-MCP-GFP. F1 females containing all transgenes were crossed with m5/m8-MS2 males; and induce removal of the FRT-STOP-FRT cassette in the germline so that NICD is produced in the eve2 pattern in F2 embryos. Controls for this experiment were obtained by crossing eve2-FRT-STOP-FRT-NICD with ovo-FLP;; His2av-RFP, nos-MCP-GFP and F1 females with m5/m8-MS2 males.To test expression from -MCP-GFP  were comαTub-Gal4::VP16 (BDSC # 7062) was combined with His2av-RFP, nos-MCP-GFP to generate αTub-Gal4::VP16; His2Av-RFP, nos-MCP-GFP. To knock down genes from the maternal germline, this was crossed with with UASp-RNAi lines detailed in UASp-w-RNAi as control (BDSC #35573). Females αTub-Gal4::VP16 / +; His2Av-RFP, nos-MCP-GFP / UASp-RNAi or αTub-Gal4::VP16 /UASp RNAi; His2Av-RFP, nos-MCP-GFP /+were crossed with m5/m8-MS2 or vnd-MS2 to obtain the experimental embryos. To combine this with ectopic NICD, m5/m8, eve2-FRT-STOP-FRT-NICD recombinants  between a semi-permeable membrane and a coverslip. The ventral side of the embryo was facing the coverslip. Videos were acquired in a Leica SP8 confocal using a 40 x apochromatic 1.3 objective, zoom x2 and 400 × 400 px size (providing an XY resolution of 0.36 µm/px), 12 bit depth, 400 Hz image acquisition frequency and pinhole of 4 airy units. 27, 29 or 32 × 1 µm slices were collected, with total acquisition time of 15–20 s per frame, depending on the experiment. Different genetic conditions are only compared in the same plot if the same microscope and settings were used. To image embryos in which nuclear levels of different factors were going to be quantified, multiple embryos were acquired at once using the multi-position setting in the microscope and a final time resolution of 1 or 1.5 min, which also helped reduce bleaching. Depending on the condition only GFP or GFP and His2Av-RFP were imaged.His2Av-RFP /+ females and males were dechorionated, glued with heptane glue to a coverslip, and covered with Voltalef medium (Samaro). Injections were performed only on pre-nc10 embryos using pulled glass needles and dextrans diluted in injection buffer  scripts that have been previously described . BrieflyGap43-mCherry , nuclei were directly segmented from the channel in which nuclear levels were going to be quantified and the average nuclear intensity quantified over time. In the other cases -GFP and H-GFP), nuclei were segmented and tracked based on the His2Av signal and the average GFP/FITC intensity quantified in each nucleus. We note that because in most cases all cells in the embryo contained these factors, the signal could not be normalized to another region to account for bleaching, and therefore the fluorescent profiles might have been reduced over time due to bleaching.Depending on which factor was being analyzed, some videos expressed only the fluorescently-tagged factor of interest (if the nuclear levels were high enough to use for nuclear segmentation) or in combination with Nuclear membranes tagged with Nup107-RFP  were segProcessing of MS2 data  has been carried out as described in our previous work . After tGal4VP16. For vnd, transcription was considered all throughout nc14. The total mRNA output (in AU) was obtained by adding all the normalized transcription values for each cell in a defined time period. In plots were cells were classified as ‘active’ or ‘active +increasing’, nuclei which increase in levels were defined if the average intensity from 15 min of the start of ME invagination was higher than the average of all active nuclei at that point and at least 1.7 times higher than the average intensity in the 15 min prior to ME invagination. Using these values, most nuclei were classified in a way that matched what could be observed by looking directly at the transcription traces.In all videos, time into nc14 was considered from the end of the 13th syncytial division. To avoid early stochastic activity, transcription was considered from 15 min into nc14 in most experiments, except for expression in the presence of maternal Gal4, in which it was considered from 30 min to exclude earlier stochastic activity induced by Start of apical constriction, start of mesoderm invagination and end of gastrulation were manually defined for each embryo from plots showing the movement of MSE cells. Transcribing nuclei in each region were selected and the average movement of their centroids in the Y axis, corresponding to the DV axis in the embryo, was calculated. This produces a plot with one or two peaks of movement. A large peak of movement is produced during ME invagination, as MSE cells move ventrally. If the embryo is mounted completely ventrally, only this peak of movement is observed. If the orientation of the embryo is tilted, the whole embryo rolls inside the vitelline membrane during apical constriction, which is detected as an earlier peak of movement of cells dorsally. After that, the second peak of movement corresponds with mesoderm invagination. The transition between the two movements, corresponds to and was used to define the start of ME invagination. Similarly, transition point in mean transcription levels was manually defined from each plot showing the mean fluorescence of selected cells, by selecting the inflection point between two different levels of transcription. Only embryos that showed a clear transition in levels were used in the correlation plots, although we note that only in most cases all embryos from each genetic condition were included.His2Av-RFP channel and the average intensity calculated.To quantify average histone intensity around the MS2 spot, spots were segmented using the Differential-of-Gaussians approach previously described . A 5 × 5In figures and figure legends, n number indicates number of embryos imaged for each biological condition. Where appropriate, n number next to heatmaps indicates total number of cells combining all embryos for each biological condition. Plots showing mean levels of transcription and SEM (standard error of the mean) combine all traces from multiple embryos from the same biological condition.https://github.com/BrayLab/LiveTrx (swh:1:rev:564d8ac59d4dcf2cd5167960e5ae97d4e8932647).The MATLAB app to track nuclei, quantify MS2 traces and define properties of gastrulation can be obtained at  Drosophila gastrulation. Through a detailed and convincing analysis of live transcriptional reporters and gastrulation-defective mutants, they report a clear example of tissue movements affecting enhancer activity. The idea that morphogenetic movements can regulate the genome through mechanical changes in the nucleus is intriguing and important and is of broad interest to cell and developmental biologists.In this manuscript, Falo-Sanjuan and Bray provide an elegant set of experiments investigating how cell movements modulate Notch transcriptional activity during  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Decision letter after peer review:[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting the paper "Levels of Notch-regulated transcription are modulated by tissue movements at gastrulation" for consideration by Comments to the Authors:eLife.We are sorry to say that, after consultation with the reviewers, we have decided that this work in its current form will not be considered further for publication by eLife. Further experimental data connecting enhancer upregulation to mechanical changes on the nucleus are needed. Currently the role of LINC complex, specifically Klar in regulating tension-dependent transcriptional activity at gastrulation, is unclear and the data are at odds with the predictions. Thus, the role of nuclear deformation and Klar’s contribution remain difficult to understand in relation to the proposed model.As you will see from their comments, all three reviewers found the subject matter interesting and timely, and considered the data documenting enhancer dynamics to be very high quality. However, the reviewers felt that the observed changes in enhancer activity correlated with cell movements were, on their own, insufficient to warrant publication in eLife aims to publish articles with a single round of revision that can be accomplished within two months. If you feel that all of the reviewer’s concerns can be addressed in a timely manner we would be open to reviewing a revised submission, but we do not want to delay publication elsewhere, if that is the preferred course of action.Thus, the reviewers felt that the work has potential, but would need extensive additional work. Please note that Reviewer #1:Drosophila embryo. Using the MS2 system to report on live transcriptional dynamics, it is convincingly shown that a burst of transcriptional activity from the Notch-dependent enhancer in cells neighboring the ventral furrow coincides temporally with gastrulation movements, and that such movements are necessary for the transcriptional burst. This gastrulation-dependent upregulation is also observed by NICD-induced transcription, suggesting the mechanism lies downstream of Notch receptor, likely in the nucleus. The idea that morphogenetic movements can affect genome regulation through mechanical changes on the nucleus is intriguing and important, and this paper represents a clear example in the context of embryonic development.This paper describes the intriguing observation that transcriptional activity of a Notch dependent enhancer depends on gastrulation movements in the Overall, the data are high quality and are presented in a clear and convincing manner. The data for the most part support the authors conclusions. Specifically, the conclusion that gastrulation movements are required for transcriptional upregulation of Notch-dependent enhancers is well supported by the data, and the use of three different genetic alterations to impair gastrulation is a strength. However, to further demonstrate a causal link between tissue mechanics and transcriptional output, it would be beneficial to also show that ectopic tissue folding or stretch is sufficient to generate an increase in enhancer activity. Currently there is only data showing a loss-of-transcriptional upregulation when tissue folding is impaired, and it is possible that some of the mutants used to block gastrulation have broader pleiotropic effects that could impact Notch signaling. Further, data connecting the observed changes in enhancer activity to mechanical changes on the nucleus are not well supported by the current data.1. To further demonstrate a causal link between tissue mechanics and transcriptional output, it would be beneficial to also show that ectopic tissue folding or stretch is sufficient to generate an increase transcription. Currently there is only data showing a loss-of-transcriptional upregulation when tissue folding is impaired, and it is possible that some of the mutants used to block gastrulation have broader pleiotropic effects that could impact Notch signaling.2. A discussion of the possible functional consequences of tissue movements on Notch-dependent transcription at this stage of embryogenesis would be helpful for the non-expert.3. Data connecting the LINC complex to m5/m8 activity are rather weak, and I'm not sure these data in their current form add much to the paper.Reviewer #2:In the study presented in this manuscript, Falo-Sanjuan and Bray explore the connection between mechanical forces and transcription during gastrulation, a process involving extensive cell movement, rearrangement as well as tension sensing and specification of different cell lineages. The overall hypothesis and ideas are interesting and valid, the experiments are well designed and data are carefully analyzed. The study starts by explaining how the tension between cells can affect Notch signaling in the mesoectoderm through monitoring MS2 transcriptional activity from the m5/m8 enhancer in control and mutant embryos in which mesoderm invagination fails due to defects in cell-cell adhesion (α-catenin) or actomyosin regulation . These results support the view that cortical cytoskeletal tension is causing an increase in Notch-dependent transcription. Then, somewhat abruptly the working model is revised when full-length Notch is shown not to be required for this increase (eve2-NICD in Notch RNAi); they hypothesize that if Notch signaling does not respond to tension at the cell membrane through ligand-receptor binding, the tension-sensing/gene upregulation at mesoderm invagination must relate to LINC complex function at the nuclear membrane. The transition to study of LINC complex function seems to be a bit jarring and needs more supportive evidence to better explain why alternative explanations were disregarded. Lastly, the title may be a bit misleading as the data support the view that "cell movements associated with gastrulation have a direct impact on transcription in the nucleus"… and that Notch-responses are not the only ones that are affected.The reviewer had specific concerns (detailed below) that relate to (i) quantitative analyses; (ii) there is an apparent contradiction between title and the fact that Notch appears dispensable for the observed phenotype; (iii) klar RNAi gives the opposite phenotype as expected (but somehow still stated as supporting their model) – this merits more explanation or investigation; and (iv) lack of attention is given to other explanations including possible connection between NICD-Twist-mechanotransduction.This is a very interesting study but the title, flow of the main text and its figures are somewhat misleading/hard to follow. In particular, the transition to study of LINC complex function seems to be a bit jarring and needs more supportive evidence to better explain why alternative explanations are disregarded. Also, the role of LINC complex, specifically klar in regulating tension-dependent transcriptional activity at gastrulation is unclear, more experiments need to be done to show how it interacts with NICD.1) MS2 spot calling and signal quantification. The reviewer realizes that the MS2 quantification has been published before but for the current focus on levels of expression, the spot analysis should be revisited. Instead of identifying the MS2 spot, the authors appear to be using the maximum intensity within a nucleus. This ignores the shape of a spot and assumes that the maximum intensity sufficiently represents the spot. A better method of spot detection would include either gaussian fitting or thresholding. Since a key point of the paper is to study an increase in signal that is correlated to gastrulation, additional attention to quantification is necessary.2) Along these lines, the authors need to provide more information about the number of cells scored for graphs that show "fraction of nuclei…".3) In Figure 2C,E, only embryos that show a clear change in mean levels of transcription are displayed. What is the total number of embryos collected for each condition? How was a "clear change in mean levels" defined?4) Figure 2, the control profiles of m5/m8 transcription activity in B, D, F appear to be very different, especially for fog mutants, is there a specific reason?5) The authors used a vnd enhancer to show that mechanical forces have a general effect on transcription at gastrulation. However, there is no further attempt to explore or discuss how vndEEE might be regulated, through another tension-sensing transcription factor or maybe also by changes in nuclear property/LINC complex? Twist seems likely (see pt 8 below).6) Figure S4, if m5/m8 is Notch-dependent, what causes the increase at time 0 in the mean profile of Notch RNAi?7) It is interesting that the ectopic expression of eve2-NICD in Notch RNAi background rescues m5/m8 transcription activity within the eve stripe , but how much of this increase relates NICD versus other tension activated factors ? Is twist upregulated in the domain of eve2-NICD expression in Notch RNAi embryos? How does this study/timing of m5/m8 upregulation at invagination relate to the mechanical signals discussed in Pouille et al., Sci Signal 2009 (which should be cited).8) The conclusion that LINC complex/Klar plays a role in tension-regulated Notch signaling activity is poorly supported. Figure 5B shows that there is an early increase in m5/m8 activity, which is, as the authors point out, opposite from the expected result. Therefore, it is necessary to better define the relationship between NICD and nuclear properties modified/influenced by LINC. How does loss of function klar (or other LINC protein) impact intracellular Notch signaling and other tension-related processes, including localization of α-catenin at the AJs, RhoGEF2 at the apical membrane, actomyosin pulsing, expression of cta and fog, etc. Depending on whether twist is found to be upregulated by eve2-NICD (see point 7 above), it might also be worth looking to see how twist expression (upregulation by NICD?) is affected by klar.9) Unlike m5/m8 enhancer which requires Notch signaling input directly to help activate gene expression, Notch input at the sim enhancer acts to 'derepress' expression by eliminating Su(H) repressor input and allowing output to be responsive to Dorsal and Twist . Therefore, the roles of Notch in m5/m8 and sim enhancers differ. In the current study, the focus is m5/m8 with little done to analyze sim enhancer properties. Interestingly, in Morel & Schweisguth, when Su(H) sites are mutated in a sim enhancer (sim.mut), reporter expression remains in N mutant embryos. It is likely that the sim.mut enhancer would also show upregulated gene expression at mesoderm invagination if tested; and if so may be a better test case for tracking the input that relates to mechanotransduction with subsequent mutagenesis. Possibly this input is Twist.10) Farge 2003 already showed that Twist increases residency in the nucleus downstream of mechanical induction. Relating to comments above (e.g. point 7 and 9), it is likely that the NICD-effect could be indirect, working to upregulate Twist. If so, this study is still interesting, but are the current results novel?Reviewer #3:11) The title is confusing as the data support the view that "cell movements associated with gastrulation have a direct impact on transcription in the nucleus"… and that Notch-responses are not the only ones that are affected.Drosophila mesoderm movements. The data are of very good quality. The authors could boost their conclusions by better replacing them in the context of their previous studies regarding the role of adherens junctions in Notch transcriptional regulation or turnover . The findings on Klar mutant conditions are highly relevant but more difficult to unit with the interplay between Notch transcriptional control and mesoderm invagination.This manuscript is of broad interest to readers in the fields of Cell and Developmental Biology. The authors provide an elegant set of experimental evidence demonstrating that Notch transcriptional activity is modulated during The major strength of the manuscript is the quantitative and original analyses of Notch (N) transcriptional levels in conjunction with morphogenetic movements in multiple genetic backgrounds. The authors could improve their manuscript by addressing the following points:1. In Figure S1, the authors have also quantified N activity with two other reporters of N transcriptional activity. Both reporters show an increase at 50 mins as described in the main text, but both reporters also show a decrease at 55 mins. This decrease is not observed for the line (II) used through the manuscript. One could therefore argue that this drop is also a relevant characteristic of Notch activation during mesoderm invagination. This data should therefore be placed in the main figure and the decrease should be mentioned in the main text. In addition, it will be important that the authors performed the some loss of function analyses  using these two other reporters to further validate their findings.2. Page 7: The following statement is confusing. "We note the mean levels of transcription from this reporter were not significantly reduced prior to gastrulation, unlike those from another reporter insertion and the endogenous genes that showed decreased levels also during cellularization . The difference likely arises because this m5/m8 [II] insertion achieves maximal transcription at lower levels of Notch activity." In the cited previous paper, the authors have shown that a-cat affects Notch transcription during cellularization. It is unclear why a difference in reporter strengths will lead to distinct conclusions. This needs to be further experimentally explored and explained.3. Page 12: "As predicted, this treatment greatly reduced transcription from m5/m8 [II] .". The reduction is 2-fold. How these fold changes compare with the ones observed in RhoGEF2 and Fog mutant conditions? The Notch mutant conditions should be shown in figure 1. Furthermore, one could argue that a large part of the increase in reporter expression does not reflect an increase in Notch activity. Please discuss.4. Figure 4. It will be important that the authors plot on the same plot with the same units the endogenous Notch activity in both wt and NotchRNAi.5. As stated by the authors, Notch transcription findings in Klar mutant conditions are distinct from the authors' prediction since transcription increases prior to mesoderm invagination. The role of nucleus deformation and the Klar contribution remain difficult to understand and insert in the authors proposed model.6. Statistics: Could the authors propose statistical tests to further support their findings regarding temporal changes in reporter levels and differences between distinct mutant backgrounds?eLife. Your revised article has been evaluated by Claude Desplan (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Notch-dependent and -independent transcription are modulated by tissue movements at gastrulation" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:Essential revisions:Overall, the manuscript is much improved and the authors have addressed most of the reviewers' major concerns. There are two remaining issues that require clarification and additional technical information related to imaging conditions and corrections for nuclear depth over the course of gastrulation, and a previously reported increase in transparency of klar embryos.1) Please provide additional information about the imaging pipeline – specifically whether any corrections are being made for spatially-dependent changes in signal due to movement of cells in the z-direction. See reviewer 2 comments for details.2) The klar mutant background is associated with a clearing of embryos, at least in older embryos at stage 8 . Please provide information regarding whether there is an increase in transparency of klar embryos at NC14 that may result in the GFP signal appearing brighter that wild type controls.Reviewer #2:The manuscript is improved. In particular, the data are more clearly presented and the authors have added more experiments, namely testing whether levels of Twist, chromatin import, chromatin compaction and/or accessibility relates to the increase in mesectoderm gene expression identified at the time of mesoderm invagination.The author's testing of Twist addresses one of the reviewer's major concerns. While it is still possible that post translational modification of Twist acts to increase its activity at invagination, their dismissal of Twist for the purpose of this study is reasonable based on the data presented.Another major concern was that the use of maximum intensity may lead to noise in the spot calling numbers. The authors state that using the maximum intensity is an accurate method because of tests they have done, however they do not elaborate on what these tests are or how they have verified this beyond the limited use of the alternate method from Garcia 2013. While using the method in Garcia 2013 does show the same general trend, the error is much larger using this method and this doesn't completely support the claim that this method proves the simpler method is correct. The fundamental concern is that single, bright pixels that occur outside of the actual MS2 spot could be skewing the data. An easy way of dealing with this is to apply a median filter, which will remove "salt and pepper" noise to the images. The authors note that they apply this filter across time to smooth the data, however this is would be selecting for nuclei that tend to be on continuously. Another approach would be to manually confirm that the maximum pixel occurs within a spot, but this would be tedious. In the methods section it is stated that "subsequent analysis allows to classify confidently when the maximum value is really representing a spot," however it is unclear how the subsequent analysis alleviates this concern. At minimum, this section of the methods needs to be expanded and made clearer so that the reader can evaluate the method being used.I do still have a worry that their imaging pipeline is not properly correcting for spatially-dependent changes in signal due to movement of cells in the z-direction. Because they have a H2A-RFP associated with the MCP-GFP MS2 movies, they should use the RFP signal to background correct . The H2A-RFP signal can vary in space in time due to position of nuclei. The authors are imaging the MS2 signal in a tissue that moves, however they don't provide a correction for this movement or rule out whether this movement is changing intensity of the signal. Specifically, when signal is lower, the MS2 is in a more dorsal location than compared to after invagination where the signal is at the ventral most point. Did the authors correct for any difference in intensity that could be occurring from differences in the position of the tissue? It is conceivable that signal from the ventral most point will be more optically accessible than from points located more dorsally, due to the curvature of the embryo and possibly the diffraction of light as it moves through tissue to reach the objective. An easy way to correct for this would be to use the signal of the H2A signal that was used to segment and track the nuclei, as any positional effects should also affect the H2A signal, and the H2A signal is already mapped to its corresponding spot.In addition, the klar mutant background is associated with a clearing of embryos such that the GFP signal may appear brighter because embryos are more transparent; this has been shown for older embryos at stage 8  but it is unclear if this is the case at nuclear cycle 14. If klar mutant embryos at these earlier stages are more transparent, could this account for the apparent increase in GFP signal associated with the klar mutant background ? Might the H2A-RFP signal serve to normalize the ctr RNAi vs klar RNAi data? Is the H2A-RFP signal also increasing in the klar RNAi? If that is the case, and the klar data is more similar to wildtype – the early phase of transcription may need to be offset vertically . In this alternative scenario, it would appear then that there is a transition late in the klar mutant background. Might the clearing of lipids affect timing of mesoderm invagination and/or when it is recognized/called?The reviewer appreciates the premise of this study but the authors would be wise to do a bit more controls/investigating to ensure that the observed upregulation in signal associated with mesoderm invagination is not an imaging artifact: (i) might positional effects on MS2 signal be responsible for the increase in GFP observed when cells invaginate/are more directly illuminated; and (ii) to determine whether klar mutants are more transparent and if so use methods to normalize signals obtained in klar in comparison with wildtype.Reviewer #3:The authors have improved their manuscript and addressed my points. They have added a lot of data related to the dynamics of the nucleus and chromatin mobility. Most of these results are negative but they contribute to the detailed characterization of the process. Overall, the findings support the authors' conclusions.Nevertheless, the role of Klar remains difficult to fit in the authors' proposed model, since Klar loss of function phenotype is distinct from the ones observed in mutants affecting the mechanical forces associated with gastrulation . I therefore doubt that Klar would be part of the mechanisms by which gastrulation associated forces modulate Notch-dependent and -independent transcriptional regulation. [Editors’ note: The authors appealed the original decision. What follows is the authors’ response to the first round of review.]Reviewer #1:Drosophila embryo. Using the MS2 system to report on live transcriptional dynamics, it is convincingly shown that a burst of transcriptional activity from the Notch-dependent enhancer in cells neighboring the ventral furrow coincides temporally with gastrulation movements, and that such movements are necessary for the transcriptional burst. This gastrulation-dependent upregulation is also observed by NICD-induced transcription, suggesting the mechanism lies downstream of Notch receptor, likely in the nucleus. The idea that morphogenetic movements can affect genome regulation through mechanical changes on the nucleus is intriguing and important, and this paper represents a clear example in the context of embryonic development.This paper describes the intriguing observation that transcriptional activity of a Notch dependent enhancer depends on gastrulation movements in the Overall, the data are high quality and are presented in a clear and convincing manner. The data for the most part support the authors conclusions. Specifically, the conclusion that gastrulation movements are required for transcriptional upregulation of Notch-dependent enhancers is well supported by the data, and the use of three different genetic alterations to impair gastrulation is a strength. However, to further demonstrate a causal link between tissue mechanics and transcriptional output, it would be beneficial to also show that ectopic tissue folding or stretch is sufficient to generate an increase in enhancer activity. Currently there is only data showing a loss-of-transcriptional upregulation when tissue folding is impaired, and it is possible that some of the mutants used to block gastrulation have broader pleiotropic effects that could impact Notch signaling. Further, data connecting the observed changes in enhancer activity to mechanical changes on the nucleus are not well supported by the current data.We are glad that the reviewer considers the results to be intriguing and important and the data to be of high quality. We appreciate the concerns, which we have addressed below.1. To further demonstrate a causal link between tissue mechanics and transcriptional output, it would be beneficial to also show that ectopic tissue folding or stretch is sufficient to generate an increase transcription. Currently there is only data showing a loss-of-transcriptional upregulation when tissue folding is impaired, and it is possible that some of the mutants used to block gastrulation have broader pleiotropic effects that could impact Notch signaling.We appreciate the concerns of the reviewer. However, because we have used a range of different methods to interfere with gastrulation, including mutations in the gastrulation specific Fog ligand, it is unlikely that these would all have the same pleiotropic effects. We have also tested a range of other mutations/disruptions including some that have quite pleiotropic effects but do not impact gastrulation, such as Zelda, and found that these do not alter the transcription profile. We have added information from some of the other genotypes tested .We also agree that the converse experiment, testing the consequences of inducing ectopic folds, would be an excellent one. However, because we can only measure the read-out in the stripe of cells where Notch is active, this makes the experiment challenging- it’s difficult to know a relevant site for the ectopic folds and to induce them in the short timescale (aprox 10 min) needed to compare transcription levels before and after the fold and before gastrulation occurs. Nevertheless, we have made extensive efforts to carry out experiments along these lines over the past 2 years. Despite many attempts, we have not been able to induce ectopic invaginations, either earlier or in the relevant places, with the tools available in our experimental set-up. We have attempted to do this again during the review period but have not been able to do so successfully.2. A discussion of the possible functional consequences of tissue movements on Notch-dependent transcription at this stage of embryogenesis would be helpful for the non-expert.Consequences of perturbing the force-augmented transcription will likely be a less precise mesectoderm boundary, which has an important role in separating the flanking neurectoderm from either side as well as in forming the midline of the CNS. We have included a comment about the possible consequences from perturbing Notch target genes at this stage.3. Data connecting the LINC complex to m5/m8 activity are rather weak, and I'm not sure these data in their current form add much to the paper.m5/m8 transcription. This result was very striking because we had tested other possible intermediates, such as the Lamin related Kugelkern and the pioneer factor, Zelda, and had seen no change in transcription. We had omitted these negative data for simplicity, but these have now been added to the manuscript to give more context and illustrate that the result with LINC is noteworthy .The perturbation of the LINC complex has a robust effect on We have also included a description of the change in nuclear morphology and an analysis of the chromatin mobility during mesoderm invagination, in relation to gastrulation and the transcriptional changes . The results show that there are substantial changes in nuclear morphology and that the chromatin becomes more mobile, a feature that is thought to be indicative of increased accessibility.Reviewer #2:In the study presented in this manuscript, Falo-Sanjuan and Bray explore the connection between mechanical forces and transcription during gastrulation, a process involving extensive cell movement, rearrangement as well as tension sensing and specification of different cell lineages. The overall hypothesis and ideas are interesting and valid, the experiments are well designed and data are carefully analyzed. The study starts by explaining how the tension between cells can affect Notch signaling in the mesoectoderm through monitoring MS2 transcriptional activity from the m5/m8 enhancer in control and mutant embryos in which mesoderm invagination fails due to defects in cell-cell adhesion (α-catenin) or actomyosin regulation . These results support the view that cortical cytoskeletal tension is causing an increase in Notch-dependent transcription. Then, somewhat abruptly the working model is revised when full-length Notch is shown not to be required for this increase (eve2-NICD in Notch RNAi); they hypothesize that if Notch signaling does not respond to tension at the cell membrane through ligand-receptor binding, the tension-sensing/gene upregulation at mesoderm invagination must relate to LINC complex function at the nuclear membrane. The transition to study of LINC complex function seems to be a bit jarring and needs more supportive evidence to better explain why alternative explanations were disregarded. Lastly, the title may be a bit misleading as the data support the view that "cell movements associated with gastrulation have a direct impact on transcription in the nucleus"… and that Notch-responses are not the only ones that are affected.We are glad that the reviewer considers the hypothesis and ideas interesting and the experiments well designed. We have addressed the queries about the alternate models below and can add substantial data to address these.The reviewer had specific concerns (detailed below) that relate to (i) quantitative analyses; (ii) there is an apparent contradiction between title and the fact that Notch appears dispensable for the observed phenotype; (iii) klar RNAi gives the opposite phenotype as expected (but somehow still stated as supporting their model) – this merits more explanation or investigation; and (iv) lack of attention is given to other explanations including possible connection between NICD-Twist-mechanotransduction.This is a very interesting study but the title, flow of the main text and its figures are somewhat misleading/hard to follow. In particular, the transition to study of LINC complex function seems to be a bit jarring and needs more supportive evidence to better explain why alternative explanations are disregarded. Also, the role of LINC complex, specifically klar in regulating tension-dependent transcriptional activity at gastrulation is unclear, more experiments need to be done to show how it interacts with NICD.We have addressed the concerns below.1) MS2 spot calling and signal quantification. The reviewer realizes that the MS2 quantification has been published before but for the current focus on levels of expression, the spot analysis should be revisited. Instead of identifying the MS2 spot, the authors appear to be using the maximum intensity within a nucleus. This ignores the shape of a spot and assumes that the maximum intensity sufficiently represents the spot. A better method of spot detection would include either gaussian fitting or thresholding. Since a key point of the paper is to study an increase in signal that is correlated to gastrulation, additional attention to quantification is necessary.We appreciate the reviewer’s concerns. However, our analysis indicates that, at this stage in embryogenesis, the method used is very applicable way to capture the spot because there is little background or extraneous spots. To verify this, we have run the alternate analysis based on differential-of-gaussian filtering that integrates the fluorescence from the whole spot  on several movies and obtained the same outcome, as illustrated in 2) Along these lines, the authors need to provide more information about the number of cells scored for graphs that show "fraction of nuclei…".This information was provided in another panel but has now been added more explicitly in the relevant panels of each figure.3) In Figure 2C,E, only embryos that show a clear change in mean levels of transcription are displayed. What is the total number of embryos collected for each condition? How was a "clear change in mean levels" defined?This information has now been added to all plots that show correlations. We note that in most cases all embryos collected in each condition have been included . We had explained how a clear change in mean levels is defined in the methods, but we appreciate it may have been quite brief. We have now expanded on the definition and included a figure for clarity.4) Figure 2, the control profiles of m5/m8 transcription activity in B, D, F appear to be very different, especially for fog mutants, is there a specific reason?There is some variability caused by the genetic background, especially those that have a Gal4 present. 5) The authors used a vnd enhancer to show that mechanical forces have a general effect on transcription at gastrulation. However, there is no further attempt to explore or discuss how vndEEE might be regulated, through another tension-sensing transcription factor or maybe also by changes in nuclear property/LINC complex? Twist seems likely (see pt 8 below).vndEEE, as this would be the most parsimonious explanation. We agree that it could be valuable to test vndEEE in other mutants, but have not done so, we focussed on demonstrating that the mechanism is not via Notch and hence points to a more general mechanism. We agree also that Twist was a prime candidate, but as discussed below, our evidence does not support that.Our hypothesis is that the same mechanisms are involved for 6) Figure S4, if m5/m8 is Notch-dependent, what causes the increase at time 0 in the mean profile of Notch RNAi?m5/m8 expression is compromised when Notch activity is perturbed by a range of different mutations . Notably, in neuralized mutants there is no expression of m5/m8 remaining. In order to carry out the experiments here, we used NotchRNAi to perturb Notch activity, as it was the most genetically tractable for the experiments . Unfortunately, RNAi is not always fully penetrant and some embryos retained residual activity. As evident from the plots in Figure 3 – supplement 1B, expression is completely or almost completely abolished in 6/8 embryos. We have opted to include all of the data for transparency, but include now also a plot from those embryos where we are confident the knock-down is effective.We have previously shown that 7) It is interesting that the ectopic expression of eve2-NICD in Notch RNAi background rescues m5/m8 transcription activity within the eve stripe , but how much of this increase relates NICD versus other tension activated factors ? Is twist upregulated in the domain of eve2-NICD expression in Notch RNAi embryos? How does this study/timing of m5/m8 upregulation at invagination relate to the mechanical signals discussed in Pouille et al., Sci Signal 2009 (which should be cited).m5/m8 transcription is dependent on Notch and that Twist is involved in priming so that the enhancer response in a sustained and robust way. Like the reviewer, we wondered whether Twist might be involved in directing the enhanced expression at gastrulation, in part based on the report that Twist is sensitive to mechanical force in other parts of the embryo, in other work from the Farge group. We have invested a large amount of time to follow up on this model but can find no evidence that Twist is at the nexus. For example, we carefully quantified Twist levels throughout nuclear cycle 14, and there is no increase at the time of mesoderm invagination. These data have now been added to the manuscript as new Figure 6. Furthermore, a mutant version of the m5/m8 enhancer that lacks Twist motifs is still upregulated at the time of mesoderm invagination. This point has been made more explicit in the text (see page 18).We have previously shown that The mechanical signal described in Pouille et al. would be upstream of the events we are investigating as it altered endocytosis of Fog, the ligand that initiates mesoderm invagination. We have made this point more explicit and included the reference as requested (see Page 16).8) The conclusion that LINC complex/Klar plays a role in tension-regulated Notch signaling activity is poorly supported. Figure 5B shows that there is an early increase in m5/m8 activity, which is, as the authors point out, opposite from the expected result. Therefore, it is necessary to better define the relationship between NICD and nuclear properties modified/influenced by LINC. How does loss of function klar (or other LINC protein) impact intracellular Notch signaling and other tension-related processes, including localization of α-catenin at the AJs, RhoGEF2 at the apical membrane, actomyosin pulsing, expression of cta and fog, etc. Depending on whether twist is found to be upregulated by eve2-NICD (see point 7 above), it might also be worth looking to see how twist expression (upregulation by NICD?) is affected by klar.We argue that LINC is required to transmit the consequences from tension regulated forces, generated by Fog and actinomyosin changes, onto the nucleus. There is no evidence that Klar would alter any of these upstream steps in the mechanotransduction. In contrast there is growing evidence that LINC is important in transducing forces to the chromatin. We have thus focussed on potential downstream changes in the nuclear properties.These additional results are reported in a new section “Changes in nuclear properties at gastrulation”. First, we have measured nuclear entry of different transcription factors, including Twist. We not find any changes in nuclear levels that can provide an explanation as shown by the new data that we have added . Second, we have quantified additional features such as nuclear morphology and chromatin motility. Both these properties change at gastrulation and could contribute to the effects seen .9) Unlike m5/m8 enhancer which requires Notch signaling input directly to help activate gene expression, Notch input at the sim enhancer acts to 'derepress' expression by eliminating Su(H) repressor input and allowing output to be responsive to Dorsal and Twist . Therefore, the roles of Notch in m5/m8 and sim enhancers differ. In the current study, the focus is m5/m8 with little done to analyze sim enhancer properties. Interestingly, in Morel & Schweisguth, when Su(H) sites are mutated in a sim enhancer (sim.mut), reporter expression remains in N mutant embryos. It is likely that the sim.mut enhancer would also show upregulated gene expression at mesoderm invagination if tested; and if so may be a better test case for tracking the input that relates to mechanotransduction with subsequent mutagenesis. Possibly this input is Twist.sim enhancer, as well as alleviating repression. We note that, although there is some expression remaining in the absence of Notch, this is very variable and weak in comparison to the wild-type. In addition, the sim enhancer shows a very similar upregulation to m5/m8 at the time of mesoderm invagination, which is lost in embryos where α-Catenin is depleted. We have added these additional data with the sim enhancer to the manuscript . As discussed above, we do not find evidence to support the hypothesis that Twist is the primary transducer.Our evidence suggests that NICD also has a positive effect on 10) Farge 2003 already showed that Twist increases residency in the nucleus downstream of mechanical induction. Relating to comments above (e.g. point 7 and 9), it is likely that the NICD-effect could be indirect, working to upregulate Twist. If so, this study is still interesting, but are the current results novel?m5/m8 enhancer in which all of the Twist motifs have been mutated (which exhibits much lower levels of expression) still undergoes a marked increase in activity at the time of gastrulation . This point has been emphasised more clearly in the text (page 18).As discussed above, we also thought Twist would be a good candidate based on Farge 2003 and have investigated this possibility quite extensively. We do not find any evidence for a change in the levels of nuclear Twist at this stage, having used a nano-body to measure the levels throughout nuclear cycle 14 . Furthermore, an 11) The title is confusing as the data support the view that "cell movements associated with gastrulation have a direct impact on transcription in the nucleus"… and that Notch-responses are not the only ones that are affected.We have modified the title to take on board the concerns and we think this better describes the results obtained.Reviewer #3:Drosophila mesoderm movements. The data are of very good quality. The authors could boost their conclusions by better replacing them in the context of their previous studies regarding the role of adherens junctions in Notch transcriptional regulation or turnover . The findings on Klar mutant conditions are highly relevant but more difficult to unit with the interplay between Notch transcriptional control and mesoderm invagination.This manuscript is of broad interest to readers in the fields of Cell and Developmental Biology. The authors provide an elegant set of experimental evidence demonstrating that Notch transcriptional activity is modulated during The major strength of the manuscript is the quantitative and original analyses of Notch (N) transcriptional levels in conjunction with morphogenetic movements in multiple genetic backgrounds. The authors could improve their manuscript by addressing the following points:1. In Figure S1, the authors have also quantified N activity with two other reporters of N transcriptional activity. Both reporters show an increase at 50 mins as described in the main text, but both reporters also show a decrease at 55 mins. This decrease is not observed for the line (II) used through the manuscript. One could therefore argue that this drop is also a relevant characteristic of Notch activation during mesoderm invagination. This data should therefore be placed in the main figure and the decrease should be mentioned in the main text. In addition, it will be important that the authors performed the some loss of function analyses  using these two other reporters to further validate their findings.The decrease noted by the referee occurs after gastrulation is completed when the midline stripes come together and the cells in the mesectoderm divide. Our evidence suggests that Notch signalling ceases around that time and that the time it takes to complete this is more variable across embryos from the same genetic condition. It is also the time when there are extensive movements and sometimes the tracking is lost. This leads to considerable variability between movies, especially in genetic conditions where gastrulation is a bit delayed or stuck. We can confirm that both the other reporters have been examined in Notch loss of function conditions, where their expression is lost. We have also tested their behaviour in embryos where α-Catenin is depleted and both show similar loss of upregulation to the main reporter used in the paper. We have added these data to the manuscript .2. Page 7: The following statement is confusing. "We note the mean levels of transcription from this reporter were not significantly reduced prior to gastrulation, unlike those from another reporter insertion and the endogenous genes that showed decreased levels also during cellularization . The difference likely arises because this m5/m8 [II] insertion achieves maximal transcription at lower levels of Notch activity." In the cited previous paper, the authors have shown that a-cat affects Notch transcription during cellularization. It is unclear why a difference in reporter strengths will lead to distinct conclusions. This needs to be further experimentally explored and explained.m5/m8 reporter gives a more robust response. We too have been perplexed by the reasons for this, although it is well known that different genomic insertion sites of the same reporter frequently yield slightly different responses. We have recently generated a new CRISPR line where the endogenous gene has been tagged with MS2 loops and verified that it shows the same transcription profile. We also find that the other enhancers yield similar results after gastrulation, making us confident that the effects are not enhancer specific, albeit sometimes the magnitude of the effect may vary. We have explained this more clearly in the text (see pages 7-8) and have added the additional data for the other enhancers .In all the experiments we have done, the second chromosome 3. Page 12: "As predicted, this treatment greatly reduced transcription from m5/m8 [II] .". The reduction is 2-fold. How these fold changes compare with the ones observed in RhoGEF2 and Fog mutant conditions? The Notch mutant conditions should be shown in figure 1. Furthermore, one could argue that a large part of the increase in reporter expression does not reflect an increase in Notch activity. Please discuss.neuralized, where there was no residual expression from the reporters . Here we used NotchRNAi to perturb Notch activity, as it was the most genetically tractable for the experiments. Unfortunately, RNAi is not always fully penetrant and some embryos retained residual activity. As evident from the plots in Figure 3 – supplement 1B, expression is completely or almost completely abolished in 6/8 embryos. We have opted to include all of the data for transparency, which is why some residual expression is seen in the mean plots, as noted by the referee. We have now included a plot from those embryos where we are confident the knock-down is effective to illustrate that very little transcription remains  The fold changes are very different from other knock down experiments, where there is transcription remaining in a significant proportion of the cells in all the embryos, as is evident in the heatmaps. We hope this clarifies the point.Here we are a victim of the variability from RNAi knock-down experiments. We have previously used several different mutations to compromise Notch activity, including 4. Figure 4. It will be important that the authors plot on the same plot with the same units the endogenous Notch activity in both wt and NotchRNAi.We are unclear what the reviewer is requesting here, and what the concerns are. In each experiment we compare with a wild type control from embryos imaged under the same conditions. For all of the Notch RNAi experiments there is a plot of the equivalent control, with control RNAi, from the same experiments. If comparing between very different experiments (e.g WT and Notch ICD overexpression), the time and imaging conditions may not be identical. For example, some experiments were conducted pre-Covid closure and others after, with changes in the microscopes between. A laser burned out at one point and some experiments were performed on a different microscope. However, where these changes have occurred, controls were always imaged in parallel. This is why there are sometimes slight differences between the control plots.5. As stated by the authors, Notch transcription findings in Klar mutant conditions are distinct from the authors' prediction since transcription increases prior to mesoderm invagination. The role of nucleus deformation and the Klar contribution remain difficult to understand and insert in the authors proposed model.We agree that the results are not 100% straightforward. However the data argue (1) that integrity of LINC complex is important for the normal levels of gene expression and (2) that the levels of transcription are no longer sensitive to the forces from gastrulation. As we have tested other factors and not found any other that prevents the gastrulation related increase, without compromising transcription overall, we therefore consider this to be a substantial result.6. Statistics: Could the authors propose statistical tests to further support their findings regarding temporal changes in reporter levels and differences between distinct mutant backgrounds?We have done this for the correlation plots and provided the results in the R-value. The mean plots are significantly different from one another, as is evident from the error bars. As discussed we always include all the data in the plots, to show the variability within a given genotype. This has been the standard approach in the current literature.[Editors’ note: what follows is the authors’ response to the second round of review.]Essential revisions:Overall, the manuscript is much improved and the authors have addressed most of the reviewers' major concerns. There are two remaining issues that require clarification and additional technical information related to imaging conditions and corrections for nuclear depth over the course of gastrulation, and a previously reported increase in transparency of klar embryos.1) Please provide additional information about the imaging pipeline – specifically whether any corrections are being made for spatially-dependent changes in signal due to movement of cells in the z-direction. See reviewer 2 comments for details.We appreciate the comments of the reviewer, because the potential for the movement of the nuclei to be a confounding factor was one that concerned us at the outset. All the movies were carefully evaluated to ascertain whether there was any relationship between the nuclear positions and the change in MS2/MCP signal intensity. We found no correlation. We have now added the graph showing this lack of correlation to the supplementary data (new figure 1S1E ). We note, as discussed in more detail below, that the strategy proposed by the reviewer, to normalize using the histones, is not feasible due to variable changes in the levels and the chromatin density etc, that complicate any such quantifications. We hope that the additional information provided addresses the concerns.2) The klar mutant background is associated with a clearing of embryos, at least in older embryos at stage 8 . Please provide information regarding whether there is an increase in transparency of klar embryos at NC14 that may result in the GFP signal appearing brighter that wild type controls.We are grateful to the reviewer for highlighting this possibility. Indeed we have now checked and there is an increase in GFP detection in the Klar mutant embryos. We have carefully quantified this difference, and corrected for it. However, the effect is a small one with a very minor impact on the overall levels that does not alter the results .Reviewer #2:The manuscript is improved. In particular, the data are more clearly presented and the authors have added more experiments, namely testing whether levels of Twist, chromatin import, chromatin compaction and/or accessibility relates to the increase in mesectoderm gene expression identified at the time of mesoderm invagination.We are glad the reviewer appreciates the additional experiments testing the role of Twist.The author's testing of Twist addresses one of the reviewer's major concerns. While it is still possible that post translational modification of Twist acts to increase its activity at invagination, their dismissal of Twist for the purpose of this study is reasonable based on the data presented.Another major concern was that the use of maximum intensity may lead to noise in the spot calling numbers. The authors state that using the maximum intensity is an accurate method because of tests they have done, however they do not elaborate on what these tests are or how they have verified this beyond the limited use of the alternate method from Garcia 2013. While using the method in Garcia 2013 does show the same general trend, the error is much larger using this method and this doesn't completely support the claim that this method proves the simpler method is correct. The fundamental concern is that single, bright pixels that occur outside of the actual MS2 spot could be skewing the data. An easy way of dealing with this is to apply a median filter, which will remove "salt and pepper" noise to the images. The authors note that they apply this filter across time to smooth the data, however this is would be selecting for nuclei that tend to be on continuously. Another approach would be to manually confirm that the maximum pixel occurs within a spot, but this would be tedious. In the methods section it is stated that "subsequent analysis allows to classify confidently when the maximum value is really representing a spot," however it is unclear how the subsequent analysis alleviates this concern. At minimum, this section of the methods needs to be expanded and made clearer so that the reader can evaluate the method being used.We apologize if the details of the methodology were not sufficiently clear. As the reviewer notes, we have applied a median filter to remove noise from background pixels that are not coming from an MS2 spot . The wording we used was perhaps misleading because the filter is only applied over 3 frames, i.e. over a period of 45-60”. Thus, it is not selecting for “continuously active spots”. The time-window was chosen because, based on transcription rates and minimum signal needed from MS2 loops, bursts shorter than 1 min would probably not be detectable so any bright pixels present for such a duration have a high probability to be background. Conversely, the “subsequent analysis” involved applying background subtraction, median filtering and selecting spots classified as ON for 5 frames (1.25-1.6 min) to identify active sites of transcription. These parameters were validated by overlaying nuclei defined by the pipeline as actively transcribing with the MS2 signal, and confirming that the two were concordant. These aspects of the analysis have been explained more clearly in the Methods (page 34).I do still have a worry that their imaging pipeline is not properly correcting for spatially-dependent changes in signal due to movement of cells in the z-direction. Because they have a H2A-RFP associated with the MCP-GFP MS2 movies, they should use the RFP signal to background correct . The H2A-RFP signal can vary in space in time due to position of nuclei. The authors are imaging the MS2 signal in a tissue that moves, however they don't provide a correction for this movement or rule out whether this movement is changing intensity of the signal. Specifically, when signal is lower, the MS2 is in a more dorsal location than compared to after invagination where the signal is at the ventral most point. Did the authors correct for any difference in intensity that could be occurring from differences in the position of the tissue? It is conceivable that signal from the ventral most point will be more optically accessible than from points located more dorsally, due to the curvature of the embryo and possibly the diffraction of light as it moves through tissue to reach the objective. An easy way to correct for this would be to use the signal of the H2A signal that was used to segment and track the nuclei, as any positional effects should also affect the H2A signal, and the H2A signal is already mapped to its corresponding spot.We acknowledge that tissue depth and position of the MS2 spot in Z might affect the intensity being detected, however we had carefully evaluated the data and had ruled out the possibility that the transition in levels is due to changes in nuclear position. We have added data to the supplementary (new figure 1S1E) to illustrate this point.We have been able to analyze the relationship between MSE nuclear depth/curvature and the MS2/MCP signal because individual embryos are mounted with slightly different positions so that each has differing changes in Z. In most cases, the embryos are not mounted completely ventrally, which means that the MSE is very close to the coverslip before as well as after gastrulation. As the embryo gastrulates, it does so within the vitelline membrane so that the MSE is captured in a similar Z position throughout time. This means that, in these cases, the signal detection is not very affected by curvature of the embryo and the depth of imaging. In other cases where the embryo is mounted more ventrally the difference in Z positions is only 5μm on average. By comparing the data from each embryo, there is no correlation between how far the position of the MS2 spots differed in Z before and after gastrulation and how much levels increased. We initially made this analysis for all the original movies  and have now performed the same analysis for the new movies we generated for the gastrulation studies  . In neiWe also note that in other genetic conditions, such as RhoGEF2 depletion, where tissues move from the same positions in DV and Z as wild type embryos, (only slower), we do not detect any increase in levels. If the increase was due to differences in Z positions, a similar increase would still be manifest in these embryos, which it is not.We appreciate the suggestion from the reviewer to utilize the His2Av-RFP intensity as a way to normalize from any changes in depth. However, in our experience this would not be a good method for several reasons. First, its levels vary over time, independently of Z position, because as nuclei increase volume the His2Av signal becomes dimmer. Second, heterochromatin begins forming during nc14, giving rise to a much more heterogeneous signal that includes differences along apical/basal polarity. Thus while His2Av is very valuable for segmenting the nuclei, its variability during nc14 makes it unsuitable for correcting on a nucleus-by-nucleus basis for the subtle changes in intensity due to changes in Z position.In addition, the klar mutant background is associated with a clearing of embryos such that the GFP signal may appear brighter because embryos are more transparent; this has been shown for older embryos at stage 8  but it is unclear if this is the case at nuclear cycle 14. If klar mutant embryos at these earlier stages are more transparent, could this account for the apparent increase in GFP signal associated with the klar mutant background ? Might the H2A-RFP signal serve to normalize the ctr RNAi vs klar RNAi data? Is the H2A-RFP signal also increasing in the klar RNAi? If that is the case, and the klar data is more similar to wildtype – the early phase of transcription may need to be offset vertically . In this alternative scenario, it would appear then that there is a transition late in the klar mutant background. Might the clearing of lipids affect timing of mesoderm invagination and/or when it is recognized/called?Klar RNAi embryos indeed exhibited higher GFP and RFP signals. We quantified this difference by measuring the average His2Av-RFP signal (15.8 % lower in control embryos) and average MCP-GFP signal in nuclei that never show MS2 activity (11% lower in control embryos). We have as a consequence corrected the MS2 signal for the “clearing effect” by reducing the signal in Klar RNAi by 11%. This correction has a very modest impact which doesn’t change the overall result. These data are now included in supplementary Figure 5S2 and commented on page 17.We thank the reviewer for bringing this reference to our attention. The reviewer appreciates the premise of this study but the authors would be wise to do a bit more controls/investigating to ensure that the observed upregulation in signal associated with mesoderm invagination is not an imaging artifact: (i) might positional effects on MS2 signal be responsible for the increase in GFP observed when cells invaginate/are more directly illuminated; and (ii) to determine whether klar mutants are more transparent and if so use methods to normalize signals obtained in klar in comparison with wildtype.We have addressed these points in detail in the two sections above.Reviewer #3:The authors have improved their manuscript and addressed my points. They have added a lot of data related to the dynamics of the nucleus and chromatin mobility. Most of these results are negative but they contribute to the detailed characterization of the process. Overall, the findings support the authors' conclusions.We are glad that the reviewer appreciates the additional data and that these have addressed many of the points they raised.Nevertheless, the role of Klar remains difficult to fit in the authors' proposed model, since Klar loss of function phenotype is distinct from the ones observed in mutants affecting the mechanical forces associated with gastrulation . I therefore doubt that Klar would be part of the mechanisms by which gastrulation associated forces modulate Notch-dependent and -independent transcriptional regulation.Klar mutants share a key element of the phenotype from α -Cat, RhoGEF2 which is to depress the increase in transcription. Klar exhibits additional changes which would suggest it has other roles besides transducing the mechanical signal.We appreciate the reservations of the reviewer, but we disagree with the premise that a difference in phenotypes a priori rules out a role in the mechanism. The