RHO) gain-of-function mutations we engineered the ZF6-DNA-binding protein (ZF6-DB) that targets 20 base pairs (bp) of a RHOcis-regulatory element (CRE) and demonstrate Rho specific transcriptional silencing upon adeno-associated viral (AAV) vector-mediated expression in photoreceptors. The data show that the 20 bp-long genomic DNA sequence is necessary for RHO expression and that photoreceptor delivery of the corresponding cognate synthetic trans-acting factor ZF6-DB without the intrinsic transcriptional repression properties of the canonical ED blocks Rho expression with negligible genome-wide transcript perturbations. The data support DNA-binding-mediated silencing as a novel mode to treat gain-of-function mutations.Transcription factors (TFs) operate by the combined activity of their DNA-binding domains (DBDs) and effector domains (EDs) enabling the coordination of gene expression on a genomic scale. Here we show that in vivo delivery of an engineered DNA-binding protein uncoupled from the repressor domain can produce efficient and gene-specific transcriptional silencing. To interfere with RHODOPSIN (DOI:http://dx.doi.org/10.7554/eLife.12242.001 Proteins called transcription factors bind to sections of DNA known as regulatory elements to activate or deactivate nearby genes. In animals, transcription factors typically have two sections: a “DNA-binding domain” that attaches to DNA, and an “effector domain” that is responsible for interacting with other proteins to regulate the gene’s expression.Rhodopsin is a gene that encodes the instructions needed to make a light-sensitive protein in the eyes of humans and other animals. Botta et al. have now used this gene as an example to investigate whether proteins that contain a DNA-binding domain – but not an effector domain – can repress gene expression.Rhodopsin gene is actually required for the gene to be expressed. Botta et al. designed an artificial protein – referred to as ZF6-DB – that is able to bind to this section of DNA. The binding of ZF6-DB to this short DNA section was sufficient to switch off a Rhodopsin gene in living pig cells, and, unlike conventional transcription factors, seemed to have minimal impact other genes.The experiments show that only a small section of the regulatory elements in the human Rhodopsin into pigs. In these animals, ZF6-DB switched off the existing copy of Rhodopsin, but not the inserted copy so the cells produced a working form of the light-sensitive protein. Further experiments were carried out in mice that have both a faulty version and a normal copy of the Rhodopsin gene. ZF6-DB switched off the faulty Rhodopsin gene, which allowed the normal Rhodopsin gene to work without any interference from the faulty copy.Next, Botta et al. used a virus to insert both the gene that encodes ZF6-DB and a normal copy of Rhodopsin can cause an eye disease that leads to severe loss of vision in humans. These new findings could now guide future efforts to develop treatments for people with this condition. It will also be important to investigate how ZF6-DB binds to the regulatory elements in the Rhodopsin gene and whether a similar strategy could be used to alter the expression of other genes.Mutations in DOI:http://dx.doi.org/10.7554/eLife.12242.002 Transcription factors (TFs) operate by entangling their DNA-binding and transcriptional activation or repression functions . Howevercis-regulatory element (CRE) of the human proximal RHO promoter region by deconstructing an engineered TF (synthetic) composed of a DBD  and the ED , which we have shown to be effective in repressing specifically the human RHO transgene carried in an adRP mouse model (RHO promoter fragment carrying the ZF6-cis deletion upstream of the eGFP reporter gene (AAV-RHO-cis-del-EGFP), after delivery to the porcine retinal photoreceptor, showed loss of eGFP expression compared to the control vector (AAV-RHO-EGFP) (RHO expression (at least for the 776 bp region used in the assay) and that binding of the synthetic ZF6-DB trans-acting counterpart of ZF6-cis, may indeed repress RHO transcription.We generated a DNA-binding protein targeted to a se model . The delse model . Genomicse model , as repose model . To studse model  in livinHO-EGFP) . This suRHO gene in a physiological genomic context, we used the porcine retina  of ZF6-DB (AAV8-CMV-ZF6-DB) resulted in a 45% decrease of porcine Rho transcript levels at 15 days post-injection  with or without the vector encoding ZF6-DB (5x1010 gc). Fifteen days after injection, the retina were disaggregated and FACS-sorted. The retina co-transduced with eGFP and ZF6-DB vector showed virtually a 'somatic knock-out' of Rho expression  experiments to evaluate binding to the ZF6-cis target genomic sequence showed occupancy by the DNA-binding protein ZF6-DB . To evale retina , which se retina . Subretinjection . Immunofnjection . Despiteme point . To furtelements ; AAV8-GN10 gc) resulted in about twenty-fold lower expression levels of the ZF6-DB and ZF6-KRAB transgenes compared to Crx and Nrl, two retina-specific TFs  and glial fibrillary acidic protein (Gfap)  from retina harvested 15 days after subretinal injection of the AAV8 vector encoding ZF6-DB . For comific TFs   . ZF6-KRAn (Gfap) . These r-DB DEGs . Consist-DB DEGs . These rfinities . These dc target  and the RHO-P347S mouse  may result in preservation of retinal function  in order to complement Rho transcriptional repression in porcine retina  generated a decrease of about 100,000 Rho RNA-seq counts after transduction  to administer to porcine retina. As an internal control the contralateral eye received the previously used ZF6-DB vector  and in concomitant replacement of the exogenous hRHO (68%), as assessed by transcripts, protein expression levels, and integrity of photoreceptor outer segments (Gnat1 transcript and protein (data not shown) levels demonstrated complementation, supporting a secondary down-regulation of Gnat1 associated with Rho repression  of ZF6-DB and the rod genomic context. The transcriptional repression mechanism of ZF6-DB binding likely relies on the interference occurring between TFs and local DNA sequence features within the RHO proximal promoter region , suggest that the molecular determinant of silencing may not be the simple displacement of key RHO TFs  or an inert DNA-binding protein harbor , but also as an intrinsically active operator of the transcriptional function. This contribution of DNA is supported by both the transcriptional repression upon the ZF6-cis deletion of 20 bp of DNA and intereference in trans by using a synthetic DNA binding protein that, not being encoded by the genome, may occupy a protein-free portion of the genome. It follows that in terms of signaling, the information source, the DNA, generates an output signal the RNA and eventually a protein (TF), whose final output functional activity is completed back by the DNA. Thus the information source, the DNA, becomes also integral part of the signaling . In this vision, to act (interfere) upstream the DNA, an external function is necessary, which is, in this study, the synthetic ZF6-DB carried by the vector.In this study, we showed that photoreceptor genomic binding of a 20 bp-long DNA sequence by a synthetic DNA-binding protein dramatically reduces r region , which w RHO TFs . We propRho transcriptional repression, which reached 85% when rods were sorted, supporting its use for diseases requiring correction of a large number of affected cells, such as adRP and other Mendelian disorders due to gain-of-function mutations. Furthermore, Rho transcriptional silencing and its complete RHO replacement support in principle the use of the DBR-R constructs for treatment of any RHO mutation including those caused by a dominant negative mechanism , 164 bp from the transcription starting site (TSS) + 5’UTR), the human rhodopsin long promoter , the human rhodopsin long promoter mutated of the ZF6-cis  and the human rhodopsin muted promoter  were generated by gene synthesis of Eurofins MWG and cloned in pAAV2.1 using NheI and NotI restriction enzymes. For the generation of DBR-R plasmid the Eurofins MWG synthetized the expression cassette RHOΔ-ZF6-DB-bGHpolyA (bovine growth hormone polyA) that we cloned in pAAV2.1 hGNAT1-hRHO using NheI restriction enzyme.The ZF6-DNA-binding domain  was amplified by PCR from AAV2.1 CMV-ZF6-KRAB  using pr2 purification , then AAV vectors were delivered sub-retinally via a trans-scleral transchoroidal approach as described by Liang et al. .ad libitum. The anesthetic and surgical procedures for pigs were previously described  female piglets were utilized. Pigs were fasted overnight leaving water escribed . AAV vecescribed . This reEscherichia coli BL21DE3 host strain. The transformed cells were grown in rich medium plus 0.2% glucose (according to protocol from New England Biolabs) at 37°C until the absorbance at 600 nm was 0.6–0.8, at which time the medium was supplemented with 200 µM ZnSO4, and protein expression was induced with 0.3 mM isopropyl 1-thio-β-D-galactopyranoside and was allowed to proceed for 2 hr. The cells were then harvested, resuspended in 1X PBS (pH 7.4), 1 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, 1 µM aprotinin, and 10 µg/ml lysozyme, sonicated, and centrifuged for 30 min at 27,500 relative centrifugal force. The supernatant was then loaded on amylose resin (New England Biolabs) according to the manufacturer’s protocol. To remove the MBP from the proteins, bound fusion proteins as cleaved in situ on the amylose resin with Factor Xa (1 unit/20 µg of MBP fusion protein) in FXa buffer  for 24–48 hr at 4°C and collected in the same buffer after centrifugation at 500 relative centrifugal force for 5 min. The supernatant containing the protein without the MBP tag was then recovered.DNA fragments encoding the sequence of the engineered transcription factors and ZF6-KRAB, to be expressed as maltose-binding protein (MBP) fusion were generated by PCR using the plasmids pAAV2.1 CMV-ZF6-KRAB and pAAV2.1 CMV-ZF6-DB as a DNA template. The following oligonucleotides were used as primers: primer 1, (GGAATTCCATATGGAATTCCCCATGGATGC) and primer 2, (CGGGATCCCTATCTAGAAGTCTTTTTACCGGTATG), for ZF6-KRAB primer 3, (GGAATTCCATATGCTGGAACCTGGCGAAAAACCG) and primer 4,(CGGGATCCCTATCTAGAAGTCTTTTTACCGGTATG) for ZF6-DB. All the PCR products were digested with the restriction enzymes NdeI and BamH1 and cloned into NdeI BamH1-digested pMal C5G  bacterial expression vector. All the plasmids obtained were sequenced to confirm that there were no mutations in the coding sequences. The fusion proteins were expressed in the hRHO proximal promoter sequence was measured by a gel mobility shift assay by performing a titration of the proteins with the oligonucleotides. The purified proteins were incubated for 15 min on ice with hRHO 65 bp duplex oligonucleotide in the presence of 25 mM Hepes (pH 7.9), 50 mM KCl, 6.25 mM MgCl2, 1% Nonidet P-40, 5% glycerol. After incubation, the mixture was loaded on a 5% polyacrylamide gel (29:1 acrylamide/bisacrylamide ratio) and run in 0.5 TBE at 4°C (200 V for 4 hr). Protein concentration was determined by a modified version of the Bradford procedure. After electrophoresis, the gel was stained with the fluorescent dyes SYBR Green I Nucleic acid gel stain  to visualize DNA. 2.5 µM of the ZF6-KRAB protein was incubated with increasing concentrations  of the duplex hRHO 65 bp, an apparent higher protein concentration (2.5 µM) was required likely because not all the protein sample was correctly folded. In the case of ZF6-DB, 1.5 µM of the protein was incubated with increasing concentrations  of the duplex hRho 65 bp. Scatchard analysis of the gel shift binding data was performed to obtain the Kd values (25). All numerical values were obtained by computer quantification of the image using a Typhoon FLA 9500 biomolecular imager .The affinity binding costant of proteins for RNAs from tissues were isolated using RNAeasy Mini Kit , according to the manufacturer protocol. cDNA was amplified from 1 μg isolated RNA using QuantiTect Reverse Transcription Kit (Qiagen), as indicated in the manufacturer instructions.The PCRs with cDNA were carried out in a total volume of 20 μl, using 10 μl LightCycler 480 SYBR Green I Master Mix  and 400 nM primers under the following conditions: pre-Incubation, 50°C for 5 min, cycling: 45 cycles of 95°C for 10 s, 60°C for 20 s and 72°C for 20 s. Each sample was analysed in duplicate in two-independent experiments. Transcript levels of pig retinae were measured by quantitative Real Time PCR using the LightCycler 480 (Roche) and the following primers: pRho_forward (ATCAACTTCCTCACGCTCTAC) and pRho_reverse (ATGAAGAGGTCAGCCACTGCC), pGnat1_forward (TGTGGAAGGACTCGGGTATC) and pGnat1_reverse (GTCTTGACACGTGAGCGTA), pArr3_forward (TGACAACTGCGAGAAACAGG) and pArr3_reverse (CACAGGACACCATCAGGTTG). humanRho_forward (TCATGGTCCTAGGTGGCTTC), humanRho_reverse (ggaagttgctcatgggctta) and eGFP_forward (ACGTAAACGGCCACAAGTTC) and eGFP_reverse (AAGTCGTGCTGCTTCATGTG). All of the reactions were standardized against porcine Actβ using the following primers: Act_Forward (ACGGCATCGTCACCAACTG) and Act_reverse (CTGGGTCATCTTCTCACGG).Triple-immunostaining for anti-HA, anti-GNAT1, and anti-Rhodopsin antibody. Frozen retinal sections were washed once with PBS and then fixed for 10 min in 4% PFA. Sections were immerse in a retrieval solution  and boiled three times in a microwave. After the blocking solution  was added for 1 hr. The two primary antibody mouse anti-HA  and rabbit GαT1 (Santacruz Biotechnology), were diluted in a blocking solution and incubated overnight at 4°C. The secondary antibodies  have been incubated for 1 hr, followed by three rinses with PBS. After the slides were incubated in blocking solution (10% NGS) for 1 hr and then incubated O.N. with primary antibody mouse- 1D4 . The secondary antibodies . Sections were photographed using a Leica Fluorescence Microscope System .Frozen retinal sections were washed once with PBS and then fixed for 10 min in 4% PFA. Sections were immerse in a retrieval solution  and boiled three times in a microwave. After the blocking solution  was added for 1 hr. The primary antibody mouse anti-HA  was diluted in a blocking solution and incubated overnight at 4°C. The secondary antibody  has been incubated for 1 hr. Vectashield  was used to visualize nuclei. Frozen retinal sections were permeabilized with 0.2% Triton X-100 and 1% NGS for 1 hr, rinsed in PBS, blocked in 10% normal goat serum (NGS), and then incubated overnight at 4°C with rabbit human cone arrestin (hCAR) antibody, kindly provided by Dr. Cheryl M. Craft  diluted 1:10000 in 10% NGS. After three rinses with 0.1 M PBS, sections were incubated in goat anti-rabbit IgG conjugated with Texas red  for 1 hr followed by three rinses with PBS. Frozen retinal sections were permeabilized with 0.1% Triton X-100, rinsed in PBS, blocked in 20% normal goat serum (NGS), and then incubated overnight at 4°C in a mouse anti-1D4 rhodopsin antibody diluted 1:500 in 10% NGS. After three rinses with 0.1 M PBS, sections were incubated in goat anti-mouse IgG conjugated with Texas red  for 1 hr followed by other three rinses with PBS. Sections were photographed using either a Zeiss 700 Confocal Microscope  or a Leica Fluorescence Microscope System . 2, 1% CHAPS, 1 mM PMSF, and protease inhibitors) and 20 µg of these lysates were separated by 12% SDS-PAGE. After the blots were obtained, specific proteins were labeled with anti-1D4 antibody anti-Rhodopsin-1D4 , and anti-β-tubulin  antibodies.Western blot analysis was performed on retinae, which were harvested. Samples were lysed in hypotonic buffer  for 15 min. Nuclei were lysed in nuclei lysis buffer  for 30 min. Chromatin was shared using Covaris s220. The shared chromatin was immunoprecipitated over night with anti HA ChIP grade . The immunoprecipitated chromatin was incubated 3 hr with magnetic protein A/G beads . Beads were than washed with wash buffers and DNA eluted in elution buffer . Then Real Time PCR was performed using primers on rhodopsin TSS, hRHOTSSFw (TGACCTCAGGCTTCCTCCTA) and hRHOTSSRv (ATCAGCATCTGGGAGATTGG), trasducin 1 TSS, hGNAT1TSSFw (CAGCCCTGACCCTACTGAAC) and hGNAT1TSSRv (CAACCGCTGACTCTGCACT), arrestin 3 TSS, hArr3TssFw (CCTGCTGTGCACATAAGCTG) and hArr3TssRv (CGTGTCCCACTCCAATCTCT), and β-tubulin TSS, hTUBTSSFw (TCCTGTACCCCCAAGAACTG) and hTUBTSSRv (GCTGCAAAATGAAGTGACGA).For ChIP experiments, HEK293 cells were transfected by CaCl10 gc)and AAV8-GNAT1-eGFP (dose 1x1012 gc) were disaggregated using Papain Dissociation System  following the manufacturers protocol. Dissociated retinal cells were analysed using BD FACSAria at IGB (Institute of Genetic and Biophysic “A. Buzzati-Traverso”) FACS Facility and sorted, dividing eGFP positive cells (rods) from eGFP negative fraction.Co-injected porcine retina with AAV8-CMV-ZF6-DB (dose 5x102 (which correspond to 1×10−5.2 to 20.0 cd*s/m2) in scotopic conditions. The log unit interval between stimuli was 0.3 log from −5.4 to 0.0 log cd*s/m2, and 0.6 log from 0.0 to +1.3 log cd*s/m2. For ERG analysis in scotopic conditions the responses evoked by 11 stimuli (from −4 to +1.3 log cd*s/m2) with an interval of 0.6 log unit were considered. To minimize the noise, three ERG responses were averaged at each 0.6 log unit stimulus from −4 to 0.0 log cd*s/m2 while one ERG response was considered for higher (0.0−+1.3 log cd*s/m2) stimuli. The time interval between stimuli was 10 s from −5.4 to 0.7 log cd*s/m2, 30 s from 0.7 to +1 log cd*s/m2, or 120 s from +1 to +1.3 log cd*s/m2. a- and b-waves amplitudes recorded in scotopic conditions were plotted as a function of increasing light intensity  as provided by ENSEMBL (SusScrofa 10.2.73). The GTF included the sequences for the 20 canonical chromosomes plus 4563 scaffolds, and counted 30.567 transcripts plus the sequences for the 3 exogenes used in the analysis (the 2 TRs and eGFP). Alignment was performed with RSEM (v.1.2.11)  according to manufacturer’s protocol. Libraries were sequenced on the Illumina HiSeq 1000 platform and in 100-nt paired-end format to obtain approximately 30 million read pairs per sample. Sequence Reads were trimmed using Trim Galore! software (v.0.3.3), that trims low-quality ends and removes adapter from reads, using a Default Phred score of 20. To obtain a precise estimation of this yet uncharacterized tissue, the 17 libraries were aligned against the full transcriptome for .1.2.11)  with defThe dataset was composed of 17 samples and 25.325 genes, divided in 3 experimental groups: 7 Controls, 4 ZF6-KRAB-treated, 6 ZF6-DB-treated.We analyzed the data following the standard Differential Expression Analysis Pipeline with DESeq2 R/Bioconductor package (v.1.8.1) , filteriWe fitted a unique Generalized Linear Model (GLM) with 1 factor and 3 levels . Differentially expressed genes were obtained out of the 2 contrasts (each treatment compared to the controls), an adjusted pvalue (FDR) of less than or equal to 0.1 was considered significant. We observed the expected upregulation of the exogenous genes used for the treatment  and for further evaluations we didn’t take into account their differential expression.cor.test, R package stats v.3.2.0)  .cor.test default function, method = 'Pearson'.Two named numeric vectors, one for each condition, containing the Log fold changes values of the 16 genes were tested for association with Sus scrofa genome were included in the list  . Plots wt-test and p-values <0.05 were considered significant. No statistical methods were used to estimate the sample size and no animals were excluded.Data are presented as mean ± Error bars indicate standard error mean (SEM). Statistical significance was computed using the Student’s two-sided  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor (Jeremy Nathans) and a Senior Editor.Thank you for submitting your work entitled "Gene-targeted transcriptional silencing by DNA-binding" for consideration by The following individual involved in review of your submission has agreed to reveal their identity: Eric Pierce (peer reviewer).The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The manuscript has been reviewed by three expert reviewers, and their assessments together with my own (Reviewing editor), form the basis of this letter.All of the reviewers were impressed with the importance and novelty of your work, and we would like to see a revision that addresses the issues raised.I am including the three reviews (lightly edited) at the end of this letter, as there are some specific and useful suggestions in them that will not be repeated in the summary here. Also, we think that this manuscript would be a better fit as a "Tools and Resources" paper rather than a regular research paper.The main issues, which I have extracted from the reviews and the subsequent discussion between the reviewers and me, are:1) Revisions to the text that focus the story on use of the ZF6-DB to suppress RHO expression. This could be accomplished by changing the title, and revising the Introduction and Discussion. We would favor moderating the claims about potential clinical use of this approach since the repression-replacement approach wasn't tested in a disease model.2) There is a question of ZF6-DB specificity and efficiency. Please provide the porcine, human, and mouse RHO TSS sequences for comparison, since it appears that the ZF6-DB recognizing hRHO TSS was used in these models regardless of any differences between the sequences. Does ZF6-DB recognize the porcine and mouse sequence? As to clinical therapeutic potential, how do the pig and human repression efficiencies compare? Is the 35% repression of Rho expression sufficient for dominant disease? These are important questions that could be addressed by supplying the sequences and the related data.3) It is unclear why cone arrestin increases in Arr3. This suggests that there may be off-target effects. Does ZF6-DB also bind to the Arr3 TSS region? ChIP data should be supplied to address off-target issues.4) 5) Regarding the repression-replacement experiment described in 6) The level of repression of the RHO gene achieved in the repression-replacement experiment described in 7) Clarity of presentation as indicated below by reviewers #2 and #3.We look forward to receiving the revised version of your manuscript.Reviewer #1:Summary: Botta et al. report a method for gene-specific transcriptional silencing using an engineered DNA-binding domain, "ZF6-DB." AAV-mediated expression of ZF6-DB in photoreceptors resulted in blockade of Rho expression, leading to therapeutic effects in animal models with gain-of-function cis- Rho mutations, such as autosomal dominant retinitis pigmentosa.Feedback:Experiment 1-showed the 20 bp in ZF6-cis is required for Rho expression via eGFP reporter assay. ChIP showed ZF6-DB binds well to CRE. Put ZF6-DB into porcine retina using AAV and measured Rho transcripts, which were decreased. Finally, they compared ZF6-DB with eGFP expression levelsCriticism: n = 2 is a low number. They should supplement their claims with additional data points.Experiment 2-Compared Rho transcription levels between ZF6-KRAB and ZF6-DB, found greater repression using ZF6-DB and less transcriptional perturbation.Criticism: They claim that ZF6-KRAB and ZF6-DB bind to same genomic targets, but they didn't explain or otherwise address the possibility that the difference in the transcription perturbations may in fact be caused by off-targeting effects or the down-regulation of RHO expression. Should perform ChIP to confirm the two are binding to the same region (as well as Crx and Nrl). Experiment 3-Compared the ERG a and b waves in the P347S RP murine model and saw much greater rescue in ZF6-DB compared with ZF6-KRAB or eGFP.Criticism: they should further explore the reason that ZF6-DB has such a greater rescue than ZF6-KRAB despite their similar binding affinity  Additional studies of the ZF6-DB approach with 1 or more additional genes would be needed to support the hypothesis that DNA binding proteins can be used broadly to regulate gene expression. Such data needs to be added to the manuscript, or the manuscript needs to be revised to focus on the specific application of this approach to the RHO gene.2) The level of repression of the RHO gene achieved in the repression-replacement experiment described in 3) Along these lines, the experiment testing the activity of the ZF6-DB in the transgenic RHO-P347S mice is helpful, but does not support the use of the ZF6-DB mediated RHO repression approach in patients, since the mutant human transgene is present on a mouse background. It would be better to test the activity of the ZF6-DB in a model with a mutant mouse allele, and include the complete repression-replacement approach in these studies.4) The ChIP experiment is helpful to show occupancy of the RHO CRE by ZF6-DB. It would be helpful to report what other sequences were detected in the ChIP experiment. That is, what else does the RHO ZF6-DB bind to? Additional ChIP data to address the question of what proteins normally occupy the RHO regulatory element would also be helpful.5) The repression of GNAT1 expression observed following treatment with the RHO ZF6-DB is interesting. Is there a similar CRE upstream of the GNAT1 gene? Or is some other mechanism responsible to the linked regulation of expression, as suggested by the data in 6) The relative specificity of the ZF6-DB activity compared to that observed using the hybrid ZF6-DB-KRAB ED protein is impressive. Other studies have used more than 30 million sequence reads to sample gene expression by the neural retina thoroughly. It would be helpful to know why 30 million reads were used in this study, and if this provides complete sampling of retinal transcripts.7) The discussion about the mechanism of ZFN-DB activity is interesting, but is beyond the data available in the current manuscript. The Discussion would be better focusing on interpreting the data in the manuscript.Reviewer #3:Botta and colleagues report on in vivo targeted silencing of rhodopsin expression by transcription factor proteins -with and without effector domain (ED)- engineered to recognize a 20 bp-cis-regulatory element of the proximal region of the rhodopsin promoter. They provide data which support the view that both engineered transcription factor proteins, with and without ED, suppresses rhodospin expression by DNA binding with similar affinities to the 20 bp-cis-regulatory element. They show that the engineered transcription factor lacking the effector domain silences rather specifically the expression of rhodopsin gene as determined by transcriptome analyses. They report that AAV-based delivery of the engineered rhodopsin-specific transcription factor protein without ED to the retina of a transgenic mouse with a human rhodopsin gene harboring a dominant negative RHO mutation prevented retinal degeneration. Finally, as a first step towards the use of this original strategy in the clinics, they nicely demonstrate that AAV-based delivery of pig-specific rhodospin silencing transcription factor protein without ED and of human Rhodopsin permitted preservation of retinal cells which deprived of pig-rhodopsin expressed the human counterpart.This is very nicely designed and executed study with real therapeutic promises. I have no major concern regarding the scientific aspects of this sound work.I have however a major critic regarding the writing of the manuscript which makes this beautiful piece of work sometimes very hard to follow. Here are some points to clarify:1) 2) 3) Anti-HA is used both in immunochemistry and CHIP analysis but it is not clearly mentioned that the constructs which were used encode this flag.4) CHIP-seq analysis: I would suggest to precise before giving the results a short sentence describing what was done, i.e. AAV-based delivery of constructs encoding eGFP or silencing transcription factor protein with or without ED in HEK293 cells, immunoprecipitation using the HA tag and RT-PCR amplification using primers flanking (where are they by the way?). The supporting figure is obscure. What are the MOCKs? Why is there no mention of the results using the CMV-ZF6-KRAB construct?In these experiments AAV2.1vectors were used whereas some others used AAV8. Not all the readers are aware of their tropism differences.5) Transcriptomic analyses. Despite the importance of this analysis I have been unable to find in the manuscript reference to what was exactly done. How were the transduced cells collected? Were the transduced regions microdissected and then the rods FACs sorted? How were the mRNA outputs from the transduced versus the untransduced regions matched?Overall, I would suggest the authors to submit their revised manuscript to a colleague that is not involved in this work to ensure clarity.eLife. Your revised article has been favorably evaluated by a Senior editor and Jeremy Nathans as the Reviewing editor. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:Thank you for resubmitting your work entitled "RHODOPSIN-targeted transcriptional silencing by DNA-binding" for further consideration at It looks substantially improved, but there is one aspect of the writing that needs work: various words or phrases that imply an absolute or extreme conclusion are over-stating the data. For example, in the Abstract is the phrase "complete gene silencing". The data do not support the use of the word "complete" – indeed, quantitative biological data only rarely support the use of that word. The best one can say of any observation of this type is that the signal is below the limit of detection. I suggest substituting the word "efficient" for "complete". Similarly, in the title of the legend to  The main issues, which I have extracted from the reviews and the subsequent discussion between the reviewers and me, are: 1) Revisions to the text that focus the story on use of the ZF6-DB to suppress RHO expression. This could be accomplished by changing the title, and revising the Introduction and Discussion. We would favor moderating the claims about potential clinical use of this approach since the repression-replacement approach wasn't tested in a disease model.We changed the title, revisited the Abstract and the Discussion to focus on RHO transcriptional repression by DNA binding. In addition, we now moderated the claim about the potential clinical use in both the Abstract and the Discussion.2) There is a question of ZF6-DB specificity and efficiency. Please provide the porcine, human, and mouse RHO TSS sequences for comparison, since it appears that the ZF6-DB recognizing hRHO TSS was used in these models regardless of any differences between the sequences. Does ZF6-DB recognize the porcine and mouse sequence? As to clinical therapeutic potential, how do the pig and human repression efficiencies compare? Is the 35% repression of Rho expression sufficient for dominant disease? These are important questions that could be addressed by supplying the sequences and the related data.We provided the porcine, human, and mouse RHO TSS sequences for comparison . As showRegarding the 35% repression of Rho expression in the whole porcine retina we agree that it might not be sufficient to treat a dominant disease such as adRP, although in singularly transduced cell (sorted rods) the repression almost reached 90%. We stated in the Discussion that the study provided a proof of concept and there is room of improvement the efficiency of the strategy on the whole retina scale in order to ensure therapeutic benefit.We added one additional retinal sample in the silencing and replacement experiment which now has n= 3 .We added one additional rod sorted sample in the silencing experiment which now has n= 3 .3) It is unclear why cone arrestin increases in Arr3. This suggests that there may be off-target effects. Does ZF6-DB also bind to the Arr3 TSS region? ChIP data should be supplied to address off-target issues.The increase of cone arrestin in Arr3 data did not show a statistically significant difference compared to other experimental groups. However, as suggested we performed ChIP analysis amplifying the proximal promoter region of Arr3 after pull down. As shown in In addition, after adding a further sample in the silencing and replacement experiment (as stated in response to comment #2) the Arr3 levels do not appear to be different from the control.4) As stated in response to comment #2, we added data on eGFP, ZF6-KRAB, and ZF6-DB treatments of wild type mice. As now shown in 5) Regarding the repression-replacement experiment described in  –RHO expression vector.We used the CMV that was used in the previous experiments  as an in6) The level of repression of the RHO gene achieved in the repression-replacement experiment described in As stated in responses to comment #1 and #2 we agree that the 35% (which has now become 38%) of whole retinal transduction may not be therapeutically effective. Accordingly, as with comment 1, we changed the claim regarding the therapeutic effect and we stated in the Discussion: “Therefore, a further development of this proof-of-concept will include optimization of the design of the silencing and replacement double construct (tuning the strength of the promoter elements), vector selection and dose, and the surgical approach”.7) Clarity of presentation as indicated below by reviewers #2 and #3.Done accordingly.It looks substantially improved, but there is one aspect of the writing that needs work: various words or phrases that imply an absolute or extreme conclusion are over-stating the data. For example, in the Abstract is the phrase "complete gene silencing". The data do not support the use of the word "complete" – indeed, quantitative biological data only rarely support the use of that word. The best one can say of any observation of this type is that the signal is below the limit of detection. I suggest substituting the word "efficient" for "complete". Similarly, in the title of the legend to According to the suggestions, we went through the manuscript carefully to mitigate absolute conclusions that may not be supported by the data.The point-by-point changes are as follow:In the Abstract: “efficient” replaced “complete”.In the section “Photoreceptor genomic binding of a 20 bp-long DNA sequence by a synthetic DNA-binding protein turns off Rhodopsin expression”:“complete” was removed, resulting in the sentence that states: “showed loss of EGFP expression compared[…]”.“complete” was removed, resulting in the sentence that states: “Immunofluorescence analysis showed depletion of Rho protein[…]”.“strongly” was removed, resulting in the sentence that states: “These data suggest that, despite the presence of an active canonical repressor domain[…]”.Rho silencing by DNA binding[…]”.“exclusively” was removed, resulting in the sentence that states: “ZF6-KRAB generated “any” was substituted with “detectable”, resulting in the sentence that states: “ZF6-DB in C57Bl/6 wild type retina did not produce detectable functional detrimental effects[…]”.Rho (38%) and in concomitant replacement of the exogenous hRHO (68%)[…]”.“balance” was removed resulting in the sentence that states: “transcriptional repression of the porcine Gnat1 transcript and protein (data not shown) levels demonstrated complementation[…]”.“full recovery” is now substituted with “complementation” resulting in the sentence that states: “Notably, In the first sentence of the discussion we substituted “turn off” with “dramatically reduces”.Rho expression (~85% decrease of Rho transcript levels)”, may appear an over-stating claim. However, we believe that “virtually” counterbalances the strong “somatic knock-out”, highlighting the impact of the finding.The following sentence: “The retina co-transduced with eGFP and ZF6-DB vector showed virtually a “somatic knock-out” of In the title of the legend to Rhodopsin transcriptional output”.We changed the sentence: “We propose a model in which the molecular features of the DNA  may contribute to the transcriptional interference observed” with: “We propose a model in which the molecular features of the DNA  may contribute to We added the following sentence that we believe completes the theoretic thinking in the Discussion:Rhodopsin transcriptional output)”.“It follows that in terms of signaling, the information source, the DNA, generates an output signal the RNA and eventually a protein, whose final output functional activity is completed back by the DNA. Thus the information source, the DNA, becomes also integral part of the signaling (Regarding the impact statement, I would favor the concise message of “turns off”: “Photoreceptor genomic binding of a 20 bp-long DNA sequence by a synthetic DNA-binding protein turns off Rhodopsin expression”.


Transcription is a highly regulated biological process, initiated through the assembly of complexes at the promoter that contain both the general transcriptional machinery and promoter-specific factors. Despite the abundance of studies focusing on transcription, certain questions have remained unanswered. It is not clear how the transcriptional profile of a promoter is affected by genomic context. Also, there is no single cell method to directly compare transcriptional profiles independent of gene length and sequence. In this work, we employ a single genetic site for isolating the transcriptional kinetics of yeast promoters. Utilizing single molecule FISH, we directly compare the transcriptional activity of different promoters, considering both synthesis and cell-to-cell variability. With this approach, we provide evidence suggesting promoters autonomously encode their associated transcriptional profiles, independent of genomic locus, gene length and gene sequence. The ability to regulate and coordinate the transcriptional process is crucial for executing gene expression properly. Much of this regulatory control is exerted over the transcription initiation process, whereby chromatin rearrangements and stepwise-interactions at or near the promoter allow for the assembly of transcriptionally-competent initiation complexes255Single molecule FISH is an effective and efficient method for measuring both transcriptional activity and steady-state mRNA levels in yeast, and has been used to address a number of biological questions58910MDN1, a 14.7 kb essential yeast gene that has been characterized previously in terms of mRNA expression levelsMDN1 transcript, signal detection using FISH probes targeted to the 5’ of the transcript was facilitated, allowing for more accurate quantification of nascent mRNAs than on short genes. Moreover, by keeping the transcribed sequence constant, the reporter represented a normalized method for assessing and comparing the specific contributions of different promoters independent of the variables that are present when comparing endogenous genes to each other.We have developed a reporter construct in which different promoters can be shuttled in and out . In eachMDN1 allele in diploid yeast , so as not to affect cell viability. In each case, the promoter was moved from its endogenous location to the MDN1 locus (Chromosome XII: 349006 – 363738). The insertion of 24 PP7 stem-loops (1200 nts) in the 5’ UTR allowed us to specifically target fluorescent probes to the altered allele and compare it to the endogenous allele to differentiate the promoter-specific effect  than POL1 (cv = 0.57). We then ranked these promoters according to transcriptional output, inferring average initiation intervals by assuming an average Pol II elongation rate of 25 bps/sec, consistent with previous reports in yeastPDR5 had a higher transcriptional output in the total population, initiating once every 2.87 minutes, while POL1 initiated once every 4.96 minutes   , with 2.88 for the endogenous PDR5 sequence and 2.62 for the reporter  . We deteal (PP7) , effectipromoter  with thaer 14YOX1 promoter exhibited a nascent transcript distribution ranging from 1 to 7, with a mean of 1.81 nascent mRNA/cell  compared to cells that underwent the temperature shift to 37 oC (cv = 0.54)  . This fuGAL1 promoter over time . In this case, the distribution of the nascent transcripts and the average transcripts per TS were identical for MDN1 and PDR5 genes, indicating that the isolated promoter recapitulates the initiation frequency of the endogenous gene. Therefore, the promoter isolation reporter provides a platform to further analyze the contribution of specific sequences to a promoter’s output, and at the same time allows for a direct comparison of different promoter behaviors.By using the MDN1 as a reporter because it is the longest gene is S. cerevisae, and therefore facilitated signal detection when studying promoters with low transcription frequencies. On a long gene, nascent mRNA will remain at the transcription site for longer periods of time and can be detected using probes binding to the 5’ on the mRNA. Therefore, it provides a better resolution of the initiation profiles for short genes. Furthermore, by keeping variables such as gene length and sequence constant, we are able to directly compare and rank the transcriptional output of different promoters in a controlled way.We chose An additional benefit of this reporter is that it inherently provides an opportunity to combine imaging approaches with biochemical data, whereby PP7 sequences can be used to isolate target mRNAs in order to assess their mRNP constituents. Thus, the promoter isolation reporter affords a means to purify associated factors that may be responsible for the transcriptional profiles observed using single molecule imaging. This important facet of our work can be appreciated by considering other studies that suggest the fate of several cell cycle and inducible mRNAs in the cytoplasm is determined by the promoter sequence, most probably by factors loaded onto the mRNA at the time of synthesis11181920212223GAL1 and HSP104, as it allows quantitative assessment of all aspects of the induction response, including cell-to-cell variability in response to an external stimulus, resulting in the temporal characterization of promoter activity. For example, the GAL1 promoter switches from an “off” state to an “on” state in which transcription is initiated more than once per minute on average. Furthermore, by measuring the highest number of nascent mRNAs for each promoter, we can determine the maximum initiation frequency of a given promoter. This system therefore allows determining various aspects of a promoter, including average strength, maximal initiation frequency, variation and timing of transcriptional output in response to different stimuli. For example, in the case of HSP104, an interesting next step could be to compare transcriptional profiles observed during heat shock with that of ethanol or sodium arsenate induced stress. Differences in these transcriptional profiles would provide evidence about the different mechanisms involved in each induction response.One of the strengths of single molecule FISH is that it provides quantitative data for single cells. This feature is even more relevant in the case of inducible promoters, like The most significant aspect of the work, however, is that we have established a system that controls for many variables long believed to influence gene activity2www.yeastgenome.org). PCR primers were designed to amplify 400–800 bps upstream of the gene. PCR products contained terminal SacI and BamHI sites, which allowed for the fragment to be cloned into a plasmid containing a his5 selectable marker, an exchangeable promoter region, 24 PP7 repeats, and flanked by regions of MDN1 homology that allow for integration by homologous recombination. Primer sequences are listed in Promoter sequences were obtained from the Saccharomyces Genome Database , 36 uL 1.0 M LiAc, and enough water to bring the final reaction volume to 360 uL. Tubes were vortexed and placed in a 42° C water bath for 40 minutes. After heatshock, cells were pelleted, resuspended in 600 uL sterile water, pelleted a second time and finally plated onto synthetic media plates lacking histidine. After two days of growth, single colonies were grown and genomic DNA was harvested using YeaStar Genomic DNA kit (Zymo Research). Colonies were then screened for proper integration of the promoter sequence by PCR.Strains were generated from W303 diploid yeast backgrounds . 50 mL cs.cerevisae are on FISH was performed on all strains using PP7V3 probes . These 518 mm circular coverslips (Fisher) were pretreated as follows: Coverslips were first boiled in mass in 500 mL 0.1N HCl (4.13 mL 12.1N HCl added to 500 mL H2O) in a beaker for 10 minutes with period and gentle stirring. Next, coverslips were washed by exchanging acid wash with 200 mL dd H2O and autoclave on standard liquid settings. Coverslips were placed on absorband sterile surfaceand dropped 150 uL of 0.01% poly-L-lysine (Sigma) on each coverslip. After 5 minutes they were washed 3 times with 150 uL dd H2O and air dried.600 of 0.4–0.8. Cells were fixed by adding 8 mL of 32% paraformaldehyde to 42 mLs of culture  and shaking at room temperature for 45 minutes. Cells were washed 3 times with 10 mL ice cold 1X buffer B (1.2M sorbitol and 100mM potassium phosphate buffer (pH = 7.5)) and resuspended in 500 μL sphereoblast buffer (1X buffer B containing 20 mM VRC (ribonucleoside-vanadyl complex from NEB) and 25U lyticase enzyme per OD of cells). Digestion of S. pombe was performed by adding 0.5 mg zymolyase and 10 mg lysing enzyme from T. harzianum to the sphereoblast buffer instead of lyticase and incubated at 30° C for 8 minutes. Next, digested cells were centrifuged at 3500 RPM for 5 minutes, resuspend in 500 uL 1X buffer B by gently pipetting up and down. One hundred and fivety uL of digested cells were added to each lysine-coated coverslip, and incubate at 4° C for 30 minutes and stored in 5 mL 70% EtOH at –20° C.Yeast cultures were grown in appropriate media to ODMounted coverslips were rehydrated by adding 2 mL 2X SSC at room temperature for 5 minutes twice. Coverslips were washed with 2 mL hybridization mix, containing 40% formamide/2X SSC at room temperature for 5 minutes. Four ng of labeled probe and 10 μL of 10 mg/μL E. coli tRNA/ssDNA (1:1) mix and dry by speed-vac were added. Twelve uL of solution F  and 38 uL dd H2O) were added per coverslip to the dried probe, and heat at 95° C for 3 minutes. A equal volume of solution H  was added to the probe mix and vortex. Twenty uL of probe-containing hybridization mix was added per coverslip, and coverslips were placed cell-side facing down onto glass plates coated with parafilm, sealed with an additional layer of parafilm to maintain moisture, cover with aluminum foil and place in a 37° C dry incubator for 3 to 12 hours. After two washes with 2 mL hybridization mix at 37° C for 15 minutes, one with 0.1% Triton X-100 in 2X SSC at room temperature for 15 minutesand two additional washes with 1X SSC for 15 minutes at room temperature, nucleus was stained with2 mL 1X PBS/0.5 μg/mL DAPI for 2 minutes at room temperature. A final wash with 2 mL 1X PBS for 5 minutes at room temperature was done before mounting on standard microscope slides by adding a small drop (~15 μL) of ProLong Gold antifade reagent (Invitrogen) and inverted coverslips, cell-side facing down.MDN1-Q670  was done in two steps. The first step with PP7-V3 probes is the same as described in hybridization (see above). After the second wash with hybridization mix, covers were incubated for 30 minutes with 10% formamide/2X SSC at room temperature. The same procedure was repeated for MDN1 probes with a final concentration of formamide of 10% instead of 40%.Because of the different annealing properties of 20-mer and 50-mer smFISH probes, two color smFISH with PP7-V3 cy3 probes and Images were acquired using an Olympus BX61 widefield epi-fluorescence microscope outfitted with a 100X/1.35 NA UPlanApo objective. Samples were visualized using the Chroma 41007 filter (Cy3), Chroma 31000 filter (DAPI), and DIC. Metamorph (Molecular Devices) was used as the acquisition software in combination with a CoolSNAP HQ CCD camera (Photometrics). Z-sections were acquired at 200 nm intervals over an optical range of 4.0 um. Exposure times for each z-section include 1200 ms (Cy3), 200 ms (Cy5) and 25 ms (DAPI). Cell volumes are then assembled by maximum intensity projections of z-sections using Metamorph. Localize, developed by Dr. Daniel Larson, was used to detect, locate and quantify intensities for fluorescent spots based on a two-dimensional Gaussian mask algorithm detailed previously5How to cite this article: Hocine, S. et al. Promoter-Autonomous Functioning in a Controlled Environment using Single Molecule FISH. Sci. Rep.5, 9934; doi: 10.1038/srep09934 (2015).


Escherichia coli K-12 MG1655. However, the genome-wide regulatory role of GadEWX is still unknown. Here we comprehensively reconstruct the genome-wide GadEWX transcriptional regulatory network and RpoS involvement in E. coli K-12 MG1655 under acidic stress. Integrative data analysis reveals that GadEWX regulons consist of 45 genes in 31 transcription units and 28 of these genes were associated with RpoS-binding sites. We demonstrate that GadEWX directly and coherently regulate several proton-generating/consuming enzymes with pairs of negative-feedback loops for pH homeostasis. In addition, GadEWX regulate genes with assorted functions, including molecular chaperones, acid resistance, stress response and other regulatory activities. These results show how GadEWX simultaneously coordinate many cellular processes to produce the overall response of E. coli to acid stress.The regulators GadE, GadW and GadX (which we refer to as GadEWX) play a critical role in the transcriptional regulation of the glutamate-dependent acid resistance (GDAR) system in  GadEWX regulons play a critical role in transcription regulation in response to acid stress. By reconstructing genome-wide GadEWX transcriptional network, here the authors show how GadEWX simultaneously coordinates many other cellular processes to produce the overall response of E. coli to acid stress. Escherichia coli K-12 MG1655, has sophisticated acid resistance (AR) systems to survive in acidic pH environments. The entire AR system of E. coli and the complexity of its regulation have been extensively studied and well described in recent reviews2341F0-ATPase, while its detailed molecular mechanism remains elusive671011Bacteria depend on maintaining pH homeostasis, as most proteins have optimal ranges of pH where they functiongadA, gadB and gadC) as well as several other genes in acid fitness islands (AFI) are mainly involved in the AR2 (GDAR) system; however, it is also under multiple levels of control. The GadE, GadW and GadX transcription factors (TFs) are at the first level of this regulatory cascade for direct regulation of these genes16172326in vitro DNA-binding experiments, mutation analysis and comparative transcriptomics161718272930313233343536in vivo GadEWX-binding events and the broader regulatory networks they comprise. A complete reconstruction of the GadEWX transcriptional regulatory networks under acidic stress would reveal the detailed mechanism of GadEWX regulation and its role in coordinating all cellular functions in response to acid stress.As mentioned above, the three genes  with lambda exonuclease digestion followed by high-throughput sequencing (ChIP-exo) for GadE, GadW, GadX and RpoS with those from strand-specific massively parallel cDNA sequencing (RNA-seq). To fully reconstruct the GadEWX regulons and RpoS involvement, we first examine the GadEWX- and RpoS-binding sites on the E. coli by in vitro DNA-binding experiments and mutation analysis161718272930313233343536in vivo of GadEWX binding has not been achieved. Therefore, we employed a recently developed high-resolution ChIP-exo method to determine the in vivo genome-wide binding profiles of these three TFs with near 1-bp resolution in E. coli under acidic stress (pH 5.5).Previously, several binding sites of GadEWX have been characterized in in vivo GadX binding sites include all of GadW binding sites as suggested in the previous comparative transcriptome studyin vitro for GadEWX with strong experimental evidence, 50% (8 out of 16) of which were also detected in this study , 16, 6 and 41 reproducible binding peaks were identified for GadE, GadW and GadX, respectively, under acidic stress . The numis study . It is uWe next assessed the widths and the genomic locations of the GadEWX-binding sites using currently available genome annotation. The widths of binding sites of each TF were 29±3.6, 30±7.6 and 28.7±8.0, respectively . The maj17E. coli151617262829303132333435P-value=4 × 10−6) and it includes gadA, gadBC, gltBD and ybaST, whose reactions are closely related with glutamate metabolism. Besides, a diverse range of COG functional categories indicate that GadEWX may play complicated roles beyond regulating genes in AFI to coordinate associated cellular processes in E. coli K-12 MG1655 as GadE has multiple roles in other pathogenic E. coli strains3839gadA and gadBC) were regulated by three additional TFs 42Currently, a total of 16 genes in 10 transcription units (TUs)  have been characterized with strong evidence as members of GadEWX regulons in gadE, ΔgadW and ΔgadX) grown under acidic stress conditions. Overall, a total of 351 genes were differentially expressed in at least one mutant. Genes with expression changes with log2 fold change≥0.5 and false discovery rate <0.01 were defined as differentially expressed. Only 25 out of 351 were differentially expressed in all mutants  (43%) were directly regulated by GadEWX under acidic stress  to determine causal relationships, we are not able to examine the autoregulation of TFs. A previous study showed that the transcription of gadE is activated by GadW and GadX when cells were grown to stationary phase at pH 5.5 in LB glucose mediagadE.  did not affect the kinetics of the promoters of core genes (gadA and gadBC) in the GDAR systemgadE deletion. Similarly, physiological studies showing that the gadE mutant was more sensitive to acid challenge than the gadX mutant also supported this observation1617A recent kinetic study of promoters and regulators in the AR2 system claimed that GadX  of those genes were mainly involved in proton consumption, generation and transport. Under acidic stress, only GadX-repressed genes involved in proton generation and influx such as speG (spermidine acetyltransferase) and dtpA (proton-dependent oligopeptide transporter)  . On the eostasis .hdeA and hdeB)asr, slp, yhiM, yhiD and hdeD) known to enhance the survival of cells under acid stress1749uspB, uspA and uspD). Interestingly, GadEWX were coordinated with other regulatory networks  indicating that GadEWX may play more complex genome-wide roles in association with other TFs. Several genes with predicted or unknown functions  were also regulated by GadE and GadX  within a remnant of a pathogenicity island  that either were activated or showed no causal relationship upon single TF knockout. Acid-stress resistance assays of knockout strains of these genes revealed that they were not directly related to GDAR, but three of them  were implicated in general AR . They were almost conserved in Escherichia and Shigella, but not in Salmonella, Yersinia and even further distant enterobacteria  in these bacteria would allow them to survive in an extreme acidic environment (pH 2) in the presence of glutamate. Interestingly, gadX as well as gadAB are conserved in some of β- and α-proteobacteria whereas gadE and gadW are not. Probably, GadX alone would be able to regulate the GDAR system in part when GadE is absent in these classes. Genes related to the proton consumption/generation  exhibited higher conservation ratio across proteobacteria even in the species that do not have the GDAR system  a total of 45 genes in 31 TUs that belong to GadEWX regulons and associations of RpoS for 28 of those genes; (ii) 19 genes in 13 TUs showing causal relationships upon single TF knockout; (iii) the negative-feedback loop pairs of proton transporters and utilization enzymes by GadEWX for pH homeostasis; and (iv) the additional roles of GadEWX regulatory networks beyond the GDAR system.We comprehensively reconstructed the GadEWX regulons at the genome scale in bo terminal oxidase (cbo) and NADH:ubiquinone oxidoreductase (ndh). Under conditions of acid challenge, E. coli increases expression of those complexes that pump protons out of the cell. Our recently developed genome-scale model of metabolism and gene expression (ME-Model) in E. coli53Using active proton transportsE. coli. One study revealed that polyamines such as spermidine, putrescine and cadaverine induce multiple components in GDAR and they are important for protection against acidic stressspeG (spermidine acetyltransferase), which consequently controls the polyamine concentration by degradation  showed significant changes in transcript level of ybaS compared to wild typeybaS can be activated in the presence of either GadW or GadX. In addition, the promoter region of gcvHTP encoding glycine cleavage system, which is known to release ammonia similar to ybaS, was also occupied by GadX Recently, two studies showed interesting observations on AR in radation . This reknockout . However by GadX . Given tE. coli K-12 MG1655 and its derivatives. The E. coli strains harbouring GadE-8myc, GadW-8myc and GadX-8myc were generated by a λ Red-mediated site-specific recombination system targeting C-terminal region of each genegadX, ΔgadW, ΔgadX and other y-genes) were also constructed by a λ Red-mediated site-specific recombination systemE. coli strains were inoculated into fresh 70 ml of M9 minimal medium  supplemented with 0.2% (w/v) glucose in 500 ml flask and cultured overnight at 37 oC with 250 r.p.m. For acidic stress, the overnight cultures were inoculated into the fresh 70 ml of M9 minimal medium at pH 5.5 (adjusted with HCl) in 500 ml flask and continued to culture at 37oC with 250 r.p.m. to OD600=0.3±0.02. At the sampling point, the pH was measured to be 5.28±0.03.All strains used are in vivo, we isolated the DNA bound to each protein from formaldehyde cross-linked E. coli cells by ChIP with the specific antibodies that specifically recognizes myc tag  and RpoS  with 1:50 dilutions, and Dynabeads Pan Mouse IgG magnetic beads (Invitrogen) were also used to capture specific antibody followed by stringent washingsf exonuclease treatment. The protein-DNA crosslink was reversed by overnight incubation at 65 °C with RNAs and proteins removal. DNA samples were used to perform primer extension and second adaptor ligation. The size-selected DNA sample was amplified and quantified. Prepared DNA library was sequenced using MiSeq (Illumina) in accordance with the manufacturer's instructions. ChIP-exo experiments were performed in biological duplicate. Sequence reads generated from ChIP-exo were mapped onto the reference genome (NC_000913.2) using bowtiehttps://code.google.com/p/chip-exo/)To identify GadEWX- and RpoS-binding maps 600=0.3±0.02 followed by purification steps using Qiagen RNeasy Plus Mini Kit (Qiagen) in accordance to the manufacturer's instruction to collect both of them. Strand-specific RNA-seq library was prepared using the dUTP methodhttp://cufflinks.cbcb.umd.edu/)Total RNAs including small RNAs were isolated using the cells treated with RNAprotect Bacteria Reagent (Qiagen) at ODThe GadEWX-binding motif analyses were completed using the MEME tool from the MEME software suite with default settingsP-value<0.05 was considered significant.The GadEWX regulons were categorized according to their annotated COG category. Functional enrichment of COG categories was determined by performing one-tailed Fisher's exact test (Hypergeometric test), and 600=0.3. These adapted cell cultures were inoculated into M9 minimal media at pH 2.5 without or with 1.5 mM sodium glutamate. The initial cell density inoculated for acid challenge was between 1 × 106 and 3 × 106 colony-forming unit (CFU) per ml. The pH 2.5 cultures were then incubated at 37 °C without shaking, and samples were collected after 2 h. Aliquots were serially diluted, and triplicates were plated onto LB agar plates. Colonies were counted after 24 h. Percent survival was calculated as followsAR assays followed the procedures of previous study with modification of usage of growth mediahttp://theseed.org) and ortholog calculation to E. coli K-12 MG1655 was also performed on RAST (Rapid Annotation using Subsystem Technology) servergadEWX and genes in GadEWX regulons were calculated from orthologs retained from RAST output.Gene annotation of strains and species were obtained from the SEED server .Supplementary Figures 1-7 and Supplementary Tables 1-5gadE deletion under acidic stressDifferentially expressed genes (DEGs) due to gadW deletion under acidic stressDifferentially expressed genes (DEGs) due to gadX deletion under acidic stressDifferentially expressed genes (DEGs) due to


Within these rigid constraints, even a slight change of distances between transcription factor binding sites (TFBS) results in loss of functionality of the enhanceosome. We hypothesized that smaller subunits of the enhanceosome may entail TF complex formation in other regulatory regions.The enhanceosome is an enhancer located upstream of the human interferon In order to verify this hypothesis we systematically searched for dimerization preferences of the TFs that have TFBS in the enhanceosome. For this we utilized our recently developed tool, TACO. We performed this computational experiment in a cell-type–specific manner by utilizing cell-type–specific DNase-seq data for 105 human cell types. We also used 20 TRANSFAC motifs comprising not only the usual TFs constituting the enhanceosome but also the architectural proteins of High Mobility Group I(Y) (HMG I). A similar experiment used 42 DNase-seq data sets for mouse cell types. We found 137 statistically significant dimer predictions in the human genome, and 37 predictions in the mouse genome, that matched the positioning on the enhanceosome with ±2 bp tolerance. To characterize these predicted TF dimers, we performed functional analysis (Gene Ontology enrichment) for sets of genes which were in the neighbourhood of predicted dimer instances. A notable feature of these instances is that (1) most of them are located in introns of genes, (2) they are enriched in regulatory states, and (3) those instances that are located near transcription start sites are enriched for inclusion in computationally predicted enhancers. We also investigated similarity of dimer predictions between human and mouse.κB did not participate in forming dimers with other proteins. Dimers predicted in mouse were fully contained in those predicted in human, with exactly the same spacing and orientation. Intriguingly, in most of the cases the enhanceosome motifs have 1 bp wider spacing than the corresponding dimers predicted genome-wide, which is likely caused by the overall 3D structure constraints of the enhanceosome-bound complex.It follows from our experiments that, except for homodimer formed by IRF proteins, the rest of the dimers were formed exclusively between one of the transcriptional activators (ATF-2/c-Jun and IRF) and a HMG I protein. NF- The online version of this article (doi:10.1186/s12918-016-0258-3) contains supplementary material, which is available to authorized users. This enhancer is located in the human genome on chromosome 9 and forms a region of length 57 bp, which is 44 bp upstream of IFN- β. The enhanceosome is one of the best studied enhancers [κB (p50/RelA), ATF-2/c-Jun and interferon regulatory factor (IRF) proteins. The first two proteins are heterodimers. All together these transcriptional activators have well characterized eight binding sites in the enhanceosome ; see [κB and ATF-2/c-Jun proteins recognize two positive regulatory domains (PRDs): PRD II and PRD IV, respectively, while IRFs recognize the other two domains: PRD I and PRD III. Notably, many neighbouring binding sites in the enhanceosome overlap.Transcriptional activation of the human interferon nhancers . It is aes); see  for a reβ is quite intricate and highly ordered as described for example in [The process of activation of interferon ample in . Since 2ample in –5. As a ample in  that theβ: even slight change in distance or orientation of TFBS results in a dramatic drop of the activity of gene expression [It was proposed by Arnosti and Kulkarni  that enhpression .κB and ATF-2/c-Jun by allosteric changes induced in the DNA [In addition to the above mentioned transcriptional activators, an important role in fine tuning the specificity in gene expression is played by architectural proteins of the mammalian high mobility group HMG I(Y) . HMG I a the DNA . Howeverβ is not precisely determined yet. For example it has been recently reported [β when performing an experiment in a transgenic mouse, but it is not so with the NF- κB subunits (p50 and RelA). In another work in the same vein [β enhanceosome region is not sufficient for maximal gene induction in response to stimulation with bacterial lipopolysaccharide (LPS). This study also identified a cluster of NF- κB sites in the 3’ downstream region of the gene.The functional role of the involved TFs on the level of expression of IFN- reported  that theame vein  it has bGiven the above remarks, we set out a hypothesis that smaller subunits of the enhanceosome may entail TF complex formation in other regulatory regions. More specifically we want to investigate which dimers (pairs of proteins) involved in enhanceosome assembly  are preferably used in other regulatory elements.In order to address these questions we applied TACO , our recAnalysis of the above mentioned computational experiment suggests the following key observations: (1) among transcriptional activators only the IRF proteins form a homodimer that is utilized on a genome-wide scale, (2) all other predicted dimers involve HMG I(Y) proteins and ATF-2/c-Jun or IRF transcription factors, (3) the majority of dimer instances is located within introns of genes.p-value <1.0e−7, Fisher’s exact test).We also repeated the same experiment with mouse genome and mouse DNase-seq data for 42 cell types. In this case, 37 predictions matched those present in the mouse enhanceosome with ±2 bp tolerance. The key observations for the mouse enhanceosome are similar to the above observations for human. A noticeable relation linking predictions in human and in mouse is that even though the latter form a subset of the former, every predicted dimer structure in mouse is also predicted in human with exactly the same arrangement. It is also noteworthy that genes that contain predicted dimer instances in human and in mouse have similar GO term annotations  had 12 different dimers predicted . In mouse the cell type with the largest number of predicted dimers (5) was Treg .Following the positions of hits in the enhanceosome we grouped our predictions into four groups: HMG-A; ATF2 , ATF2; HMG-B , and IRF-B; HMG-C . Figure Table It is interesting to notice that in both cases when a given dimer type was predicted in the largest number of cell types  the cell types related to a given dimer are very much the same. Table In order to check whether positions of instances of predicted dimers are enriched in chromatin states associated with regulatory function, we used the data deposited from the ENCODE Project for Broad ChromHMM . In thisPredictions are referred to by their hypothesis_id number. For a given cell type the data from ChromHMM for that cell type assigns, to each position in open chromatin, one of the 15 possible HMM states that describe predicted state of activity for that position. A dimer instance may have more than one state assigned to the genomic locus it covers. In order to assign a unique state to each instance, we decided to choose the state of the midpoint of the instance as representative.p-value of obtaining at least that many occurrences in G-plus group, this could be considered as a binomial process. A more detailed statistics is given in . On average, the number of informant species supporting a given Billboard prediction was 2.24. Additional file As an additional sanity check, we selected all 2663 human genes that contain within 10 kb for their Transcription Start Site (TSS) at least one instance of a predicted dimer. We restricted our attention to only those genes that contain at least three instances within the 10 kb distance from their TSS. In this way we reduced the number of genes to 70. Next we applied a computational tool, Billboard , to predselected by a dimer instance if it is the closest gene to that instance: either the instance is located within the gene, or else the distance between the midpoint of the instance and the closest nucleotide position within the gene is minimal (upstream or downstream). We group the selected genes, for a given dimer, into 7 sets: those that contain an instance of the dimer within its introns (denoted Q0), and those located within 100, 500, 1,000, 5,000, 10,000, and 50,000 bp from an instance of the dimer are denoted Q1, Q2, Q3, Q4, Q5, and Q6, respectively.In order to elucidate a potential regulatory function of predicted dimers we investigated genes which are located in the neighbourhood of dimer instances. A gene is said to be x=0,1,…,6) set union of all sets Qx over all considered cell types.In addition to this, we also performed for each dimer a global analysis consisting of taking as Qx , all of them being blood microvascular endothelial cells. The biological process (BP) enriched terms for Q0 genes selected by dimers of this type are: ‘regulation of Rho protein signal transduction’, ‘focal adhesion assembly’, ‘regulation of phosphate metabolic process’, ‘establishment or maintenance of cell polarity’, and ‘actin cytoskeleton organization’. For cellular component (CC) domain the enriched terms include: ‘focal adhension’, ‘cytoskeleton’, ‘cell cortex’, ‘neuron projection’, and ‘cell-cell junction’. Finally, for molecular function (MF) domain we have the following enriched terms: ‘cytoskeletal protein binding’, ‘Ras/Rho guanyl-nucleotide exchange factor activity’, and ‘protein tyrosine kinase activity’. It is also noticeable that the set of genes located within 500 bp of a dimer instance (Q2 query) has enriched a BP term ‘positive regulation of tyrosine phosphorylation of STAT protein’. As an illustration, Fig. An interesting collection of enriched terms is for genes selected by dimers of type ATF2; HMG-B. For BP domain the enriched terms include: ‘axon guidance’, ‘cell junction assembly’, ‘regulation of signal transduction’, ‘transmembrane receptor protein tyrosine kinase signalling pathway’, ‘wound healing’, and ‘actin filament organization’. Enriched GO terms for CC domain include: ‘neuron projection’, ‘adherence junction’, and ‘actin cytoskeleton’, while terms for MF domain are: ‘actin binding’, and ‘Rho/Ras guanyl-nucleotide exchange factor activity’.The third dimer type, IRF-A; IRF-B, does not have a very pronounced set of enriched terms for Q0 genes. Instead it has an interesting set of enriched terms for Q2–Q3 sets of genes. Notably, the BP enriched term ‘ISG15-protein conjugation’ is enriched for all queries Q1–Q5. A BP term ‘negative regulation of type I interferon production’ is enriched for many cell types. Likewise the BP terms ‘defense response to virus’, ‘defense response to other organism’, and ‘NLS-bearing protein import into nucleus’ are enriched for Q2 and Q3 queries. Also query Q6 has a very wide spectrum of BP enriched terms. These are mainly involved in defense response and in the adaptive immune system.D that was predicted in human and mouse cell types we consider all pairs of cell types  such that D was predicted in human cell type CH, as well as in mouse cell type CM. For i=0,…,6, let i selected genes that are also selected by dimer D in CH. In a similar way we define genes CM. Let In order to assess similarity of enriched terms between human and mouse we performed the following experiment. For a given dimer p-values for Fisher’s exact test for global instances for each dimer and each pair of cell types  that have the best p-value for the given dimer. It clearly follows from this table that Q0 selected genes for dimers of type HMG-A; ATF2 exhibit higher similarity between human and mouse than for dimers of type IRF-A; IRF-B. However, for the latter dimers similarity between selected genes is still quite clearly visible. Additional file p-values assigned to Fisher’s exact test for each dimer and each pair of human-mouse cell types.Table κB) did not show up in this analysis. It should be kept in mind that dimers predicted by TACO are only those that show overepresentation in terms of binding instances in a particular cell type over the background. Hence, if a certain dimer was not predicted it does not mean that this dimer configuration is not plausible.Of the 16 predicted dimers in the human enhanceosome, 7 dimers were constructed from an architectural protein of High Mobility Group I(Y) (HMG I) and one of the two transcriptional activators: ATF-2/c-Jun and interferon regulatory factor (IRF). In total these heterodimers fall into three types, depending on their binding location in the enhanceosome: the leftmost enhanceosome binding position of HMG I (HMG-A) with ATF-2/c-Jun, ATF-2/c-Jun with the middle enhanceosome binding position of HMG I (HMG-B), and IRF with the rightmost enhanceosome binding position of HMG I (HMG-C). The remaining 9 dimers constituted homodimeric arrangement of IRF with itself. It is remarkable that nuclear factor kappa-light-chain-enhancer complex of activated B cells , three from ENCODE Tier 2 , and the rest of cell types are from ENCODE Tier 3. Most of the cells with predicted dimers are from blood tissues: monocytes, B cells, B-lymphocyte, T cells, T regulatory cells, and T helper cells – all of them of normal karyotype, but also some diseased cells, mainly leukemia . It is noticeable that for all types of dimers, except ATF2; HMG-B, the predicted cell types that contain the dimer are mainly blood cells. For both dimers of type ATF2; HMG-B the predicted cell types are leukemia (K562), prostate , and epithelium .We also performed a similar analysis in mouse cell types. We have found 7 dimers present in 14 cell types. It is remarkable that the structure  of all these dimers matched exactly the structure of dimers predicted in human. It is also noticeable that the 7 dimers predicted both in human and in mouse exhibit the highest number of human cells containing them.Interestingly, 13 dimer structures predicted in human out of 16, were characterized by a motif spacing 1 bp tighter than in the original enhanceosome. What is more interesting is that the remaining 3 dimers  are all of the same type HMG-A; ATF2 and are therefore located on the enhanceosome leftmost side of the sequence. All of the above mentioned 13 dimer structures are located inside the enhanceosome. An additional support that we are not discussing here is that the above mentioned 3 dimers are predicted in mouse with exactly the same offset, and the other 4 mouse dimers, having the same offset as in human, turn out to be 1 bp tighter than in the original mouse enhanceosome. A possible mechanistic explanation of this phenomenon is that a 1 bp wider offset between TFs that are located inside of the big protein complex which binds the enhanceosome is caused by the overall three-dimensional structure of the complex, with weaker constraints imposed on the sides of the enhanceosome complex.As a sanity check we investigated ChromHMM states assigned to genomic dimer instances. In an overwhelming number of cases these instances were assigned to an enhancer or promoter state. More than half of the instances are located within a gene (in introns). Moreover, using an enhancer annotation tool, Billboard, we searched the ±10 kb area around TSS of human genes that contain at least three dimer instances. With help of five informant species, we concluded that in 89 % of the 205 instances they were included in a predicted enhancer. This supports the hypothesis that the predicted dimers, being part of the human enhanceosome, are also utilized in the genome by other enhancers.In order to analyze genes which contain in their vicinity a dimer instance, we defined 7 groups, which we call queries: genes containing a dimer instance (Q0), and queries Q1 through Q6 that consist of genes that the closest distance to a dimer instance is less than 100, 500, 1000, 5 000, 10,000, and 50,000 base pairs. Next, for each dimer we performed Gene Ontology analysis for each of the above mentioned queries. We have also compared enriched GO terms for similarity between human and mouse. The performed Fisher’s exact test suggests that the enriched terms for queries Q0 and Q6 are significantly similar between human and mouse. Even stronger similarity is observed for comparison of human/mouse genes selected by these two queries. For this we adopted Fisher’s exact test by utilizing the orthology relation between human and mouse genes.We have performed a comprehensive search of overrepresented dimers composed of transcription factors that constitute the human enhanceosome. We searched over many cell types, both in human and in mouse. The predicted dimers were grouped into 4 types, according to their binding position in the enhanceosome: HMG-A; ATF2, ATF2; HMG-B, IRF-A; IRF-B, and IRF-B; HMG-C. Here HMG-{A, B, C} represents three binding sites of the architectural protein HMG I in the enhanceosome. No other combinations of TFs and/or architectural proteins were predicted. The majority of cell types in which these dimers were discovered were various kinds of blood tissues.p-value <2.6e−05).Despite the fact that we allowed for dimer spacing variability up to ±2 bp comparing to the enhancesome sequence, most of the predicted dimers were exactly 1 bp wider than suggested by the motifs within the enhanceosome. The remaining 3 dimer predictions were of the same type HMG-A; ATF2, located on the side of the enhanceosome. Since in most of the cases the predicted dimer binding sites were associated with a state of regulatory character, we assumed that the role of these dimers is being a part of enhancers other than the enhanceosome. We observe that dimer instances are preferentially located within introns of genes and that those instances that are located near TSS of a gene are preferentially included in computationally predicted enhancers . Search of a homologous piece in the mouse genome reveals a region of very high similarity on chromosome 4  that is located 101 bp upstream of mouse interferon beta 1 gene Ifnb1 (Ensembl ID: ENSMUSG00000048806). We took for the analysis a sequence from the mouse genome (assembly mm9) of the same length (57 bp). Human and mouse enhanceosomes are almost identical: except for the first position and the last four positions, they differ by only two mismatches.We took as the human enhanceosome the 57 bp piece of DNA located on chromosome 9 (positions 21077989 to 21078045 in genome assembly hg19), which is 44 bp upstream of the human interferon beta 1 gene Human:TAAATGACATAGGAAAACTGAAAGGGAGAAGTGAAAGTGGGAAATTCCTCTGAATAGMouse:AAAATGACAGAGGAAAACTGAAAGGGAGAACTGAAAGTGGGAAATTCCTCTGAGGCAHere are the corresponding enhanceosome sequences: κB dimer (p50+RelA). We took these motifs without any further slicing. Also motifs for the two related TFs: IRF3 and IRF7 were difficult to discriminate, so we took all such motifs as representing any of the two TFs. In addition to this, we also considered High Mobility Group (HMG) proteins that function in the enhanceosome as architectural proteins which remove the intrinsic bend of the double helix and therefore allow binding of transcriptional activators [It should be noted that in TRANSFAC motif database, some TF motifs of binding sites already represent dimeric complexes. In the case of the enhanceosome, this happens for ATF-2+c-Jun dimer, as well as NF- tivators .κB complex; and 4 (out of 6) representing HMG motifs. TRANSFAC identifiers of the selected 20 motifs are reported in Additional file A set of 27 motifs was initially selected for further analysis. We scan the human enhanceosome sequence with each motif with the motif score threshold set in such a way as to give the balanced threshold of 100, i.e. the threshold that makes the ratio of false positive rate to false negative rate at the level of 100 . It turnWe collected DNase I hypersensitivity data for human  and for mouse . This data was generated at the University of Washington and obtained from the ENCODE Project, Genome Browser track wgEncodeUwDnase. The list of DNase I files for human and mouse and the list of corresponding cell types is given in Additional file Standalone tool TACO  was provEach dimer binding site was classified as either located within a gene , or else a distance to the nearest gene (both upstream and downstream) was calculated. Here we take the midpoint of the dimer instance and calculate its distance to the nearest position of a gene (it would be a Transcription Start Site (TSS), if the gene is located downstream, or the end position of a gene, if that gene is located upstream). Then, for a given distance x  and for a given dimer binding site we report all genes such that the calculated distance from the dimer binding site is less than x.Gene Ontology analysis for the selected genes was performed with help of Biopython  and Bio.GH and GM be selected genes in human and mouse, respectively. Let RO be a many-to-many gene orthology relation between human and mouse. We iteratively build partitions of GH and GM as follows. Initially all partition blocks in GH and GM are one-element and the orthology between blocks is given by RO. Iteratively, we join two partition blocks to form a new block (say X) in human (or in mouse) when they are linked by the orthology relation to a common block (say Y) in the other species. The new block X is linked by new orthology relation to Y. The process terminates when no new blocks to be jointed exist.In order to assess similarity of selected genes between human and mouse we have to preprocess the data. Let Fortunately, when we run this procedure on the data it terminates very quickly with only very few blocks having more than one element.