ERAP1 and ERAP2 are implicated in a variety of immune and non-immune functions. Most studies however have focused on their role in shaping the HLA class I peptidome by trimming peptides to the optimal size. Genome Wide Association Studies highlighted non-synonymous polymorphisms in their coding regions as associated with several immune mediated diseases. The two genes lie contiguous and oppositely oriented on the 5q15 chromosomal region. Very little is known about the transcriptional regulation and the quantitative variations of these enzymes. Here, we correlated the level of transcripts and proteins of the two aminopeptidases in B-lymphoblastoid cell lines from 44 donors harbouring allelic variants in the intergenic region between ERAP1 and ERAP2. We found that the presence of a G instead of an A at SNP rs75862629 in the ERAP2 gene promoter strongly influences the expression of the two ERAPs with a down-modulation of ERAP2 coupled with a significant higher expression of ERAP1. We therefore show here for the first time a coordinated quantitative regulation of the two ERAP genes, which can be relevant for the setting of specific therapeutic approaches.The Endoplasmatic Reticulum Aminopeptidases  ERAP1 and ERAP2 are ubiquitous, zinc-dependent multifunctional enzymes involved in immune activation and inflammation3, blood pressure regulation5 and antigenic peptide repertoire shaping. ERAP1 is a type II integral membrane enzyme, whereas ERAP2 is not. The two aminopeptidases share ~49% sequence homology. ERAP1 and ERAP2 genes are both regulated by interferons (IFNs) and Tumour Necrosis Factor-α (TNFα)11. Whilst differing for the preferred substrates, they have similar proteolytic activities and might even form heterodimers in order to generate optimal peptide repertoires. When overactive, the ERAPs can destroy the putative HLA class I ligands by reducing their length below the threshold of 8–10 amino acids. Loss of both enzymes is a frequent event significantly associated with the lack of HLA class I surface expression17. While the general rules governing the role of the ERAP genes in antigen processing are well established, several studies suggest that the effects on individual HLA class I alleles and/or epitopes may differ18.The ER-resident aminopeptidases ERAP1 and ERAP2 genes reside in the same cluster located on the long arm of chromosome 5 (5q15), where they lie contiguous and oppositely oriented. Both genes are polymorphic with strong linkage disequilibrium across the chromosome 5q15 locus and many functional variants appear to primarily affect their enzymatic activity. In particular, it has been shown that a splicing variant in the ERAP2 gene (rs2248374)19 leads to nonsense-mediated decay (NMD) of the mRNA. Since this variant is maintained by a balanced selection to a frequency of approximately 50%, it follows that 25% of the population fails to express the ERAP2 protein20.The ERAP1 and ERAP2 in the onset of several immuno-mediated diseases (IMD), such as ankylosing spondylitis (AS), Behçet’s disease (BD), psoriasis (Ps), inflammatory bowel disease (IBD), juvenile idiopathic arthritis (JIA), birdshot chorioretinopathy (BSCR), type I diabetes mellitus (T1D) and multiple sclerosis (MS)29. In particular, the epistatic interactions observed between ERAP1 and HLA-B27 in AS, HLA-Cw6 in Ps and HLA-B51 in BD, strengthen the central role of antigen presentation in the so-called “MHC-I-opathy”, although the allelic variants of ERAPs specifically associated with each disease may be different30. In several studies, ERAAP deficient mice resulted in significant changes in the MHC-peptide repertoire thus activating CD8+ T lymphocytes and Natural Killer cells32. These findings suggest a direct interaction of the two ERAP proteins with precursors of HLA class I epitopes, which affect innate and specific immunological surveillance.GWAS have suggested a role for both ERAP1, could determine the production of an “atypical” peptide repertoire that predisposes to IMDs33 as well as the failure in rejecting tumours and in fighting pathogens34.Overall, an impaired activity and expression, especially of ERAP genes does exist36. Indeed, the combination of the ERAP polymorphisms can be termed “allotypes” since they are often co-inherited as complex haplotypes, suggesting that this can be the outcome of a selective pressure by pathogens. The SNPs found associated with AS and with other IMDs, as well as with cervical cancer and other pathological conditions39, probably have a synergistic effect with other variations present in the same haplotype41. Although there are examples of a direct effect of single SNPs on enzymatic activity and substrate binding43, the data concerning haplotype combinations are conflicting49. In addition, the functionality of different allotypes may be closely dependent on the quality and quantity of the peptide substrates42, as well as on the amount of the enzyme itself. Therefore, in some cases, SNPs associated with pathologies may not be causative of the condition themselves, but in linkage disequilibrium with allelic variants impacting on mRNA transcription, maturation and translation50. This could explain why the association between the two ERAP genes and the different IMDs may rely on distinct or even alternative polymorphisms harboured by different haplotypes in different ethnic groups51. Furthermore, many risk variants in ERAP1 and ERAP2  have been reported as expression quantitative trait loci (eQTL) in most tissues53 and their association could derive from quantitative variations and/or from linkage with other causal SNPs. To date, however, there are no relevant genetic studies in the ERAP1 and ERAP2 gene promoters and very little is known about the mechanisms that regulate their expression.Although most of the studies have been focused on the function of individual SNPs, it must be considered that extensive linkage disequilibrium within and around the ERAP1 and ERAP2 in a panel of (EBV)-transformed lymphoblastoid cell lines (B-LCLs) known to express high levels of these aminopeptidases54. We found an inverse correlation in the expression level of the two aminopeptidases depending on a genetic variant mapping in the intergenic region. It is noteworthy that this SNP is in strong LD with an ERAP1 variant found associated with several IMDs.In this study, we have performed a quantitative analysis of ERAP1 and ERAP2 gene expression. All B-LCLs were genotyped for SNPs rs30187 and rs27044 in the ERAP1 gene and rs2248374 in the ERAP2 gene  and then normalized to β-Actin . Interestingly, among individuals heterozygous at rs2248374, 6 out of 24  which induces NMD20. However, ERAP2 protein was not detectable in the B-LCLs (A → H group) whereas present in all the other samples genotyped at rs2248374 as A/A or A/G.Western blot Fig.  confirmeERAP1 and ERAP2 transcripts showed that the six B-LCLs heterozygous for rs2248374 and lacking ERAP2 protein, had a higher ERAP1/ERAP2 mRNA ratio, even above the ratio shown by the G/G homozygous in which the ERAP2 mRNA encoded by both haplotypes was degraded by NMD  (region named M1). As for the CA repeats, we analysed a total of 400 DNA samples from Sardinia and observed a Gaussian distribution of allele frequencies around 32 repeats (data not shown). We therefore arbitrarily named as “L” the alleles with a number of repeats equal or higher than 32 and as “S” the alleles with a number of repeats lower than 32. The frequency in our panel of 44 B-LCLs was consistent with that of Sardinian population (76% “S” and 24% “L”). As for the two SNPs, rs75862629 and rs2911138, their frequency in the Sardinian population was similar to that reported for Italy (https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/) with 13% G and 77% T, respectively.To assess whether this variability could be due to gionFig. . Three pERAP1 or ERAP2. The G variant at rs75862629 instead, was carried by all those cell lines that, although heterozygous for the functional rs2248374 variant, did not express ERAP2 protein  and Z (A/A at rs2248374) B-LCLs  (20 and our observation). As expected, a lower level of ERAP2 mRNA was expressed by the haplotype identified by G at rs2248374 (ratio A–A/A–G > 1). Unexpectedly, the presence of G at rs75862629 makes the haplotypes ratio G–A/A–G or G–A/G–G close to the unit. Furthermore, NMD inhibition induced an increase of the ERAP2 transcript carrying the A–G haplotype of about 8 times, whereas the presence of a G at rs75862629 (G–A or G–G) correlates with a lower susceptible to NMD inhibition , which falls within the ion Fig. .Figure 5These data indicate that, as to the ERAP2 transcripts, the G allele at rs75862629 tags the haplotypes whose transcripts are susceptible to NMD. However, the greater effect of the NMD inhibition on the haplotype A–G rather than G–A (rs75862629-rs2248374), makes plausible the hypothesis that the G at rs75862629 negatively affects ERAP2 transcription by itself. This was strengthened by a further relative allele specific real-time PCR analysis in which the cDNAs corresponding to G–A and A–A haplotypes from emetine-treated B-LCLs were normalized to the A–G haplotype Fig. . Cell liERAP1 expressors carrying the G variant at rs75862629, are preferentially within a haplotype that includes the variant T at rs30187 (pc = 1.5 × 10−8)  Table .Table 1FERAP1 and ERAP2 in B-LCLs genotyped for rs30187 (K528R) and rs27044 (Q730E) SNPs in ERAP1 and rs2248374 in ERAP2 gene. The analysis of the ERAP1 and ERAP2 mRNA levels in the presence or absence of emetine, a NMD inhibitor, pinpointed the SNP rs75862629, mapping in the ERAP2 promoter, as a marker that correlates with a double effect on ERAP2 mRNA, that is, lower transcription and higher degradation. Interestingly, this parallels with a higher ERAP1 transcription suggesting a concerted, opposite regulation.In this study, we have analysed the expression of 23. The major allele C (Arg528) instead reduces ERAP1 activity and, while protective for AS and psoriasis, is a risk factor for Behçet’s disease. It will be interesting to investigate the interplay between the promoter polymorphism rs75862629 that controls the quantitative level of ERAP1 and its activity as regulated by rs30187 in different populations and different diseases. Of note in Sardinia, the G allele at rs75862629 is part of a haplotype that includes the T allele at rs30187 and that characterizes individuals with high ERAP1 expression. This is in agreement with the observation that the T variant at rs30187 correlates with a higher ERAP1 mRNA transcription and protein expression, particularly in B-LCLs and monocytes-derived dendritic cells36. Other studies have suggested that ERAP1 expression traits may be under a cis-regulatory control and that rs30187 shows the strongest association with cis-regulation tag frequency-derived haplotype frameworks56. Since the lack of ERAP expression was not associated with DNA methylation in the promoter of ERAP1 or ERAP257, our findings suggest that this latter phenomenon could be due to the presence of variants that, falling into cis-regulatory elements, would favour the hijacking of specific factors towards the ERAP1 rather than ERAP2 transcription. Although our study has been limited to few markers and therefore other cis-elements can be responsible of the observed phenomenon, it is interesting that the rs75862629 maps within the ERAP2 gene promoter region flanked by putative TATA-box sequences thus making plausible that rs75862629 itself may affect gene transcription resulting in a modification within the putative transcription factor binding site of IRF-11. In this context, it is remarkable that a constitutively inverse mRNA expression pattern was also found for ERAP1 and ERAP2 in melanoma cell lines compared to melanocytes57. A further possibility is that ERAP1 grabs transcription factors that control the expression of both genes and this results in a poor ERAP2 transcription. A lower production of negative regulators (i.e. non-coding RNA), whose genes could overlap with the ERAP2 gene is also conceivable. In this regard, the ERAP2 mRNA, regardless of rs2248374 allelic variant and in contrast to ERAP1 mRNA, is particularly influenced by emetine treatment, suggesting the existence of a higher number of alternatively spliced mRNA isoforms undergoing NMD degradation.The presence of T at rs30187 (Lys528) has been found associated with AS, non-AS spondyloarthritis, type I diabetes, psoriasis, multiple sclerosis, anterior uveitis and enthesitis-related arthritisERAP1, counterbalanced by a lack of expression of ERAP2, can have in the shaping of the peptidome and on the numerous functions of these enzymes. It must be considered that the ERAPs may influence pathological processes other than those strictly involving cell-mediated immunity. Indeed, ERAP1 role in angiogenesis and in the regulation of blood pressure arises the possibility that in some IMDs, vascular alterations taking place during inflammation as well as vessel remodelling and stability, can contribute to the pathologies and, in this context, the quantitative variations of the ERAP1 expression can play a relevant role58.These findings provide a new tool to evaluate the functional consequences that a higher expression of ERAP genes. Overall, a deeper knowledge of the possible consequences of ERAPs quantitative variations, particularly of ERAP1, and of factors controlling their expression, may well be fundamental to the design of novel and specific therapeutic approaches in different pathologies.In conclusion, we demonstrated the existence of a regulatory intergenic region marked by the SNP rs75862629 that balances the level of the two Peripheral Blood Mononuclear Cells (PBMC) were collected from forty-four Sardinian donors recruited in the Department of Medical Sciences, University of Cagliari, Italy. Informed consent was obtained by all subjects and the study was approved by the local ethics committee . All experiments were performed in accordance with relevant guidelines and regulations.59, and grown in an atmosphere with 5% CO2 at 37 °C in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 units/ml), streptomycin (100 μg/ml) and amphotericin B (Invitrogen). B-LCLs were cultured at 5 × 105/ml. For the inhibition of Nonsense Mediated Decay (NMD), cells were transferred to a new flask and kept in culture for 7 h in the presence or absence of emetine (Sigma) at concentration of 100 μg/ml.PBMC were isolated from 30 ml of freshly drawn blood by Ficoll density-gradient centrifugation (StemCell Technologies). B-lymphoblastoid cell lines (B-LCLs) were established from Epstein-Barr virus (EBV) transformed B lymphocytes according to the standard protocolGenomic DNA was obtained from B-LCLs or from EDTA-treated peripheral blood samples using QIAamp DNA Blood mini kit  according to the manufacturer’s protocol. SNPs genotyping was performed by quantitative Real-Time Polymerase Chain reaction (qRT-PCR) with functionally tested TaqMan Allelic Discrimination Assay . The analysis of the genomic regions reported as M1, M2 and M3 Fig.  and the ERAP1 gene was determined by fragment length analysis. Briefly, the microsatellite region was amplified by PCR with fluorescent (6-FAM) −5′-end labelled primers (Table The number of CA repeats (hereinafter named as “size”) in the promoter region of rs Table . PCR proTotal RNA from B-LCLs was extracted using Trizol Reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. RNA was quantified by a Nanodrop spectrophotometer ND-1000 (Thermo Scientific) and its quality was assessed by 1% agarose/Tris–Acetate–EDTA (TAE) gel electrophoresis. 1 μg of RNA underwent reverse transcription reaction using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems).ERAP1, ERAP2 and β-Actin mRNA absolute quantification was performed by qRT-PCR using the standard curve method. RT-qPCR was performed with gene specific primer sets  and used to generate cDNA as already described. Haplotype-specific ERAP2 cDNA was quantified in triplicate using an allele-discriminating TaqMan genotyping assay for the rs2287988 coding diagnostic SNP20. Briefly, we generated a standard curve consisting of serial dilutions of DNA samples homozygous for A or for G allele. We used a heterozygous genomic DNA sample to validate the regression equation, in which a mean allelic ratio of 1.0 was expected.B-LCLs heterozygous at SNP rs2248374 were treated with 100 μg/ml of emetine (Sigma) for 7 hours to inhibit NMDRelative quantification of rs75862629-rs2248374 G–A and A–A haplotypes after emetine treatment was performed using the TaqMan genotyping assay for the rs2287988 coding diagnostic SNP. The expression level of mRNAs is indicated as ratio of the targets normalized to the A-G haplotype using the 2−ΔΔCt method, as ratios of fold change relative to the calibrator. The 2−ΔΔCt method assumes that the amplification efficiency of the reaction is ideal and constant for each sample.6 cells were harvested, washed twice in 1x PBS and lysed for 30 min on ice in 30 μl of 1% NP40 protein lysis buffer containing 100 U/ml of phenylmethylsulfonyl fluoride (PMSF), 1 μg/ml of aprotinin and 0.5% sodium deoxycholate proteinase inhibitors. Proteins were obtained from the cell lysates by centrifugation at 16363 g for 15 min at 4 °C, and their concentrations determined using the Biorad protein assay kit  with bovine serum albumin (BSA) used as standard. 40 μg of protein extract for each sample were separated on a 4–12% NuPage Bis-Tris gel (Invitrogen) at 125 V for 100 min in NuPage MES SDS Running Buffer (Invitrogen), and transferred to nitrocellulose membranes. The membranes were incubated ON with mouse anti-ERAP1 mAb antibody , mouse anti-ERAP2 mAb  and mouse anti-β-Actin mAb . The membranes were washed twice in TBST, incubated with horseradish peroxidase–conjugated secondary Ab  and revealed by ECL Western blotting detection system (Amersham). The proteins were visualized by ChemiDoc XRS+ System .Approximately 3 × 10ERAP1 and ERAP2 transcripts. For comparison of two groups, two tailed Mann–Whitney U-test or paired t test were used. Statistical analyses were performed using GraphPad Prism 5 software . Haploview version 3.2 (http://www.broad.mit.edu/mpg/haploview/) was used to analyse the patterns of haplotype blocks . P values underwent Bonferroni correction for multiple comparisons.In the scatter plots the mean ± standard error of the mean (SEM) are indicated. Pearson’s test (one tailed) was employed for the correlation analysis of Supplementary Information


Drosophila H3K9-specific histone methyltransferases, SU(VAR)3-9 and SETDB1. We asked whether SETDB1 is required for targeting of SU(VAR)3-9. Using DamID-seq, we obtained SU(VAR)3-9 binding profiles for the chromosomes from larval salivary glands and germline cells from adult females, and compared profiles between the wild type and SETDB1-mutant backgrounds. Our analyses indicate that the vast majority of single copy genes in euchromatin are targeted by SU(VAR)3-9 only in the presence of SETDB1, whereas SU(VAR)3-9 binding at repeated sequences in heterochromatin is largely SETDB1-independent. Interestingly, piRNA clusters 42AB and 38C in salivary gland chromosomes bind SU(VAR)3-9 regardless of SETDB1, whereas binding to the same regions in the germline cells is SETDB1-dependent. In addition, we compared SU(VAR)3-9 profiles in female germline cells at different developmental stages (germarium cells in juvenile ovaries and mature nurse cells). It turned out that SU(VAR)3-9 binding is influenced both by the presence of SETDB1, as well as by the differentiation stage.H3K9 methylation is known to play a critical role in gene silencing. This modification is established and maintained by several enzymes, but relationships between them are not fully understood. In the present study, we decipher the interplay between two  Eukaryotic genomes are formed by a complex mosaic of single copy genes and repeated sequences. Some genes are active across all cell types and developmental stages, whereas other genes become activated only in a specific tissue or for a narrow period of time. Likewise, transcription of repeated DNA sequences is typically tightly controlled, and most of the time they must remain silent. Maintenance of the appropriate chromatin state, either active or inactive, occurs via epigenetic mechanisms and relies on the balance of epigenetic marks such as methylated DNA residues, as well as various post-translationally modified amino acid residues in the context of histone molecules.DNA methylation has been found in most organisms ranging from bacteria to mammals . InteresPlanococcus ; 1473/TSTLeggy w attP18[mini-hsp70-Δt-Su(var)3-9-Dam]; 1473/TSTLeggTo analyze the material from third-instar larval salivary glands, animals from the following stocks lacking the stop-cassette (Δt) in the transgene were used:TSTL balancer allows easy discrimination between homozygous egg mutant and heterozygous larvae.These animals express Dam or SU(VAR)3-9-Dam ubiquitously, and presence of the dominantly marked hsp70 promoter and a stop-cassette (t). The latter was removed by crossing to the flies expressing nanos-driven CRE:y w attP18[full-hsp70-t-Dam]; Df(2R)Dll-MP/Cyy w attP18[full-hsp70-t-Su(var)3-9-Dam]; Df(2R)Dll-MP/Cyy w; nanos-1473CRE egg/CyFor germline-focused experiments, we used the flies carrying a full-length nanos-CRE transgene was recombined onto the 1473egg chromosome by conventional crosses, whereas Df(2R)Dll-MP  overlapping the egg gene was introduced into the stocks with Dam/SU(VAR)3-9-Dam transgenes. Females lacking Cy marker at the age of 1–2 days post-eclosion were selected among the progeny of the crosses. These females were hemizygous for egg, and expression of the Dam/Su(var) 3-9-Dam transgenes was restricted to the germline.In the female progeny obtained, removal of the stop-cassette occurred exclusively in the germline cells, but not in the somatic cells of the ovary. To keep the mutant background, egg mutant females, ovaries fail to develop normally and lack egg chambers with nurse cells ; +; +y w; nanos-CRE; +In se cells ,22. For All flies were kept at 18 °C on standard fly food. Organs were excised and collected in PBS. For each sample, 25 salivary glands or 30 whole ovaries were collected. For salivary glands, three biological replicates for Dam and SU(VAR)3-9-Dam samples were done. For germline, two biological replicates were done.Drosophila genome. The latter protein associates with the chromatin either specifically via its SU(VAR)3-9 part, or non-specifically, via Dam. In both Dam and SU(VAR)3-9-Dam, Dam part functions to methylate adenines in the GATC sites nearest to the position where binding has occurred. In Drosophila, cytosine or adenine methylation is only present in trace amounts in embryos, and adenine methylation takes place out of the GATC context [We followed the tissue-specific DamID protocol described in detail in . Below w context ,34. Henc10(p) scale). Black horizontal lines above the X axis (see appropriate figures below) denote threshold values with false discovery rate (FDR) <5%. Only the peaks exceeding the threshold value were used in all of our downstream analyses. Notably, the profiles shown have distinct threshold values, but for comparative purposes a common threshold value, —the largest among the samples compared—was used.The collected material  was processed for genomic DNA isolation using standard phenol/chloroform extraction, followed by the special protocol for selective amplification of the fragments that are flanked by methylated GATC sites on both ends. The DamID libraries obtained were sequenced using Illumina MiSeq platform according to the manufacturer’s instructions. DamID-seq profiles have been deposited in the GEO under accession number GSE134250.egg (SG egg), and compared it to the distribution of SU(VAR)3-9 in wild type chromosomes (SG wt) reported earlier [egg females do not develop and lack nurse cells [egg ovaries (OV egg). For this purpose, juvenile ovaries from freshly eclosed females were used (OV wt), as they are largely composed of the germarium cells.In order to understand to which extent SETDB1 is needed for SU(VAR)3-9 binding to chromatin, we used five types of samples representing different tissues and genotypes. Specifically, we constructed SU(VAR)3-9 binding profile in the salivary glands of females that were mutant for  earlier . We also earlier , howeverse cells ,22, we negg mutation does result in SU(VAR)3-9 re-localization. To help correctly resolve the situations with nested genes, targets analyzed here were defined as protein coding genes with at least one coding exon significantly enriched by SU(VAR)3-9 regardless of the binding status of the intronic sequences and UTRs.Genome-wide, all SU(VAR)3-9 profiles appear similar across all samples and have most of the peaks in the pericentric heterochromatin, with significantly fewer peaks in the euchromatin . Detaileegg mutants, the binding is reduced to 287 genes, of which 216 correspond to the gene targets in SG wt, and 71 representing novel ones. SU(VAR)3-9 binding is abrogated for the genes residing in the region 31B-E of the chromosome 2L 3-9 gene targets are SETDB1-dependent in salivary gland and female germline chromosomes, respectively 3-9 was significantly bound to 629 genes. In osome 2L . This isectively .egg is still noticeable. In mutant background, the regions of significant SU(VAR)3-9 binding may re-distribute within the gene unit. To illustrate this, sls gene has two SU(VAR)3-9 binding regions: in SG wt, these include common 5′-end exons and in OV wt these include alternative exons on the 3′-end of the gene. In egg mutants, both regions are SU(VAR)3-9-positive in salivary glands and germline (trol gene has a single SU(VAR)3-9 peak next to the two alternative exons, whereas in egg mutants additional SU(VAR)3-9 signals appear in the common exons found at the 3′-end of the gene, where it is normally absent. Yet another gene, rg is targeted by SU(VAR)3-9 both in the wild type and mutant backgrounds, however in the latter case, numerous binding sites within the gene become detectable. In contrast, SK gene displays reduced density of SU(VAR)3-9 peaks in egg animals 3-9 is differentially affected by egg. H3K9me2 profiles were retrieved from the published dataset for salivary glands, a tissue displaying relatively uniform cellular composition [egg mutants vs. wild type animals. Values below and above the X axis indicate that methylation levels are decreased/increased in the mutant background. All three sets of genes analyzed are significantly different from the genome-wide average  as well as from each other 3-9 only in egg mutants (“new in egg” category), H3K9me2 is increased. Overall the dynamics of H3K9me2 is positively correlated with the presence of SU(VAR)3-9, although this does not apply for the SETDB1-independent gene dataset.We analyzed H3K9 methylation status in position . Taking Su(var)3-9 mutants  have very similar distributions, unlike the remaining two groups. Yet, despite the visual similarity, the box-plots for “all genes” differ significantly (p << 0.001), whereas for the “lost in egg” group the difference does not reach significance 3-9). By further subtracting “Su(var)3-9 value” from “egg value”, we observed that for most of the “lost in egg” genes the results centered around zero 3-9 depletion is caused by Su(var)3-9 or egg mutations, H3K9me2 is altered approximately the same way. This contrasts the situation observed for the “common wt egg” gene set. The differences between the mutants are uniformly distributed from −1 to +4 without forming a distinctive peak (Su(var)3-9 animals, the decrease in H3K9me2 level is far more pronounced.We then focused on the analysis of the groups “lost in  diagram b, each gund zero . Namely,ive peak . ConsequIn order to understand which features are common to SETDB1-dependent targets, wild type SU(VAR)3-9-bound genes were split into three groups, depending on their chromosomal localization, i.e., residing in the euchromatin, heterochromatin, or the chromosome 4. Next, the genes were categorized as either SETDB1-dependent or -independent, and their enrichment at the particular chromosomal location was analyzed a. We obsegg mutation is probably best illustrated by the heterochromatin of the X chromosome in germline cells 3-9 is found associated with the genes in the middle part of heterochromatin only in the presence of SETDB1 (grey frame in The degree to which SU(VAR)3-9 localization is sensitive to the chromatin context and ne cells . Distal-egg mutation on SU(VAR)3-9 binding were also analyzed for the sequences located at the border between eu- and heterochromatin. This border between two chromatin domains is clearly seen as the sharp transition from regions having few if any SU(VAR)3-9 peaks to the regions with high density of peaks in each chromosomal arm and across all the cell types analyzed. Importantly, the position of this border was cell type independent and was unaffected by the meiotic arrest genes bam, aly, and can in Drosophila males [egg mutant background, and the border moves more proximal. This shift is pronounced to a different extent across the chromosome arms, and is overall stronger in the germline cells, compared to the salivary gland chromatin. For instance, in the chromosome 2R, the shift is ~50 kb for salivary glands, whereas it constitutes ~300 kb in the germline ovarian cells. In the chromosome 3L from salivary glands, the eu/heterochromatin border remains unaffected, whereas it shifts some 200 kb in the germline , and at a later differentiation stage, i.e., in mature nurse cells of the ovary (NC wt). Several SU(VAR)3-9 binding patterns became apparent 3-9 at both differentiation timepoints regardless of the presence of SETDB1. Other examples, such as CG8008 and nearby genes, are also bound by SU(VAR)3-9 throughout the entire development of the ovary, but this binding is SETDB1-dependent. CG9780 gene is found associated with SU(VAR)3-9 only in the germline cells of juvenile ovaries, but this binding is progressively reduced throughout differentiation. Interestingly, Su(var)3-9 gene itself is the target of the SU(VAR)3-9 protein, and this binding is restricted to mature nurse cells, as no SU(VAR)3-9 binding to the Su(var)3-9 gene is observed in the germarium. Two more unusual patterns of SU(VAR)3-9 distribution are worth noting, exemplified by the genes CG2750 and Ir47a. In both instances, SU(VAR)3-9 is absent from these regions in the juvenile ovaries, unless egg mutation is present. Unlike CG2750 that becomes targeted by SU(VAR)3-9 at a later differentiation stage in the wild type situation, Ir47a is not a normal target of SU(VAR)3-9 at any stage of ovarian development. In total, 73 genes were identified whose SU(VAR)3-9 binding was restricted to germarium, 126 genes that are nurse cell-specific, and 85 genes that bind SU(VAR)3-9 at both stages. Thus, the SU(VAR)3-9 profile undergoes differentiation-associated changes in germline cells, and the number of SU(VAR)3-9 targets increases as differentiation proceeds (To trace the dynamics of SU(VAR)3-9 binding throughout female germline cell differentiation and to see how this process may be affected by SETDB1, we proceeded to the comparisons of SU(VAR)3-9 profiles at the earliest developmental stage, when the germline tissue is largely composed of the undifferentiated germarium cells 3-9-positive exclusively in the germarium, constitute a group that is also nearly always SETDB1-dependent. Of the gene targets that are present regardless of the differentiation stage, the fraction of SETDB1-independent genes is increased (SU(VAR)3-9 distribution pattern appears both stage- and ncreased .Su(var)3-9 mutation: some targets remain unchanged, some are up- or down-regulated [One of the key processes controlled by SETDB1 in the female germline is suppression of transposable elements activity. Multiple piRNA clusters scattered throughout the genome are central to this process . Whereasegulated . It was egulated .flam, and 20A, —in wild type vs. egg mutants, and matched the data with H3K9me2 profiles in wild type, Su(var)3-9, and egg mutants. Use of H3K9me2 distribution data restricted to salivary glands has been explained above. Clusters of piRNA display different features of organization, activity, and localization. 38C and 42AB map to the autosomal euchromatin, and show dual-strand piRNA expression, whereas 20A and flam are found in the heterochromatin of the X chromosome, and display uni-strand expression. Furthermore, expression of the clusters 38C and 42AB is restricted to the germline, flam is expressed exclusively in the somatic cells of the ovary, whereas 20A is expressed in both germline and the soma [We focused our analysis of SU(VAR)3-9 distribution across the four large piRNA clusters—38C, 42AB, the soma .egg mutants, SU(VAR)3-9 binding remains unchanged in the salivary glands, but is virtually absent in the germline (egg mutants (Su(var)3-9 mutants 3-9 associates with the clusters 38C and 42AB in both salivary glands and germline a,f. In egermline b,g. H3K9 mutants c,e, but  mutants c,d. Thisflam and 20A, in the wild type salivary glands and the germline, SU(VAR)3-9 is found associated with flam and ignores the 20A (egg mutant background increases SU(VAR)3-9 binding at these sites in both cell types. Specifically, in the cluster 20A, 1-2 significant binding peaks become visible 3-9-positive in both cells types, and this binding is weakly affected by the egg mutation. Cluster 20A is active in the germline, however, unlike 38C and 42AB, it has uni-strand expression, and so it is under a distinct mechanism of transcription control [egg mutation at the 20A, which is opposite to the effect observed at the clusters 38C and 42AB.To summarize, one can conclude that piRNA clusters have cell type specific regulation. It is possible that SU(VAR)3-9 binding (with or without SETDB1) and the nature of the protein complex responsible for H3K9 methylation are dependent on their transcriptional activity. Clusters 38C and 42AB are inactive in salivary glands and essentially behave as typical repeated sequences of the pericentric heterochromatin. So, in salivary glands, SU(VAR)3-9 binding and H3K9 methylation are both SETDB1-independent at these sites. In contrast, these clusters have a very specific function in the germline, and so their H3K9 methylation must be compatible with transcription rather than with silencing. This is now under the control of a distinct enzyme, SETDB1, and SU(VAR)3-9 has a downstream function of spreading of the methylation mark. The same logic can be applied to explain the SU(VAR)3-9 binding at the  control . This maDrosophila tissues and demonstrated the important role of SETDB1 in binding of SU(VAR)3-9 to its targets. Previously, we speculated that SU(VAR)3-9 may have a context-dependent role. In euchromatin, it may be required for fine-tuning the expression of select genes, whereas in heterochromatin, piRNA clusters and the chromosome 4 it rather acts as a domain-wide regulator [C. elegans, where SET-25 (SU(VAR)3-9 ortholog) is responsible for H3K9 tri-methylation in retrotransposons and pseudogenes, whereas MET-2 (SETDB1 ortholog) controls H3K9 di-methylation in the satellites [In this work, we continued our earlier efforts to analyze SU(VAR)3-9 distribution on the chromosomes from different egulator . Our pretellites .egg mutants, SU(VAR)3-9 binding over the chromosome 4 decreases, but given the initial strong enrichment, most of the SU(VAR)3-9 targets remain unaffected and continue to bind SU(VAR)3-9. This is unlike the situation observed for the chromosome 4 in the germline, where SU(VAR)3-9 targets are no longer detectable upon depletion of SETDB1 3-9 enrichment in salivary glands, but not in other cell types analyzed . In egg TDB1 see . As for Drosophila embryos, and the subsequent maintenance step is controlled by SU(VAR)3-9 [1473egg allele characterized by the in-frame deletion of the entire SET domain [Exactly how SETDB1 influences SU(VAR)3-9 recruitment to its targets, remains an open question. Several options can be imagined. One could be similar to the mechanism described for piRNA clusters, where SU(VAR)3-9 operates to spread the methyl mark . In this(VAR)3-9 . The sec(VAR)3-9 . In thisT domain , one canegg mutant background. Nonetheless, several novel SU(VAR)3-9 binding sites have been observed to appear as well. Upon closer inspection of this group of sites, several possible reasons why this may occur became apparent. First, our data processing algorithm allows for a 5% FDR, therefore, some of these novel SU(VAR)3-9 targets in egg mutants could be the result of statistical error. Second, most of the heterochromatin-embedded genes are known to have short exons and large introns hosting repeated sequences. In such heterochromatic genes, SU(VAR)3-9 is typically found associated with introns rather than exons. In egg mutants, a certain degree of SU(VAR)3-9 spreading may occur, and so the intron-mapping peak may “touch” the adjacent exon. This in turn results in classifying this gene as a novel target, but in fact is a mere consequence of its localization in the heterochromatin. Finally, the third reason may be related to the intrinsic peculiarities of these genes, that truly become targeted by SU(VAR)3-9 upon depletion of SETDB1, as exemplified by the gene Ir47a 3-9 3-9 binding is abrogated for most genomic targets in the r47a see  as well )3-9 see . It may egg background, respectively. It must be underlined, that this trend is merely a correlation rather than a dependence. egg mutants approximately 20% of “lost in egg” genes have a positive value of H3K9 methylation change, indicating that the disappearance of SU(VAR)3-9 has led to increased H3K9 methylation.Changes in SU(VAR)3-9 binding are expected to correlate with altered H3K9me2 levels, and our analyses confirm this is indeed the case. Reduced and increased methylation of SETDB1-dependent genes is directly associated with the loss and acquisition of SU(VAR)3-9 in egg vs. Su(var)3-9 mutants) of the box-plots for “lost in egg” and “common wt egg” gene sets (see egg mutation affects SU(VAR)3-9 binding but also in the relative contribution of the methyltransferases into H3K9 methylation. Genes belonging to the “lost in egg” category lose SU(VAR)3-9 in both egg and Su(var)3-9 mutants. It is unclear whether these genes are normally associated with SETDB1 but in egg mutants neither SETDB1 nor SU(VAR)3-9 are found at these genes. By definition, in Su(var)3-9 animals, these genes are not targeted by SU(VAR)3-9, and the status of SETDB1 binding is not known. Nonetheless, in both mutants the level of H3K9me2 is shifted approximately to the same extent. Consequently, H3K9 methylation of genes from the “lost in egg” gene set is either primarily controlled by SU(VAR)3-9, or both enzymes contribute equally. This is unlike the situation observed in the “common wt egg” gene set. Here, in the absence of SETDB1, SU(VAR)3-9 persists, yet this results in lower levels of H3K9 methylation. Hence, this methylation is in all likelihood mediated by SETDB1. In Su(var)3-9 mutants, these genes lack SU(VAR)3-9, the status of SETDB1 association is unknown, and H3K9 methylation level decreases even further. We believe that “common wt egg” genes constitute a category wherein histone methylation is dependent on both proteins, more on SU(VAR)3-9, and less so on SETDB1. Unfortunately, our speculations are limited by the lack of SETDB1 distribution data, and more experiments are needed to uncover the relative contribution of each enzyme into H3K9 methylation.By performing pairwise comparisons 3-9 in wild type, yet become SU(VAR)3-9-bound in egg mutants, which predictably leads to up-regulated H3K9 methylation. However, the amount of H3K9me2 drops in Su(var)3-9 background for these genes, contrary to the expectation that it would remain unaltered. This small gene set is formed by a composite mix of genes, as described above, and each gene in this category may have its own regulation pattern without a common trend.H3K9me2 pattern in the “new in egg-dependent, SETDB1 may also be involved in this regulation. Presently, the details of SETDB1 distribution within genes are not known and it is unclear whether it has a certain predilection to alternative exons. Indirect evidence indicating this may be the case exists. For instance, in the context of chromosome 4 in Drosophila, SETDB1 is physically associated with the protein POF, and POF association with genes where the two proteins co-localize is known to be SETDB1-dependent [Our previous report demonstrated that the coding parts of the genes are significantly enriched and introns are significantly depleted for SU(VAR)3-9 in the context of euchromatin-residing genes. Furthermore, moderate, yet significant SU(VAR)3-9 enrichment is also observed for the alternatively spliced genes . In humaependent ,29. Notaependent . Also, iependent , where tependent .egg and Su(var)3-9 mRNA levels found in the germarium and mature egg chambers, respectively [egg- and differentiation-associated changes can be attributed to the differential activity of the underlying genes. Su(var)3-9 mRNA levels increase significantly throughout differentiation, and so does the number of SU(VAR)3-9 targets. At the same time, Su(var)3-9 mRNA levels in the germarium are relatively low, yet sufficient for the strong recruitment of SU(VAR)3-9 to the pericentric heterochromatin. We observed that the vast majority of targets whose association with SU(VAR)3-9 is restricted to the germarium  are SETDB1-dependent, and their percentage drops as differentiation proceeds (see Both SU(VAR)3-9 and SETDB1 are active in the female germline and function sequentially throughout differentiation, with maximum ectively . We hypoeeds see . ApparenptnSu(var)3-9 and Su(var)3-9 overexpression result in massive heterochromatinization, whereas in Su(var)3-1JIL-1 hypermorphic mutants heterochromatin expansion is repressed [The topics of where the molecular border delimiting eu- and heterochromatin is located, whether it is permanent and, if not, how it is controlled have been a subject of active research in the past. Sharp changes in H3K9me2 levels are associated with the transition from eu- to heterochromatin. These changes are clearly related to the ratio of unique vs. repeated DNA sequences, as both repeats and H3K9me2 are increased at the border . Intriguepressed . In the


Master transcription factors have the ability to direct and reverse cellular identities, and consequently their genes must be subject to particular transcriptional control. However, it is unclear which molecular processes are responsible for impeding their activation and safeguarding cellular identities. Here we show that the targeting of dCas9-VP64 to the promoter of the master transcription factor Sox1 results in strong transcript and protein up-regulation in neural progenitor cells (NPCs). This gene activation restores lost neuronal differentiation potential, which substantiates the role of Sox1 as a master transcription factor. However, despite efficient transactivator binding, major proportions of progenitor cells are unresponsive to the transactivating stimulus. By combining the transactivation domain with epigenome editing we find that among a series of euchromatic processes, the removal of DNA methylation (by dCas9-Tet1) has the highest potential to increase the proportion of cells activating foreign master transcription factors and thus breaking down cell identity barriers. Master transcription factors dominantly direct cell fate and barriers ensuring their tissue specific silencing are not clearly defined. Here, the authors demonstrate that inefficient targeted transactivation of Sox1 in neural progenitor cells is surmountable through targeted promoter demethylation using dCas9-Tet1. These proteins are temporally and spatially highly restricted in their expression. The erroneous expression of developmental master transcription factors is a permanent threat to the integrity of any multicellular organism, as they are able to impose their transcriptional programs outside of their natural context. This issue has been exemplified by the so-called reprogramming factors, which are master transcription factors able to overwrite already established cellular programs by enforcing foreign cell identities2. Because cell identity switches rarely  occur under normal circumstances, it is likely that special measures restrict the activation of master transcription factors in somatic cells.Cellular features, including identity, behavior, and potency, are initiated during development by transcriptional programs, which are triggered by individual master transcription factorsAlthough it is obvious that the most effective barriers against cell identity changes would control the expression of master transcription factor genes and that several chromatin processes  have been implicated in this process, causalities were extremely difficult to establish. This is due to the fact that until recently the lack of experimental options for site-specific manipulation made it challenging to generate unambiguous experimental evidence for the causality of local chromatin features. But even with recent advancements in the field of DNA targeting, most experimental reprogramming approaches still rely on vector-mediated overexpression of fate determinants, bypassing the primary activation of the endogenous reprogramming factor gene. Thus, the potentially most relevant cell identity barriers have evaded identification and functional analysis.5, as well as epigenome editing, which is the site-specific manipulation of epigenomic features, for which a number of options have been developed recently7. Gene induction by transcriptional engineering aims for the targeted manipulation of gene expression of endogenous genes8. While the epigenomic and/or transcriptional effects are defined by the specific dCas9 fusion protein used, the target locus is specified in the protospacer sequence encoded in the short synthetic guide RNAs (gRNAs). The system most frequently used to date to achieve gene activation is a fusion of an enzymatically dead version of Cas9 with four copies of the transactivator domain of the viral transcription factor VP16 (dCas9-VP64)9. Transcriptional engineering has been successfully used for transcription activation of a number of genes, including master transcription factors like MyoD, Ascl1, and Sox211. Although examples remain sparse and we lack comprehensive approaches, it emerges already that endogenous master transcription factors behave in a particularly rigid manner compared to control genes13.However, two experimental strategies, both based on adaptations of the bacterial CRISPR complex, have recently enabled the circumvention of these two issues and can be used to investigate the presence and identity of such functional barriers: transcriptional engineering, which is the manipulation of gene expression by targeting artificial transcription factors to gene promotersSox1) is amongst the first transcription factors induced during neural development in xenopus14, chicken15, rodents16, and humans17. In mice, Sox1 shows strong and specific expression in neuro-epithelial cells19, the progenitors of all neural cells. Sox1 has some sequence identity (51%) to Sox2, but unlike its paralog it is not expressed in pluripotent cells and only becomes detectable in the newly formed neuroectoderm around the onset of somitogenesis20. Shortly thereafter, Sox1 expression disappears, except in the adult neural stem cell niches of the hippocampus and the adult subventricular zone, where it marks a population of progenitor cells with long-term neurogenic potential21. Interestingly, Sox1-positive NSCs can be propagated only poorly in vitro, as cultured cells irreversibly lose Sox1 expression22. This conversion coincides with a progressive loss of neuronal differentiation potential and parallels the natural development in vivo. Despite its undeniable relevance as an early lineage marker, the functional roles of Sox1 are, compared to its paralogs Sox2, Sox9, Sox10 and Sry, still poorly understood23.During neurogenesis, a temporal sequence of master transcription factors controls the progression of neural development. The sex-determining region Y-box transcription factor 1   do not express the neural stem cell factor Sox1. First, we tested whether transcriptional engineering can be used to significantly activate this early lineage marker in NPCs. For this, we generated clonal NPC lines stably expressing the transcriptional trans-activator dCas9-VP64 that can be targeted to specific genomic loci through simultaneous delivery of gRNAs. The cells continued to produce mostly glial progeny when differentiated (see below). To test the capacity of these cells for targeted gene activation, we used an expression construct containing two gRNAs (A1-9). Those were designed to target with high predicted specificity the promoter of es) Fig. . To induSox1 promoter. Moreover, to rule out that the specific choice of gRNAs is the source of the insufficient Sox1 induction, we generated seven different lentiviral constructs, each targeting a different site in the Sox1 promoter . Here, GFP has been knocked in-frame into the endogenous copy of the transcription factor Sox1, leaving the promoter unchanged18. NPCs heterozygous for the GFP reporter (Sox1wt/Sox1GFP) and stably expressing dCas9-VP64 were used henceforth to analyze transcriptional induction by flow cytometry or to separate GFP-positive and -negative cells by fluorescence activated cell sorting (FACS). As expected, cells lacking dCas9-VP64 or Sox1 targeting gRNAs appeared almost exclusively GFP-negative in flow analysis , while the majority of cells remained unaffected for all gRNA combinations used  cells and GFP–negative (subsequently referred to as Sox1GFP negative or non-responsive) cells and analysis by qPCR showed that the Sox1GFP positive population is the main source of Sox1 induction, as significantly more Sox1 mRNA is found in those cells , 482 genes downregulated . The highest induced factors (>10-fold) contained many rarely studied genes, some of which have been shown to functionally contribute to early brain development 28). Among those transcripts significantly induced (4–30-fold) were several genuine neural stem cell factors  or FACS-sorted neural rosettes (NRs), see methods)  and did not induce Prominin (Cd133), the cells tended to cluster and induced several neural stem cell markers that were absent in NPCs, including Occludin (Ocln), and zona occludens 1 (Zo-1) and elevated others  that were already weakly detectable in control NPCs  Fig. . Downregds) Fig. ; howeverSox1GFP-positive and -negative NPCs by FACS and tested their differentiation potential. Under differentiation conditions, non-targeted cells or cells with control gRNAs gave rise almost exclusively to glial progeny ; however, Sox1GFP-positive cells produced significantly more neuronal (as indicated by the marker for young neurons Tuj1) and less glial progeny  was absent. These data support the notion that the activation of the stem cell factor gene Sox1 induces transcriptional NSCs programs, which are sufficient to release NPCs from their glial commitment and restore neuronal differentiation potency.Since transcriptomes and functional markers indicate at least a partial reversion toward a neural stem cell identity, we asked whether these changes translate into the restoration of cellular potency. To that end, we separated eny Fig. . When giSox1 targeting. Immunoblot analysis revealed that Sox1GFP-positive and -negative populations exhibit comparable levels of dCas9-VP64 protein . As depicted in Fig. Sox1 in gRNA co-delivery experiments . As depicted in Fig. Sox1 promoter at comparable levels in Sox1GFP -positive and –negative cell populations, strongly suggesting that processes downstream of transactivator binding interfere with the activation of Sox1 in non-responsive cells.Recent publications indicate that the compaction of chromatin at the target locus might influence the binding of Cas9Sox1 is a particularity due to the use of dCas9-VP64, we employed a composite CRISPR activator, VPR, additionally equipped with trans-activator domains of the NF-kappa-B p65 subunit and the R transactivator of Epstein–Barr virus (RTA)34. Since VPR has been shown to be significantly more potent than VP64 on a series of promoters3, this experiment also allowed us to test whether stronger activation signals would overcome the incomplete Sox1 induction. Flow cytometry and qPCR revealed that although more GFP mRNA can be detected in the bulk population when using dCas9–VPR, the responsible Sox1GFP positive fraction is not elevated compared to dCas9-VP64  and lysine 27 (H3K27me3). As indicated in Fig. Sox1 promoter is marked by H3K9me3 to a similar degree as repressed control genes  in unresponsive cells, while NPCs with induced Sox1 expression have significantly lower H3K9me3 levels, almost to the degree of a strongly expressed housekeeping gene (Gapdh). However, H3K27 methylation did not show modulation between Sox1-expressing and non-responsive cells  on the Sox1 promoter  the responding Sox1GFP-positive population had greatly reduced DNA methylation levels , indicating that the detected differences are likely specific for the Sox1 promoter and do not represent a global cellular reduction of DNA methylation . All teion Fig.  but not ion Fig. . Taken tSox1 transcription and whether they functionally hinder the activation of the master transcription factor. For this, we combined dCas9-VP64 with targetable chromatin enzymes at the Sox1 promoter in NPCs . While none of these factors are expressed in NPCs . Through immunocytochemistry we observed that only one of the master transcription factors, NeuroD4, is upregulated in a significant proportion of NPC cells (ca 50%) in response to transcriptional engineering; the other three remain silent in most cells. To investigate whether (as for Sox1) targeted de-methylation can break down a barrier of transactivation, we combined transcriptional engineering with epigenome editing. While dCas9-dTet1 has minimal effects, dCas9-Tet1 multiplies the proportion of responding cells  induction and makes their genes insensitive even to potent transcriptional activators. However, manipulating these modifications accordingly can release the functional block and make cells undergo cell fate changes.To test whether these findings are a peculiarity of PCs Fig. , only twlls Fig. , but onlSox1, Nkx2-2 and Oct4, that the induction of developmental transcriptional programs must overcome functional barriers. These are constituted (at least in part) by chromatin features on the promoter of the master transcription factor itself. We found that some of these epigenetic features have the potential to decouple the effects of a bound transcription factor on the transcription of another (and thus play important roles in cell identity robustness). It is clearly evident that the targeted activation of transcription varies in the order of three to four magnitudes depending on the gene, but the molecular basis for this variation and to what extent cellular or experimental heterogeneity might contribute are currently unknown.Here we show for several example master transcription factors, such as Sox1 overexpression in vivo has been shown to bias neural progenitors to neuronal commitment, while progenitors from Sox1-deficient mice generate fewer neurons19. Thus far, few examples have been reported in which cell identity could be manipulated using the transcriptional activation of an endogenous gene39. We find that with transcriptional engineering strong Sox1 protein induction can be achieved but that only small subsets of cells are able to respond to the transactivation signal. This is to our knowledge the first study examining the sources of heterogeneity in cellular responses during transcriptional editing, separating experimental from biological heterogeneity. Due to the widespread use of over-expressing exogenous gene copies in cellular reprogramming models, the concept of cell identity barriers has mainly concentrated on the targets of reprogramming factors41.Here, we use transcriptional engineering of the widely used neuro-epithelial marker Sox1 to increase the potency of neural progenitor cells. In line with our results, MyoD42. In contrast to this and other publications employing dCas9-Tet1, DNA de-methylation alone had limited impact on transcription of Sox1. Only when additional transcriptional stimuli were applied, absence or presence of DNA methylation really made a significant difference. If this is as common among master transcription factors, as our data indicates, it could solve the paradox that although DNA methylation is needed for development, repressed developmental genes are rarely activated when DNA methylation is eliminated43.Here, we investigated instead the chromatin processes interfering with the activation of an endogenous master transcription factor, thereby protecting the existing cell identity. For this we employed epigenome editing, a technique with numerous applications whose effectiveness along the lines of cell identity has recently been demonstrated on an enhancer element of the muscle reprogramming factor Sox1 transcription start site promoter in responsive NPCs. Secondly, critical roles for DNA methylation during neural cell fate choices, in particular in glial vs. neuronal commitment, have been suggested44. Thirdly, we were able to reproduce the effects of the DNA de-methylase Tet1 with a fully independent approach, applying an inhibitor of DNA methyltransferases. Importantly, we could detect similar principles when activating Oct4 and Nkx2-2, showing that the observed effects are not unique to Sox1 and have broader implications.With this approach, we were able to separate cause from consequence and investigate, which chromatin features functionally interfere with the consequence of a bound trans-activator. Although our results suggest that several tested chromatin processes might form an intrinsic barrier against cellular conversion , we concentrated further on DNA methylation for three main reasons: firstly, we found the significant reduction of DNA methylation around the Sox1 promoter45, some of which repeatedly overlap CpG sites  transcription factors are predicted to bind sites in the tes Fig. . SeveralIt is currently unclear how many of the myriad of individual chromatin features have functional roles, but it is not unlikely that many of those are only relevant when new conditions arise, may these be local (a new transcription factor binding event) or global (cellular identity changes). We show that transcriptional engineering and epigenetic editing can be combined to investigate such conditional causalities, paving the way to comprehending the complex interplay between transcription factor binding and effects, chromatin features and barriers, as well as cellular identity and heterogeneity.To generate expression plasmids and/or lentiviral vectors containing dCas9 fusion proteins, first a hygromycin resistance cassette was added to pMLM3705 (Addgene plasmid 47754) by cutting with SgrDI  and MluI . The cassette was amplified from Addgene Plasmid 41721 using the primers Hygro_1fwd and Hygro_1rev and a SV40 polyadenylation sequence from the Addgene Plasmid 13820 using primers Hygro_2fwd and Hygro_2rev. Inserts and backbone were mixed in molar ratios of 3:3:1 and combined with Gibson assembly Mastermix  for 30 min at 50 °C to generate dCas9-VP64-Hygro. To introduce chromatin modifying domains , dCas9-VP64-Hygro was cut with PstI  and PmeI  and chromatin modifier domains were amplified from available templates listed in supplementary table www.benchling.com) that employs a published algorithm for gRNA binding efficiency51. Specificity scores above 40 were set as requirements, and only sequences with a 5’G were considered in order to fit the requirements of the human U6 promoter. gRNA sequences were designed in the region starting 250 bp upstream of the transcription start site (TSS) up to the TSS. For the STAgR constructs, the gRNAs of each pair were required to be at least 100 bp apart to avoid spatial hindrance. STAgR plasmids were generated using the protocol provided by Breunig et al., 201824. For lentiviral gRNA plasmids, the vector pLKO.1 (Addgene plasmid 10878) was modified as described in Koeferle et al.52. For subcloning of STAgR constructs into the modified pLKO1 backbone, the gRNA cassettes were amplified by PCR using the amp_gRNA_pLKO_fwd and amp_gRNA_rev primers. 25 μl Phusion High-Fidelity PCR Master Mix with HF Buffer , 0.5 μl of each primer (100 μM), 1 ng template in a total reaction volume of 50 μl. The backbone was digested with AgeI-HF . Insert and backbone were gel-purified and mixed in a molar ratio of 3:1, and 2.5 µl of the mix were incubated in 2.5 µl 2x Gibson Mastermix for 30 min at 50 °C. For cloning of single gRNAs into the lentiviral backbone, gRNA sequences were ordered as strings  as described53) and cultured in NeuroCultTM Proliferation kit  media. For expansion of NPCs the media was supplemented with 10 ng ml−1 human recombinant bFGF , 20 ng ml−1 human recombinant EGF  and 1 µg ml−1 Laminin . Cells were grown in a monolayer in Laminin coated cell culture dishes at 37 °C and 5% CO2. For the establishment of clonal dCas9-VP64 expressing cell lines, 300000 cells were seeded into 6-well plates. In total 2.5 µl Lentivirus were added to the medium. After 3 days, selection with 8 µg ml−1 Blasticidin S  was started and kept throughout the duration of experiments. After seven days, resistant cells were seeded as single cells in 96-well plates. Clones were expanded and dCas9 expression verified by qPCR and western blot. For transfections, 350000 cells were plated into six-well plates and 2 µg or 3.2 µg of STAgR plasmid and/or dCas9 expression plasmid respectively were transfected using Lipofectamin® 2000  according to the manufacturer’s protocol. dCas9 expression from plasmids was selected with 150 µg ml−1 Hygromycin B . For transduction of gRNAs, 250000 cells were plated into T25 cell culture flasks. A volume of lentivirus equivalent to 1 × 106 particles was added to the medium. To select for gRNA expression, 0.08 µg ml−1 Puromycin  was added to the medium. qPCR primer and antibodies are listed in Supplementary Table Murine Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


Arabidopsis SSB, WHIRLY1, by using loss- or gain-of-function lines. We show that WHIRLY1 binding to the promoter of WRKY53 represses the enrichment of H3K4me3, but enhances the enrichment of H3K9ac at the region contained WHIRLY1-binding sequences and TATA box or the translation start region of WRKY53, coincided with a recruitment of RNAPII. In vitro ChIP assays confirm that WHIRLY1 inhibits H3K4me3 enrichment at the preinitiation complex formation stage, while promotes H3K9ac enrichment and RNAPII recruitment at the elongation stage, consequently affecting the transcription of WRKY53. These results further explore the molecular actions underlying SSB-mediated gene transcription through epigenetic regulation in plant senescence.Single-stranded DNA-binding proteins (SSBs) are assumed to involve in DNA replication, DNA repairmen, and gene transcription. Here, we provide the direct evidence on the functionality of an  Plant senescence is the last stage of plant development. It is a controlled process that plants response to internal factors (age and hormone) and external factors (abiotic stress and biotic stress), in which plants remobilize nutrients from source leaves to developing tissues. In the past decades, molecular components underlying the onset of senescence have been studied, however, the age-dependent mechanisms that control the onset of senescence remains opening.WRKY22 also showed accelerated and delayed senescence phenotype in dark condition (wrky70 mutant showed aggravated senescent phenotype during development and dark treatment (via gibberellin (GA)-mediated signaling pathway (WRKY is a major transcription factor (TF) family of plants, in which many of them are the central players in gene regulation during leaf senescence. WRKY6 was found to activate several senescence-associated genes during leaf senescence , 2002. Mondition . WRKY53 ondition , while wreatment . WRKY54 reatment . Additioreatment  and WRKY pathway . Mutual  pathway .WRKY53 gene is activated by GATA4 . It has been verified that WHIRLY1 bind to the promoter of WRKY53 to repress expression of WRKY53 and WRKY33 in Arabidopsis leaf senescence  Heynold ecotype Columbia 0 background, WHIRLY1 T-DNA insertion line Salk_023713 (why1) were provided by the European Arabidopsis Stock Centre, while WHIRLY1 overexpression mutants (oepnWHY1 and oenWHY1), WHIRLY1 complementary line (PWHY1) which harbors its own promoter, and WHIRLY1 CDS plus HA target were constructed at previous research (th rosette leaves from PWHY1 mutants at 6th week were collected for treatment with 3-Deazaneplanocin A (Dzenp) to detect the influence of WHY1 binding affinity and enrichment of H3K4me3, H3K9ac.All the plants used were research . Seeds wresearch . 5–8th rChlorophyll fluorescence of leaf 5 from different developmental plants was measured using a Pocket PEA Chlorophyll Fluorimeter (Hansatech) after 15-minimum dark incubation. The average Fv/Fm value of leaf 5 from at least 12 individual plants was calculated. Chlorophyll concentrations of leaf 5 from 12 different developmental plants were measured by Dualex 4. Three points of each leaf were detected.th to 8th rosette leaves was isolated according to the manufacturer’s protocol of TransZol UP (TRANSGEN), and was then treated with RNase-free DNase I . First-strand cDNA was generated from 1 μg portion of total RNA using RevertAid First-Strand cDNA Synthesis Kit , following the instruction. PCR was performed to analyze the expression of genes. To determine the relative expression rate, data were normalized to the expression level of wild-type or of 5-week-old plants (which were set to 1) after normalized to the internal control of GAPC2. Additionally, three technical replicates of three biological replicates and the determination of a melting curve of the amplified PCR products were carried out.Total RNA from 5Supplementary Material. The immunoprecipitated DNA was isolated by Universal DNA purification kit . Purified DNA was analyzed by real-time PCR with specific primers (see Supplementary Tables). Relative histone modification levels in WT during leaf development were therefore normalized to the input, while the enrichment in WHIRLY1 mutants was normalized to WT again, referring to ΔΔCt method1.The ChIP assay was performed with a modified method . About 0E. coli as described by The recombinant WHY1 protein was expressed in 2. pG5ML with promoter of WRKY53 including mutated WRKY53II [mutant (GTNNNGGT) m1 or mutant (CTNNNNAAAT) m2 WHIRLY1 binding motif] . The levels of H3K4me3, H3K9ac are given relative to the total H3 levels. In vitro ChIP to detect the effect of WHIRLY1 on transcription stage was performed in the presence or absence of 0.01% sarkosyl, which inhibits PIC assembly but does not affect elongation by pre-formed complexes . The expression of RBCS, a gene encoding the small subunit of ribulose 1,5-bisphosphate carboxylase, was downregulated at the 6th week while the expression of SAG12 gene encoding a cysteine protease which expressed only in senescent tissues, was drastically induced at 8th week in current case . Therefore, we inferred that 5th to 7th week was the initiation and early stage of leaf senescence. WHIRLY1 was phosphorylated by calcineurin B-like-interacting protein kinase14 (CIPK14) and accumulated steady in the nucleus after 5W . Although occupancy of WHIRLY1 on the promoter of WRKY53 was conversed with the accumulation of nuclear form WHIRLY1, the expression level of WRKY53 strikingly increased from 5th week to 6th week while the occupancy of WHIRLY1 on the promoter of WRKY53 declined . Taken together, these results demonstrate that WHIRLY1 chronologically repressed WRKY53 transcription and expression via binding to its promoter at senescence initiation stage from 5th week to 7th week.In previous work, we performed ChIP-Seq and WHIRLY1 was found binding on the promoter of l manner . Leaves after 5W  , and H3K27me2   using 5- to 8-week-old wild-type plants. The ChIP assay showed that enrichments of H3K4me2 and H3K4me3 at WRKY53P region which was downstream of WHIRLY1 binding domain at the promoter of WRKY53 and at WRKY53II region which contained WHIRLY1 binding domain with TATA-box , were at high level in 5-week-old plants, then significantly declined from 5th to 6th, and started to increase from 6th to 8th week, and significantly climbed up to 20% high level at 8th week . The enrichment of H3K4me2, H3K4me3 at the residual detected regions of WRKY53 promoter were at low level compared to that of WRKY53P. H3K27me2 was the lowest at all the detected regions of WRKY53 gene . Although the enrichment of WHIRLY1 at WRKY53 promoter decreased gradually, still maintained at 40% level at 7th week , and WRKY53 expression level increased rapidly from 5th to 6th week then declined at 7th week . Taken together, these results suggest that the occupancy of WHIRLY1 protein at WRKY53 promoter may be associated with the enrichment of H3K4me2 and H3K4me3 at WRKY53. However, WHIRLY1 occupancy did not repress WRKY53 expression via directly correlated with H3K4me2 and H3K4me3.To investigate whether WHIRLY1 binding on the WRKY53 in wild-type plants during leaf aging. The H3K9ac at promoter and P regions of WRKY53 maintained high level at 5th and 6th week but decreased sharply from 7th week . H3K9ac has been reported to positively correlate with the recruitment of RNAPII . The enrichment of H4ac at promoter and P region of WRKY53 showed a constant low level during leaf senescence in our study . Taken together, these results indicate that histone modifications at the promoter and translational start region of WRKY53 are dynamic from 5th to 8th week and H3K9ac seems more closely related to the transcription of WRKY53.We also detected the pattern of H3K9ac, H4ac, and RNAPII occupancy at f RNAPII . ExpecteWRKY53 promoter during the initiation period of plant senescence from 5th to 7th week. The results showed the ratio pattern of H3K9ac/H3K4me2-3 enrichment was chronologically associated with the occupancy of WHIRLY1 on the WRKY53 promoter  and the transcription level of WRKY53Supplementary Figures S1D,E and Figure 1F) during the initiation period of plant senescence, which suggests that H3K9ac/H3K4me2-3 synergistically control WRKY53 transcription in plant developmental manner.A combinatorial interplay between posttranslational modifications on the same histone was proposed based on the patterns of H3 methylation and acetylation at promoters of specific target genes . We calcWRKY53, we detected the pattern of H3K4me2, H3K4me3, and RNAPII recruitment at WRKY53II and WRKY53P regions of WRKY53 in WHIRLY1 knockout line (why1), overexpression nucleus-located WHIRLY1 line (oenWHY1), WHIRLY1 complementary line (PWHY1) as well as WT plants , but RNAPII recruitment did not change . Interestingly, overexpression nuclear isoform WHIRLY1 . The results suggest that loss of WHIRLY1 enhances the H3K4me2 and H3K4me3 enrichment at the 5′ untranslated region and translational start region of WRKY53 (WRKY53P), while the occupancy of WHIRLY1 seems to inhibit the enrichment of H3K4me2 and H3K4me3 at its binding site on WRKY53 at senescence initiation stage.To further investigate whether the binding of WHIRLY1 affects the enrichment of H3K4me2 and H3K4me3 at promoter and P regions of T plants . The ChI WHIRLY1  did not WRKY53. We treated 6-week-old PWHY1 plants with different concentrations of 3-Deazaneplanocin A (DZnep), which is a S-adenosyl homocysteine hydrolase inhibitor and has been reported to affect H3K4me3 enrichment at the promoters of 3% genes in zebrafish . However, DZnep had no effect on the occupancy of WHIRLY1 at WRKY53 promoter . Interestingly, DZnep treatment resulted in increase of H3K9ac at WRKY53II region of WRKY53 . Therefore, H3K4me3 marks at WRKY53 promoter did not directly affect the occupancy of WHIRLY1 at WRKY53 promoter, but H3K4me3 marks correlate with H3K9ac.H3K4me3 at promoters and 5′-end regions have been reported to significantly correlate with active gene expression , and theebrafish . The resWRKY53, we performed ChIP experiment in why1, oenWHY1, PWHY1, and WT plants with anti-H3K9ac and anti-RNAPII antibody . As showed in Figure 3 and Supplementary Figure S5, the loss of WHIRLY1 led to an increase in H3K9ac at WRKY53II region of WRKY53 at 6-week-old and 7-week-old wild-type plants, and enhanced the H3K9ac enrichment at WRKY53P region of WRKY53 at 7-week-old wild-type plants. H3K9ac enrichment at WRKY53P region of WRKY53 was inhibited at 6-week-old and 7-week-old oenWHY1 mutant, while H3K9ac enrichment at WRKY53II region of WRKY53 was only inhibited at 7-week-old oenWHY1 mutant . Interestingly, significant enrichment of RNAPII at WRKY53II and WRKY53P regions of WRKY53 were also detected in the 7-week-old why1 mutants. Overexpression of WHIRLY1 reduced the RNAPII recruitment at WRKY53II and WRKY53P regions of WRKY53 at 7-week-old PWHY1 . As shown in the Figures 3C,D, higher transcript level of WRKY53 and SAG12 in the why1 mutant and lower transcript level of WRKY53 and SAG12 in the oenWHY1 were detected, showing high proportion of yellow leaves and less chlorophyll content in the why, reversely, less proportion of yellow leaves and high chlorophyll content in the oenWHY1 line. These results demonstrated that WHIRLY1 binding on WRKY53 accelerated the deacetylation of H3K9ac and repressed RNAPII recruitment at promoter and translational start region of WRKY53 to determine WRKY53 transcript level and senescence-related parameter in a developmental manner.To determine whether WHIRLY1 occupancy affects H3K9ac level and RNAPII recruitment at WRKY53 promoter directly impacts H3K4 methylation, H3K9ac and WRKY53 transcription initiation, we recruited a cell-free transcription system  . Micrococcal nuclease digestion showed that all templates are chromatinized equivalently . In vitro ChIP was performed with assembled chromatin. Chromatin was incubated with or without recombinant WHIRLY1 protein expressed in E. coli  . Further the transcript run-on assay and quantitative RT-PCR were used to detect the transcript elongation and accumulation of TBP report genes. Surprisingly, the results showed that report gene TBP transcript elongation and accumulation were increased with the time course in the presence of WHIRLY1, while TBP transcription did not process when pWRKY53II were mutated or in the deficiency of WHIRLY1 . Together, these results indicate that WHIRLY1 binding on WRKY53II in vitro represses the enrichment of H3K4me3 and enhance the enrichment of H3K9ac and RNAPII recruitment, and activate the TBP transcription.To confirm occupancy of WHIRLY1 protein at the gure 4A) . Chromat E. coli  and immugure 4C) . InteresWRKY53 promoter impact H3K4me3 before or after PIC formation, therefore, 0.01% sarkosyl, which inhibits PIC assembly but does not affect elongation by pre-formed complexes . ChIP–qPCR results showed that neither sarkosyl nor B2 RNA affected the enrichment of WHIRLY1 at the WRKY53 promoter. However, sarkosyl treatment decreased the enrichment of H3K4me3 at WRKY53 promoter while B2 RNA treatment inhibited enrichment of H3K9ac and RNAPII recruitment at WRKY53 promoter . Occupancy of WHIRLY1 on WRKY53 promoter also inhibited the enrichment of H3K4me3 in the presence of B2 RNA and enhanced the enrichment of H3K9ac and RNAPII recruitment in the presence of sarkosyl . The accumulation of TBP transcript was shown in the presence of sarkosyl . These results suggest that WHIRLY1 binding to the WRKY53 promoter represses the enrichment of H3K4me3 at the preinitiation stage of PIC formation, while promotes the enrichment of H3K9ac and RNAPII recruitment during the elongation by pre-formed complexes, consequently promotes the TBP transcriptionH3K4me3 has been reported to involve in the preinitiation complex (PIC) assembly during transcription . We wondomplexes , or B2 Romplexes , was useWRKY53 promoter in transcriptional level, differentially expression genes related to histone modifications were picked from the dataset of RNA-seq between whirly1 and wild-type plants . SWI3D, HD2D, JMJ22, NFA2, and HTA4 were selected to be further analyzed in 6-week-old why1, oenWHY1 mutants as well as wild-type plants by qRT-PCR. Surprisingly, our qPCR results showed the expression of HD2D and JMJ22 in both why1 mutants and oenWHY1 mutants was lower than that of WT , which contradicts with the previous RNA-seq data. Overexpressing or knocking out WHIRLY1 has no effect on gene expression of SWI3D, NFA2, and HTA4. Moreover, the expression levels of histone methyltransferase ATX1 (Arabidopsis Trithorax-like protein 1), ATX2, SUVH2, and histone deacetylases HDA15, HDA6, HDA2, HDA5, and HDA9 were also detected by qPCR. ATX1 and ATX2 play roles for trimethylating and dimethylating K4 of histone H3, respectively . Hence, WHIRLY1 is likely mediated histone methylation by regulating expression of histone methyltransferases ATX1. HDA2, HDA5, HDA6, HDA9, and HDA15 which has been shown highly expression in leaves based on Arabidopsis eFP database3 were also selected for qPCR. Interestingly, the loss of WHIRLY1 decreased the transcription level of HDA2, HDA5, HDA6, and HDA9, while overexpressing WHIRLY1 only increased HDA6 and HDA9 expression. These results suggest that WHIRLY1 eFP database might involve in regulating gene expression of histone deacetylases to remove the H3K9ac marks at the promoter of WRKY53.To investigate whether WHIRLY1 regulates H3K4me3, H3K9ac, and RNAPII recruitment at the ectively , and SUV  as well as gene body (from +367 bp to +549 bp) of WRKY53 locus increased in senescent leaves which consisted with the expression of WRKY53 . The observation that the level of both H3K4me2/3 and H3K9ac at selected regions (WRKY53II and WRKY53P) of WRKY53 from 5th to 7th week correlated with the enrichment of WHIRLY1 protein binding on WRKY53 promoter. It indicates that WHIRLY1 spatio-temporally affect H3K4 methylation and H3K9 acetylation at WRKY53 during leaf aging. In vitro assay further confirms WHIRLY1 binding on specified motifs (GNNNAAATT) of WRKY53 promoter repressed enrichment of H3K4me3 and potentiated enrichment of H3K9ac, RNAPII recruitment, and TBP report gene transcription , implicating that WHIRLY1 involves in modification of local chromatin states.H3K4me2/3 is high at promoter or around the transcription start site (TSS) regions of active or poised genes in animals, while H3K4me2/3 is enriched at the proximal promoter and TSS site of genes in plants . Both inf WRKY53 . Howeverpression . WRKY53  WHIRLY1 , or whic WHIRLY1 . In thisWRKY53 promoter affected H3K4me3 and H3K9ac in vivo and in vitro. We utilized two transcription inhibitor sarkosyl and B2 RNA to address this question .Both H3K4me3 and H3K9ac are closely associated with active genes and play an important role in transcription. In human cells, H3K9ac potentiates the interaction of H3K4me3 and basal TF TFIID . There aquestion . Sarkosyquestion , while Bquestion . ResultsWRKY53 promoter was upstream and close to TATA box, in addition, the structure analysis of transcription initiation by RNA polymerase II showed PIC form around TATA box . Enrichment of H3K4me3 at WRKY53II was inhibited by histone methylation inhibitor DZnep, while the enrichment of H3K9ac was improved by Dznep treatment, in turn, the enrichment of H3K9ac at WRKY53II region decreased in oehada15 mutant plants while H3K4me3 level at WRKY53II region increased in them. Many protein complexes and epigenetic modifications in the local chromatin environment are necessary for the progress of RNAPII-mediated transcription . Occupancy of WHIRLY1 on WRKY53 promoter mainly occurs as WRKY53 is being shutdown and decreases followed by aging. However, in vitro ChIP assay WHIRLY1 increased H3K9ac enrichment and RNAPII recruitment and upregulate downstream target gene transcription. It is a possibility that WHIRLY1 protein not only acts as a block of WRKY53 transcription before senescence initiation, inhibits activated transcription, but also acts as an activator of other downstream genes such as PR10a in potato or S40 in barley . Further, what signals or factors promoted and controlled the action of WHIRLY1 on WRKY53 promoter during leaf senescence are still speculated. The accumulation of WHIRLY1 in nucleus is mediated by CIPK14 kinase which was promoted by light conditions, sugar, cytokinin, and calcium–calmodulin signal (Conclusively, we propose a model that WHIRLY1 binding on n signal . Indeed,n signal , furtherin vitro ChIP assay. DH and BD performed ChIP–qPCR, Western blots, and phenotyping. WL performed qRT-PCR. DL crossed double mutant and screen them, phenotyping. WfL and YR for qRT-PCR and RNA-seq data analyses. DH and YM analyzed the data and wrote the paper.YM designed the study and performed The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


BMPR1B gene. In this study, we identified the ovine BMPR1B gene promoter and demonstrated that its transcription was regulated by Smad4. In sheep ovarian follicles, three transcriptional variants of BMPR1B gene with distinct transcription start sites were identified using 5′ RACE assay while variants II and III were more strongly expressed. Luciferase assay showed that the region −405 to −200 nt is the PII promoter region of variant II. Interestingly, two putative Smad4-binding elements (SBEs) were detected in this region. Luciferase and ChIP assay revealed that Smad4 enhances PII promoter activity of the ovine BMPR1B gene by directly interacting with SBE1 motif. Furthermore, in the ovine granulosa cells, Smad4 regulated BMPRIB expression, and BMPRIB-mediated granulosa cells apoptosis. Overall, our findings not only characterized the 5’ regulatory region of the ovine BMPR1B gene, but also uncovered a feedback regulatory mechanism of the canonical BMP/Smad signaling pathway and provided an insight into the transcriptional regulation of BMPR1B gene and sheep prolificacy.BMPR1B is a type 1B receptor of the canonical bone morphogenetic protein (BMP)/Sma- and mad-related protein (Smad) signaling pathway and is well known as the first major gene associated with sheep prolificacy. However, little is known about the transcriptional regulation of the ovine  The transformation growth factor-β (TGF-β) cytokine superfamily is one of the protein families with the largest number of members (more than 33 proteins) exhibiting the most diverse functions in organisms ,2. BasedBMP5, BMP6, BMP7, BMP15, and BMPR1B, have been identified as candidate genes responsible for reproductive traits through genome-wide association analysis (GWAS) and whole-genome sequencing (WGS) [BMP15, BMPR1B, and GDF9) of the four major genes  identified to affect the high fecundity of sheep are core components of the BMP/Smad signaling pathway [BMPR1B is the first major gene identified for ovine high fecundity traits, including ovulation rate (OR) and litter size (LS), and A746G variant  is the only major effector mutation found in BMPR1B gene at present [FecB gene have higher LS and OR, fewer apoptotic granulosa cells, and higher ovarian BMPR1B levels [The BMP/Smad signaling pathway is known to be involved in follicular development, ovulation, and female reproduction, especially fecundity in domestic animals. In pigs, multiple core members of this signaling pathway, such as ng (WGS) ,9. In sh pathway ,11. BMPR present . MechaniB levels ,14. A moB levels .BMPR1B, of the canonical BMP/Smad signaling pathway, were significantly different between ovaries of high- and low-fecundity Hu sheep [BMPR1B gene. In this study, we identified and characterized the transcription start sites and the core promoter of the ovine BMPR1B gene, and investigated the transcriptional regulation of its 5′ regulatory region. Our previous report showed that the expressions of core components, including Hu sheep . HoweverBMPR1B gene 5′ regulatory region, we first identified the transcription start sites of the ovine BMPR1B gene using 5′ RACE assay. The first-strand was obtained using cDNA from follicles of Hu sheep ovaries as a template. Nested PCR was performed using first-stand as a template, and P1 as specific primers. Interestingly, clone sequencing showed that three sequences with different nucleotide sequences were identified within the amplification fragment  were isolated from the 5′ regulatory region of transcript variant II, and then, inserted into luciferase reporter vector pGL3-basic. Subsequently four deletion constructs were successfully generated, and termed as P1-200, P2-405, P3-921, and P4-1322 , 40 T (19.51%), 49 C (23.90%), and 53 G (25.85%) bases (Capra hircus) and cattle (Bos taurus) BMPR1B gene, respectively. The multiple putative binding sites for transcription factors, such as STAT6 , BRCA1 (Breast cancer 1), FOXL1 (Forkhead box protein L1), Smad4 (Sma-and mad-related protein 4), ELK1 (Ets-like protein-1), KLF4 (Kruppel-like factor 4), and Sp1 (Specificity protein 1) were determined in the PII core promoter region of the ovine BMPR1B gene. They were predicted using an online tool with a relative score of more than 82.0  bases . The nuchan 82.0 , indicatBMPR1B gene, we generated the luciferase reporter vector of PII core promoter  of the target promoter ,15. Notapromoter B, and copromoter C,D; howepromoter E,F. ThesBMPR1B gene, we constructed the luciferase reporter vector of the individual SBE motif-mutant type PII promoter  [BMPR1B gene remains unclear. Although Yao et al. [BMPR1B level by directly targeting its 3′-UTR, only the partial sequence of its 3′-UTR was isolated and characterized. Here, we identified, for the first time, the transcription start sites of the ovine BMPR1B gene, and elucidated the characteristics of its 5′-UTR, which might provide a foundation for further research on its transcriptional regulation.on (MAS) ,16. Unfoo et al.  demonstrCYP11A1, CYP17, IRS-2, and RGS, by direct binding to a consensus GC-rich motif within the promoter of these targets, in mammalian ovary [BRCA1 is known as a tumor suppressor gene for breast and ovarian cancer, and belongs to the family of ataxia-telangiectasia-mutated-mediated DNA double-strand break (DSB) repair genes and plays a critical role in homology-directed repair of DSBs [BMPR1B gene, indicating that ovine BMPR1B transcription may be regulated by DNA methylation. A recent report showed that BMPR1B is one of the differentially methylated region-related genes in ovaries between high- and low-fecundity Hu sheep using deep whole-genome bisulfite sequencing (WGBS) [Importantly, we also identified and characterized the PII core promoter, and a large number of transcription factor binding sites (TFBSs) were found in this region. Notably, some transcription factors, such as Sp1, BRCA1, and ELK1, are involved in female reproduction, similar to BMPR1B . Sp1 belan ovary ,19,20,21 of DSBs . BRCA1 i of DSBs . ELK-1 b of DSBs ,25. In ag (WGBS) .BMPR1B gene, and demonstrated that Smad4 controls BMPR1B transcription in the ovine granulosa cells, which acts as a transcription factor. In fact, binding with promoter regions of important functional genes and regulating their transcription are the main way for SMAD4 to exert its biological functions [CYP19A1, a key gene in E2 production, has been shown to be directly regulated by SMAD4 at the transcriptional level, in granulosa cells [miR-143-targeted FSHR (a marker of granulosa cells) signaling pathway by direct binding to the promoter of miR-143 gene [miR-425 and miR-1306, are also direct transcriptional targets of SMAD4 in porcine granulosa cells [Interestingly, we found that two SBE sites are located within the PII promoter of the ovine unctions ,27,28. Hunctions ,29. For sa cells . In porc143 gene . In addisa cells ,29. In cBMPR1B, an upstream signal molecule of SMAD4, in the ovine granulosa cells. This suggested that Smad4 can regulate the canonical BMP/Smad signaling pathway via positive feedback. The feedback regulatory mechanism has been proved to exist widely in the TGF-β family signaling pathway, especially in the canonical TGF-β/Smad signaling pathway [miR-520e-TGFBR2 axis [miR-425-TGFBR2 axis [miR-122-5p-TGFBR2 axis [miR-1306-TGFBR2 axis [miR-17 family-BMPR2 axis and Noggin-BMP4 axis mediated feedback regulation of this positive feedback factor in the neuron [SMAD4 is the final signal molecule of the canonical BMP/Smad signaling pathway. In this study, we showed that SMAD4 is a transcription activator of  pathway . SMAD2 aBR2 axis , interacBR2 axis ,34. SmadBR2 axis . The enhBR2 axis , miR-122BR2 axis , and miRBR2 axis . For thee neuron  and in de neuron , respectin vivo, both SMAD4 and BMPR1B have been shown to be strongly involved in follicular atresia of domestic animals including sheep [Functionally, we showed that Smad4 could rescue the granulosa cell apoptosis induced by BMPR1B silencing, demonstrating that this feedback regulation is involved in ovine granulosa cell apoptosis. It is well known that the canonical BMP/Smad signaling pathway is associated with fecundity in domestic animals, and is essential for gonadogenesis, steroidogenesis, follicular development, and ovulation ,10,38. Mng sheep , pigs [4ng sheep . In concBMPR1B gene contains multiple transcriptional star sites, and several regulatory elements were identified within its PII core promoter region. We also showed that SMAD4 functions as a transcription factor, feedback induces BMPR1B transcription, and then, regulates BMPR1B-mediated cell apoptosis in the ovine granulosa cells. Overall, our findings suggested, for the first time, that feedback regulation exists within the canonical BMP/Smad signaling pathway in ovarian granulosa cells, and revealed that this feedback mechanism regulates granulosa cell apoptosis and provides a new insight into the mechanism of granulosa cell apoptosis, follicular development, and female reproduction in mammals.In summary, we reported that ovine Ear tissues and fresh ovary samples of Hu sheep were collected from Xilaiyuan sheep breeding farm . Ear tissues were stored in 75% ethyl alcohol, and used for DNA extraction. Ovary samples were kept in physiological saline, and used to isolate medium follicles and total RNA. Animal experiments were reviewed and approved by Nanjing Agricultural University Animal Care and Use Committee and performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals .For the ovine granulosa cell culture, fresh ovaries were obtained from Hualing Slaughterhouse , immersed into sterilized saline at 37 °C, and then transported back to the laboratory within 2 h. Granulosa cells were isolated and cultured as previously described . KGN andGAPDH was used as an internal control. The 2−ΔΔCT method was used to analyze relative levels of target genes. DNA was extracted using the conventional phenol–chloroform extraction method. TRIzol reagent  was used to isolate total RNA from the ovine ovarian follicles and granulosa cells. Then, cDNA was prepared using a Prime Script™ RT Master Mix . Quantitative RT-PCR (qRT-PCR) was carried out in triplicate using a kit , and ® RACE 5′/3′ Kits (TaKaRa) were used to obtain the 5′-UTR sequence of Hu sheep BMPR1B gene, according to the manufacturer’s instructions. Briefly, total RNA from follicles was reverse transcribed into cDNA using enzyme terminal deoxynucleotidyl transferase (TdT) which has the ability to add a string of identical nucleotides to the 5′ end of the cDNA, followed by PCR amplification with specific primers designed by BMPR1B reference sequence. PCR products were detected by gel electrophoresis and the purified fragments were cloned into pUC-19 vector. After sequencing and alignment, the full-length sequence of Hu sheep BMPR1B 5′ end was obtained. The primers used are listed in SMARTerhttps://www.ncbi.nlm.nih.gov/nuccore/) was used to obtain the ovine reference BMPR1B gene (NC_019463.2 for Texel sheep). The putative promoter region of Hu sheep BMPR1B gene was predicted using an online software Promoterscan (http://www-74bimas.cit.nih.gov/molbio/proscan/). The TFBSs were predicted using the online database Jaspar (http://jaspardev.genereg.net). NCBI database  and sub-cloned into the pGL3 luciferase reporter vector which was then transferred into E. coli to construct the luciferase reporter plasmids. The plasmids were extracted using an Endo-free plasmid mini kit II50 , and all plasmids were validated by sequencing. TaKaRa MutanBEST Kit (TaKaRa) was used to generate SBE mutation-typed plasmids.Smad4 expression vector, pcDNA3.1-Smad4, was prepared in our laboratory . For ideectively . PCR proHEK293T and KGN cells were cultured into 12-well cell culture plates for 24 h. Transfection of reporter plasmids and measurement of luciferase activity were done as described in our previous report .Extraction and quantification of total protein in granulosa cells was performed using conventional methods and BCA Kit . Western blotting was performed as described previously . PrimaryThe apoptosis of granulosa cells in this study was determined using the Annexin V-FITC/propidium iodide apoptosis kit , according to the manufacturer’s protocol. Apoptosis rate of granulosa cells was calculated and analyzed using Flowjo software .® Plus Enzymatic Chromatin IP Kit . The anti-SMAD4 antibody  was used as a specific antibody for ChIP assay, while IgG (Origene) was used as control. The primers for ChIP-PCR are listed in Chromatin immunoprecipitation (ChIP) assay was performed using the SimpleChIPp <0.05 was considered as statistically significant.Data were expressed as the mean ± S.E.M. of at least three independent experiments. Statistics analysis was performed using SPSS v20.0 software .