Transposable elements (TE) are commonly regarded as “junk DNA” with no apparent regulatory roles in the human genome. However, a growing body of evidence demonstrates that some TEs exhibit regulatory activities in a range of biological pathways and diseases, with notable examples in bile metabolism and innate immunity. TEs are typically suppressed by epigenetic modifications in healthy somatic tissues, which prevents both undesirable effects of insertional mutagenesis, and also unwanted gene activation. Interestingly, TEs are widely reported to be dysregulated in epithelial cancers, and while much attention has been paid to their effects on genome instability, relatively little has been reported on their effects on gene regulation. Here, we investigated the contribution of TEs to the transcriptional regulation in breast cancer cell lines.We found that a subset of TE subfamilies were enriched in oncogenic transcription factor binding sites and also harboured histone marks associated with active transcription, raising the possibility of these subfamilies playing a broad role in breast cancer transcriptional regulation. To directly assess promoter activity in triple negative breast cancer cell lines, we identified four breast cancer-associated genes with putative TE-derived promoters. TE deletion confirmed a contribution to promoter activity in all cases, and for two examples the promoter activity was almost completely contained within the TE.Our findings demonstrate that TEs provide abundant oncogenic transcription factor binding sites in breast cancer and that individual TEs contain substantial promoter activity. Our findings provide further evidence for transcriptional regulation of human genes through TE exaptation by demonstrating the regulatory potential of TEs in multiple breast cancer cell lines.The online version of this article (10.1186/s13100-019-0158-3) contains supplementary material, which is available to authorized users. Transposable elements (TE), or transposons, are repetitive genetic elements that are ubiquitous in eukaryotic genomes , 2. WhenThe regulatory activity of TEs is derived from the cis-regulatory elements within their sequences, which include internal promoters and binding sites that can be recognised by host transcription factors (TF) and RNA polymerases (RNA Pol) , 15–17. Human TEs harbour a substantial number of TF binding sites (TFBS). On average, TEs are estimated to contain ~ 19% of the total TFBSs within the human genome . These bBAAT, which encodes an enzyme involved in bile metabolism [HYAL-4 and FUT5, which are involved in hyaluronan catabolism and cell adhesion respectively [Furthermore, an early study by Jordan et al. shows that 25% of the > 2000 human promoters documented in The Human Promoter Database contain TE-derived DNA sequences . This fitabolism , 22. Furectively . These eWhile these cis-regulatory sequences have endowed TEs with the potential to regulate human gene expression, the transcriptional activity of the majority of TEs are suppressed heavily in somatic tissues , 24. ThiIncreased DNA methylation is often associated with gene silencing, and most TEs are heavily suppressed by DNA methylation and thus rendered inactive in somatic tissues –29. UnliHowever, the suppressive regulations over TEs, such as DNA methylation, are often alleviated in some disease states, and the transcriptional activation of TEs is commonly observed in tumours , 31, 32.SYT1, UCA1, AK4 and PSAT1 contributed promoter activity in TNBC cell lines.In this study, we aimed to investigate the transcriptional activity of TEs in breast cancer, particularly their interactions between TFs and contribution to promoter activity, using breast cancer cell lines as a model. We analysed genome-wide binding sites for three breast cancer-associated TFs  in MCF7 cells, and confirmed that a substantial fraction of these TFBSs resided in TEs. We showed that these TFBSs were distributed across a diverse range of TEs, and identified enriched TE subfamilies with a potentially important role in modulating breast cancer transcription. We found that the binding of breast cancer-associated TFs to TEs was correlated with active histone modifications, further supporting the transcriptional activity of these TEs. We subsequently identified individual TEs within the promoters of breast cancer-associated genes and confirmed that TEs located upstream of To investigate the prevalence of TEs in breast cancer-associated TFBSs, we mapped the genome-wide binding sites for C/EBPβ, E2F1 and MYC in MCF7 breast cancer cells by re-analysing publicly available chromatin immunoprecipitation sequencing (ChIP-seq) datasets. These TFs were selected for analysis based on literature evidence on their oncogenic capacity in cancer , 41–47. p < 4.3E-5 indicated statistical significance). LTRs were observed to contribute more enriched subfamilies than any other class of TEs for all TF datasets investigated, and represented approximately 30% of enriched TE subfamilies  and repressive (H3K9me3 and H3K27me3) histone tail modifications in MCF7 cells –53. For Next, we sought to identify breast cancer-associated genes with putative TE-derived promoters. The Cancer Genome Atlas Network (TCGA) had previously identified 3662 genetic elements as being differentially expressed in breast cancer , 55. FroWe then identified genes and lncRNAs with putative TE-derived promoters by intersection with TSSs and TFBSs. Of the ~ 380,000 FANTOM CAT CAGE clusters, ~ 76,800 clusters were located nearby a breast cancer-associated TFBS (window = 300 bp). Amongst these CAGE clusters, 6727 overlapped with TEs. Finally, 56 TE-harboured CAGE clusters were located within the putative promoters for 41 breast cancer-associated genes (identified by 79 GENCODE mRNA transcripts) and 8 FANTOM CAT lncRNAs . To estimate the contribution of TEs to the promoter activity, TEs hypothesised to harbour promoter activity were removed from the wild-type promoters, and the remaining DNA sequences within the promoters were tested for promoter activity by luciferase assays. TE deletion resulted in reduced activity of all four promoters in at least one cell line.For SYT1 resulted in near complete ablation of expression in MDA-MB-468 and MDA-MB-231 cells, where only 2.6 and 11.2% of promoter activity remained after deletion respectively . Reduced methylation in tumours was not observed for the other TEs of interest. Investigating published DNase-seq data as an indicator of open chromatin, we observed significantly higher DNase sensitivity in L1PA2-SYT1 and LTR7C-UCA1 elements in MCF7 cells, compared to HMEC control cells , while the remaining TEs showed no statistically significant increase in DNAse sensitivity in MCF7 cells.To further characterise these TE-derived promoters, we investigated whether they displayed differential methylation in breast cancer tumours and whether they resided in open chromatin in breast cancer cell lines, by analysing published whole-genome methylation capture sequencing and DNase-seq datasets , 65  to chromatin remodelling e.g. CHD1 and CHD2), as well as those with specialised roles in cellular functions (e.g. IRF and STAT involved in innate immunity) , 10. Her and CHD2TEs can be categorised hierarchically into classes, families and subfamilies based on their sequence features . AlthougThe activity of TEs often occurs at the cost of host genomic stability, as active TEs can generate large mutations or drive unwanted gene expression , 32. TheSYT1, UCA1, AK4 and PSAT1) for in vitro validation. These genes were prioritised based on existing literature describing oncogenic characteristics in various cancers [Next, we sought to validate the promoter activity of TF-bound TEs in breast cancer cell lines. The intersection of breast cancer-associated genetic elements with CAGE clusters, TFBSs in MCF7 cells and TEs provided a subset of putative TE-derived promoters. We selected the putative TE-derived promoters of four candidate genes  located in the first 500 bp of the TE. Similar oncogene activation by L1-ASP has been reported in other cancer types, such as MET activation in bladder cancer [SYT1 is a protein-coding gene found to facilitate the export of the oncogenic growth factor FGF-1 [SYT1 in breast cancer cell lines coincided with its increased DNAse sensitivity and decreased DNA methylation, suggesting the L1PA2-SYT1 element can promote expression of the proto-oncogene SYT1. It remains unknown whether this promoter activity is tumour-specific, and whether it contributes to SYT1 expression under normal physiological conditions. Nonetheless, SYT1 expression is likely mediated by the L1PA2-SYT1 transposon in the context of breast cancer cell lines.The L1PA2-r cancer , 78, 79.or FGF-1 . The proUCA1 element also contributed essential promoter sequences to the UCA1 promoter  and FASTQ respectively , 81 , then counting ChIP-seq peaks that intersected with a TE using a custom python script. The average count of peaks intersecting with each TE subfamily was divided by the total number of ChIP-seq peaks for each TF to produce the expected possibility of TFBSs being located in TEs by random chance. The observed number of TF peaks overlapping each TE subfamily was counted with no rotation applied. A binomial test was used to identify significantly enriched TE subfamilies. To investigate the association between oncogenic TF binding and the epigenetic status of TEs, we analysed the epigenetic profiles of top enriched TEs bound by oncogenic TFs in MCF7 cells using published ChIP-seq datasets    were converted to hg38 by LiftOver , 91. TSSFor all molecular cloning, plasmids were purified using QIAprep Spin Miniprep kit (QIAGEN) following the manufacturer’s protocol. Gel extractions were performed using 1% agarose gels, and MinElute Gel Extraction Kit (QIAGEN) following the manufacturer’s protocol, unless otherwise specified.SYT1. Amplicons were analysed by agarose gel electrophoresis and extracted.Wild-type promoters were amplified from HeLa genomic DNA by polymerase chain reaction (PCR) using the Kapa HiFi PCR kit (Kapa Biosystem)  using T4 DNA ligase (New England Biolabs) (37 °C for 2–16 h), and transformed into SYT1, only the first ~ 500 bp of L1PA2 was deleted, as this region was previously shown to be critical for the L1 promoter activities [TEs were deleted from mutation-free wild-type promoters via PCR-driven overlap extension  Fig. 4)4), usingtivities    and transformed into E.coli DH5α cells, followed by ampicillin selection. Colonies were screened for inserts in the correct orientation via colony PCR using RVprimer3.Wild-type and TE-deleted promoters were excised from the pCR Blunt vectors by restriction enzymes digestion  per construct were subjected to plasmid extraction. Plasmid DNA of each set of wild-type and TE-deleted constructs, which would be directly compared against each other in luciferase assays, was extracted simultaneously. Plasmid DNA was confirmed for identity and supercoiling by restriction enzyme digestion and gel electrophoresis. Only predominantly supercoiled plasmid DNA was used for transfection.2, and passaged with Trypsin-EDTA (Gibco) at 80–90% confluency. Cell passaging at a 1:6 ratio was performed twice a week for MDA-MB-231 cells, and weekly for the other cell lines.TNBC cells lines used in this study were MDA-MB-468, MDA-MB-231 and BT549. All cell lines were cultured in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific) supplemented with 10% foetal bovine serum (Thermo Fisher Scientific). Cells were incubated at 37 °C and 5% COCells were plated in 24-well plates to give a density of 80–90% at 24 h . At 48 h post-transfection, medium was removed and cells were lysed using 50 μL of luciferase reagent diluted with 50 μL of PBS. After a 10-min shaking incubation, 90 μL of cell lysate from each well was transferred to a White Opaque 96-well microplate (PerkinElmer). Firefly luminescence was measured at 25 °C on a DTX880 Multimode Detector (Beckman Coulter) (MDA-MB-468 and MDA-MB-231 cells), or CLARIOstar (BMG LABTECH) (BT549 cells). Renilla luminescence, averaged amongst the triplicates and normalised to the positive control.The relative luciferase activity of each construct was calculated as the ratio of firefly: P < 0.05 indicated statistical significance.For each gene, luciferase assays were replicated in three independent experiments. Relative luciferase activities were normalised to the mean wild-type activity across the replicates for each gene. Using R, the relative luciferase activities of TE-deleted constructs were compared against the wild-type with a one-tailed t-test assuming equal variances, to test our hypothesis that the deletions would reduce promoter activities. To investigate the epigenetic status of the candidate TEs in TNBC, their methylation states were evaluated using published whole-genome methylation capture sequencing data . This teP < 0.05 indicated statistical significance.As TFBSs were associated with transcriptional activities and thus likely to be epigenetically regulated , reads mTE epigenetic states were also evaluated by analysing ENCODE DNase-seq datasets from MCF7 and HMEC  cell lines  Additional file 2:CAGE clusters and genes mapped to putative TE-derived promoters: contains the list of CAGE clusters and genes overlapping TE-derived promoters (DOCX 54 kb)Additional file 3:TEs with putative promoter activity: contains the list of genomic locations for TEs with putative promoter activity in breast cancer (XLSX 19 kb)Additional file 4:Supplementary Tables: contains supplementary tables referenced in the main manuscript (CSV 2 kb)


IL33 locus are associated with increased susceptibility to asthma. Large-scale genome-wide association studies have identified minor “G” allele of the single-nucleotide polymorphism rs928413, located in the IL33 promoter area, as a susceptible variant for early childhood and atopic asthma development. Here, we demonstrate that the rs928413(G) allele creates a binding site for the cAMP response element-binding protein 1 (CREB1) transcription factor. In a pulmonary epithelial cell line, activation of CREB1, presumably via the p38 mitogen-activated protein kinases (MAPK) cascade, activates the IL33 promoter containing the rs928413(G) allele specifically and in a CREB1-dependent manner. This mechanism may explain the negative effect of the rs928413 minor “G” allele on asthma development.Cytokine interleukin 33 (IL-33) is constitutively expressed by epithelial barrier cells, and promotes the development of humoral immune responses. Along with other proinflammatory mediators released by the epithelium of airways and lungs, it plays an important role in a number of respiratory pathologies. In particular, IL-33 significantly contributes to pathogenesis of allergy and asthma; genetic variations in the  Interleukin 33 (IL-33), a member of the interleukin 1 (IL-1) cytokine family, is expressed by endothelial, epithelial, and fibroblast-like cells in barrier tissues and in the sites of inflammation . This cyNumerous studies have demonstrated the particular importance of IL-33 in respiratory and allergic diseases ,9. IL-33IL33 locus includes several single-nucleotide polymorphisms (SNPs) associated with asthma development in different populations [IL33 gene, and its minor “G” allele was identified as a susceptible variant for early childhood asthma [Large-scale genome-wide association studies demonstrated that ulations ,14,15,16d asthma  developmd asthma . The rs9d asthma , also poIL33 gene. In this paper, we demonstrate that the presence of rs928413(G) allele in the IL33 promoter indeed results in its increased activity in a human lung carcinoma cell line due to binding of the cAMP (cyclic adenosine monophosphate) response element binding protein 1 (CREB1) transcription factor. Our results offer a tentative explanation for the negative effect of rs928413 on asthma development.Based on the available data, we hypothesized that the rs928413(G) allele may be mechanistically linked to asthma susceptibility via transcriptional activation of the IL33 transcription in different cell types is rather complex, and the available data is rather limited [IL33 gene available through UCSC Genome Browser (Available online: http://genome.ucsc.edu) is consistent with the activity of the most distant promoter in a broad range of tissues, with epigenetic features which are characteristic of regulatory elements such as DNase-I hypersensitivity sites, major histone modifications , and the presence of transcription factors (TF) binding sites spanning the 2,5 kb region upstream of the transcription start site (TSS)  A. Singleclopedia . The repIL33 promoter variants containing alternative rs928413 alleles is likely associated with the emergence or disappearance of specific transcription factor binding sites (TFBS). We used the PERFECTOS-APE software  [http://hocomoco11.autosome.ru) and JASPAR databases (Available online: http://jaspar.genereg.net) to generate a list of candidate transcription factors with the best scores, which included CREB1 (cAMP response element-binding protein 1), FOXF2 (forkhead box protein F2), HNF1A (hepatocyte nuclear factor 1 homeobox A), and E4F1 (E4F transcription factor 1). Of these four, three proteins had no apparent connection to the pathogenesis of asthma: Foxf2 has been shown to play a role in embryogenesis [IL33 transcription may provide a mechanistical explanation for this association.The difference in the activity between  Russia)  with TFBogenesis , in partogenesis  and secoogenesis , HNF1A iogenesis  and in gogenesis , and E4Fogenesis ,30. On togenesis . Importaogenesis ,33,34. Hogenesis . We hypoIL33 promoter. We created four variants of the IL33 gene promoter region (−2500/−2300), containing either common “A” or risk “G” rs928413 allele, and either the original or mutated CREB1 site (IL33 promoter containing risk rs928413(G) allele.We used a pull-down assay with nuclear extracts from NCIH-196 cell line to investigate the influence of rs928413 on CREB1 binding to the EB1 site . The proActivation of CREB by phosphorylation at serine 133 is promoted by various type of kinases, including mitogen-activated protein kinases (MAPKs) ,36; p38 CREB1 mRNA in NCIH-196 cells  allele and functional CREB1-binding site. Both the presence of rs928413(A) allele and the CREB1 site mutation made the reporter unresponsive to TNF. Activation was also completely abrogated by the addition of CREB1-specific siRNA. These results indicate that the potentially negative role of the minor rs928413(G) allele in asthma may be mechanistically explained by IL33 promoter stimulation with CREB1 transcription factor, that can be further activated by TNF.In order to determine the role of CREB1 in IL33 in the airway and lung epithelium is supposed to be associated with the maintenance of mucosal inflammatory conditions and the aggravation of pathological changes during asthma.The epithelium of airways and lungs is one of the most important physical barriers between external environment and the human organism. Due to permanent interaction with pathogenic microorganisms and inhaled antigens, lung epithelial cells (ECs) act as mediators of the host’s defense , and oftIL33 gene. We demonstrated that the risk rs928413(G) allele is associated with the presence of the CREB1 binding site and increased IL33 promoter activity, which can be enhanced by TNF, presumably via p38 MAPK cascade. As already noted, increased activation of this pathway was observed in the asthmatic airways; the extent of the activation correlated with the severity of the disease [IL33 promoter created by the rs928413(G) allele is consistent with the observation of more pronounced clinical asthma symptoms in patients carrying both homozygous and heterozygous rs928413(G) variants [In the present work, we characterized a possible functional role of SNP rs928413 located in the distal promoter area of the  disease ,38. Furt disease . Elevate disease ,69 and t disease . In the  disease . CREB1 bvariants .In conclusion, our data suggest that a mechanism that may explain the observed association of rs928413 polymorphism with asthma susceptibility. Elevated production of IL-33 by damaged or stressed cells of the barrier epithelium could promote airway hyper-responsiveness and development of inflammatory response during the initiation and persistence of asthma. The proposed mechanism corroborates the critical role of epithelium in asthmatic pathogenesis reported in an increasing number of studies. In the future, it is important to assess the correlation between CREB1 binding to the rs928413(G) allele with IL-33 production in primary cells, and with disease symptoms in asthmatic patients.IL33 expression, as measured by real-time PCR. Therefore, this treatment regiment was used in further experiments.A NCIH-196 human lung carcinoma cell line was obtained from American Type Culture Collection . Cells were cultured in RPMI-1640 medium  supplemented with 10% fetal bovine serum . Preliminary experiments with various concentrations of recombinant human TNF (tumor necrosis factor)   for 24 and 48 h demonstrated that 100 ng/mL TNF for 24 h yielded the strongest increase in The Scientific Council of the Engelhardt Institute of Molecular Biology declared no ethical approval requirements for experiments performed in this study, because only commercially available cell lines were used.IL33 promoter region containing nucleotides −2512 to +79 bp from the transcription start site (TSS) was amplified by PCR using genomic DNA isolated from NCIH-196 cells and specific oligonucleotide primers containing HindIII/NcoI restriction sites. IL33 promoter variants containing the minor (G) allele of rs928413 and mutations of the CREB1 binding site were amplified by overlap PCR, and verified by Sanger sequencing. Variants of IL33 promoter were cloned into pGL3-basic luciferase reporter vector  using HindIII and NcoI restriction sites. The sequences of the primers are indicated in The human Сells were transfected with 1.8 μg of plasmid DNA and 0.2 μg of pRL-CMV Renilla luciferase control vector using X-treme Gene HP DNA Transfection Reagent  according to manufacturer’s protocol. Firefly luciferase activity was measured as described , and norIL33 promoter (−2500 to −2300 from the TSS) containing rs928413 were amplified by PCR using DNA templates containing combinations of the A and G rs928413 alleles with either intact or mutant CREB1 binding site. An irrelevant DNA fragment that did not include any predicted CREB1-binding sites was used as a negative control. All PCR products were verified by sequencing; sequences of PCR primers are represented in Two hundred bp fragments of the CREB1 mRNA level by RT-PCR was performed as described [CREB1 mRNA level. The sequences of the siRNA and the CREB1-specific PCR primers are represented in Commercially synthesized single-stranded RNAs  were annealed as previously described . NCIH-19escribed . Of threescribed , one medProtocols of total cell lysates preparation, electrophoresis, and transfer to the nitrocellulose membrane were previously described . Membrant-test. Data were represented as mean ± SEM.We used Microsoft Excel for statistical analyses. Statistical significance was determined using two-tailed unpaired Student’s


In this study, Takahashi et al. show that ZNF445/ZFP445 binds imprinting control regions (ICRs) in mice and humans. In mice, ZFP445 and ZFP57 act together, maintaining all but one ICR in vivo, whereas earlier embryonic expression of ZNF445 and its intolerance to loss of function mutations indicate greater importance in the maintenance of human imprints. ZFP57 mutations in humans and mice suggests additional effectors. We reveal that ZNF445/ZFP445, which we trace to the origins of imprinting, binds imprinting control regions (ICRs) in mice and humans. In mice, ZFP445 and ZFP57 act together, maintaining all but one ICR in vivo, whereas earlier embryonic expression of ZNF445 and its intolerance to loss-of-function mutations indicate greater importance in the maintenance of human imprints.Genomic imprinting is an epigenetic process regulated by germline-derived DNA methylation, causing parental origin-specific monoallelic gene expression. Zinc finger protein 57 (ZFP57) is critical for maintenance of this epigenetic memory during post-fertilization reprogramming, yet incomplete penetrance of  In general, they function to recruit repressive epigenetic states to transposable elements in a species-specific manner , but evisferases . ZFP57 isferases , but onlant mice . In humas (DMRs) . Moreoves (DMRs) . We hypoSupplemental Fig. S1A; Supplemental Table S1). We confirmed these data through chromatin immunoprecipitation (ChIP) combined with high-throughput sequencing (ChIP-seq) of hESCs overexpressing either protein , finding ZFP57 associated with 17 and ZNF445 associated with eight germline DMRs . In contrast to most KZFPs, ZNF445 was not significantly associated with transposable elements . Interestingly, the DMRs most affected in ZFP57-mutated human subjects  . The two KZFPs generated overlapping binding profiles at maternal DMRs (such as KvDMR and MEST), whereas, on paternal DMRs, they occupied distinct genomic positions, especially in the case of the DLK1-DIO3 imprinted locus, where ZNF445 was bound to the human germline-inherited DMR IG-DMR, whereas ZFP57 was found at the somatic DMR at the MEG3 promoter , and, accordingly, the recruitment of KAP1 to the DMRs matched that of ZNF445 rather than ZFP57 in these cells  in hESCs, suggesting that the binding of the KZFP responsible for its genomic recruitment is dependent on DNA methylation .We first screened our database of human KZFPs genomic binding sites in HEK293T cells  for thosd GRB10)  were notpromoter A. ZFP57 se cells A. Furthehylation . BindingMus musculus domesticus (C57BL/6J) and M. musculus Castaneus (CAST/Ei) strains , allowing the parental origin of alleles to be determined. We compared our results with ZFP57-binding data from the same reciprocal hybrid cells . About one-third of the Zfp445 zygotic mutants survived to adulthood, a much milder phenotype than observed with zygotic Zfp57 mutations . Furthermore, Zfp445 mutants did not exhibit any loss of methylation imprints at ICRs analyzed in the brain and liver at embryonic day 12.5 (E12.5), including those bound by ZFP445 in mESCs . Consistent with no change in the methylation imprints, expression of imprinted genes was unperturbed in Zfp445 mutants . We conclude that the absence of ZFP445 alone has no impact on imprinting during mouse early development, perhaps due to compensation by high levels of ZFP57 , while zygotic depletion induced total loss of DNA methylation at only three ICRs  and partial loss at another eight ICRs. To determine whether compensation by Zfp445 might explain the persistence of some imprinting in these Zfp57 mutants, we generated Zfp445–Zfp57 double-mutant mice . Homozygous zygotic mutations for both genes caused embryonic lethality and showed no gross morphological abnormalities but significant reduction in size and weight at E11.5 . Correspondingly, Zfp57/Zfp445 zygotic inactivation resulted in more severe loss of imprinting at 15 ICRs than solo Zfp57 zygotic mutation . In addition to the three ICRs that were devoid of methylation in Zfp57 zygotic mutants, 11 more completely lost their imprints in the double mutants . Thus, zygotic ZFP57 and ZFP445 are enough to protect methylation at 14 out of the 19 ICRs analyzed. Interestingly, Peg10 was the only ICR found to be entirely unaffected in embryos homozygous for both mutations.Maternal–zygotic deletion of murine at E11.5 D, a phenIG-DMR, Impact, Grb10, and KvDMR) in the double-zygotic mutants suggested that maternal ZFP57 might attenuate the phenotype of the double-zygotic mutants, and thus we assessed the impact of Zfp445 inactivation in a maternal/zygotic Zfp57 knockout mouse . These Zfp57MZ(−/−)/Zfp445het mutants exhibited a more severe imprinting defect at nine ICRs compared with maternal–zygotic Zfp57 mutants and at two ICRs (IG-DMR and Impact) compared with double-zygotic Zfp57/Zfp445 mutants . Interestingly, only H19, which is one of the most ancient ICRs conserved in marsupials (Zfp57MZ(−/−)/Zfp445het than the double-zygotic mutant, indicating that H19 has a stronger dependency on ZFP445 compared with other ICRs. Finally, we were able to generate a Zfp57MZ(−/−)/Zfp445Z(−/−) embryo despite the high level of embryonic lethality of the required Zfp57Z(−/−)/Zfp445het mutant mother . Indeed, only two surviving female Zfp57Z(−/−)/ Zfp445het mice could be obtained from 134 litters . The first female had five conceptuses at day 11.5 of gestation, with one being the sought-after Zfp57MZ(−/−)/Zfp445Z(−/−) mutant; this appeared to have stopped developing around E10 . The second female had nine conceptuses at day 10.5 gestation, and none of them was a Zfp57MZ(−/−)/Zfp445Z(−/−) mutant . We analyzed methylation of the Zfp57MZ(−/−)/ Zfp445Z(−/−) mutant using both yolk sac and embryo and confirmed that methylation was maintained at Peg10 but was negligible or completely lost at all of the ICRs previously shown to be resistant to zygotic loss of ZFP57 with ZFP455 (Peg10) during mouse embryonic development.Partial loss of methylation at four ICRs  showed binding of both ZNF445 and KAP1 in hESCs  and thus are more likely to remain controlled by the two proteins in this cellular model. We knocked down ZNF445 expression in hESCs by RNAi using two different shRNAs . This resulted in loss of KAP1 binding and H3K9me3 enrichment at ZNF445/KAP1-bound imprinted DMRs  and a drop in DNA methylation and up-regulation at the imprinted genes MEG3 and H19  . Deregulation of MEG3 and H19 imprinted genes and loss of H3K9me3 were fully rescued by overexpressing a shRNA-resistant form of ZNF445 and could be only partially compensated for by forced expression of ZFP57 . Interestingly the IG-DMR, which is the ICR most consistently affected upon ZNF445 knockdown even when ZFP57 is overexpressed , is not perturbed in human patients with ZFP57 mutations, correlating with the presence of only one ZFP57-binding motif in its sequence . These data confirm the functional ability of ZNF445 to (1) bind human ICRs, (2) maintain DNA methylation at the IG-DMR and H19 DMR, (3) recruit KAP1 and trigger deposition of H3K9me3, and (4) regulate expression of a subset of imprinted genes in hESCs. Contrary to the mouse model, removal of ZNF445 alone was sufficient to affect the epigenetic status of ICRs and the expression of imprinted genes.In contrast to mice, human tivation A. This ltivation . We thusZNF445 and ZFP57 in mice and humans, we sought to retrace their evolutionary history. We used a previously described approach based on homologies in the so-called “zinc fingerprint” of KZFPs, predictive of their DNA-binding specificity . Marsupials have been shown to have imprinted expression of a subpopulation of the genes that are imprinted in eutherians, including H19-Igf2 and Peg10 . Intriguingly, Peg10 is a neogene derived from a Ty-3 Gypsy retrotransposon of the Sushi-ichi class  and Zfp57MZ(−/−) mice . The Shet score . It suggests that mutations in ZNF445, albeit rare in the human population, might constitute the basis for severe multilocus imprinting disorders and for unexplained cases of infertility and miscarriage.To further probe a potential role for database . We foundatabase  to have et score , which eZFP57 mutations on imprints argue for a less prominent role of this protein in imprinting maintenance. The expression profile of ZNF445, its intolerance to loss-of-function mutations, and the ability of its product to bind and instate heterochromatin at ICRs strongly suggest that ZNF445 is a major factor in human early embryonic imprinting maintenance. Our evolutionary analysis further suggests that ZNF445 might have been the first KZFP that evolved to control imprinting. In humans, it has retained a central role, while ZFP57, which likely emerged later, became functionally more prominent in rodents. Thus, our study uncovers the parallel evolution of two KZFPs involved in the regulation of an essential mammalian epigenetic process and portrays how different evolutionary lineages have balanced the relative functional impact of these two factors and differentially modulated their transcriptional regulation during early development.In conclusion, our study reveals an important role for the evolutionarily conserved ZNF445/ZFP445 in the regulation of imprinting. In mice, ZFP57 plays the predominant role in imprinting maintenance, while, in its absence, ZFP445 is required for preserving methylation at a subset of ICRs. In humans, the lack of maternal ZFP57 and the mild effects of tm1a(EUCOMM)Hmgu] were obtained from the International Mouse Phenotyping Consortium. ZFP57 mutants . The Zfp57-expressing vector was obtained from previous work .mESCs were cultured in 2i + LIF medium, and the hESC line  was cultured in mTeSR1 medium (Stem Cell Technologies) on hES-qualified Matrigel (BD Biosciences) and in the presence of ROCK inhibitor (Y-27632). Cells deficient for d Dnmt3b  were obtous work . Zfp445 7 cells with Dynabeads (Thermo Fisher) in immunoprecipitation buffer  overnight. Antibodies used were anti-HA.11 (Covance), H3K9me3 , KAP1 , and ZNF445 . ChIP samples were used for SYBR Green qPCR (Applied Biosystems) or library preparation for sequencing. All primers sequences are listed in Supplemental Table S3. Libraries of immunoprecipitated chromatin and total input control from ChIP were generated with paired-end adaptors as described previously , and immunoprecipitations were performed with chromatin from 1 × 10eviously . SequencSupplemental Table S3.RT-qPCR was described previously . All priR version 3.1.2 or Graphpad Prism version 4.0 was used for statistical analyses.ChIP-seq analyses in human cells: For previously published data sets, raw data are available at GSE57989 (KAP1 hESCs) and GSE78099 (KRAB-ZFPs in HEK293Ts). Reads were mapped to human assembly hg19 using Bowtie2 short read aligner  using th6) the number of aligned reads. Visualization of ChIP-seq data was performed with the Washington University Epigenome Browser  with Bowtie2 . Potential PCR duplicates were removed with Picard tools (“MarkDuplicates” function). Peak calling was performed with MACS2 (version 2.1.0)  with the Browser .P-values from Fisher exact test.Enrichment analysis on genomic features was performed with BEDtools software to generate intersection, shuffle tracks, and calculate For RNA sequencing, human and murine early embryonic development data were taken from GSE36552 and GSE45719, respectively, and reanalyzed as described previously .For human genetic analyses, genomic and variant data were obtained from 123,136 unrelated human exomes and 15,496 whole human genomes from gnomAD (release 170228) . Only vahet scores were obtained from a previous study . However, both scores were calculated on the basis of the observed number of protein-truncating variants in the same 60,706 exomes from gnomAD, although Shet also excluded frameshift variants from the statistical model.The pLI scores were obtained from the first published gnomAD data set containing 60,706 exomes, with the absence of additional or novel loss-of-function variants that could influence the original pLI scores confirmed using the latest release with 123,136 exomes. The Sus study . The pLI2H2 zinc finger domains were identified using HMMER 3.1b1. The positions of the specific amino acids within these domains were computationally annotated. Canonical transcripts of each gene and KRAB domains were obtained from the ENSEMBL database. The z-scores for the ZNF domains and DNA fingerprint positions were calculated with the number of SNVs normalized to the genes number of ZNF domains, with x being the normalized number of SNVs within the zinc finger domains of each KZFP. The z-scores were calculated on the basis of the whole-genome sequencing cohort from gnomAD only to maximize the number of KZFPs included and avoid any coverage bias, as some KZFPs contain exons that are badly covered with exome sequencing.The CAll raw and processed data have been submitted to the Gene Expression Omnibus database (accession no. GSE115387).


PCC 6803 lacking all group 2 σ factors. The high RNAP-SigA holoenzyme content directly induces transcription of a particular set of housekeeping genes, including ones encoding transcription and translation machineries. In accordance with upregulated transcripts, ΔsigBCDE contain more RNAPs and ribosomal subunits than the control strain. Extra RNAPs are fully active, and the RNA content of ΔsigBCDE cells is almost tripled compared to that in the control strain. Although ΔsigBCDE cells produce extra rRNAs and ribosomal proteins, functional extra ribosomes are not formed, and translation activity and protein content remained similar in ΔsigBCDE as in the control strain. The arrangement of the RNA polymerase core genes together with the ribosomal protein genes might play a role in the co-regulation of transcription and translation machineries. Sequence logos were constructed to compare promoters of those housekeeping genes that directly react to the RNAP-SigA holoenzyme content and those ones that do not. Cyanobacterial strains with engineered transcription and translation machineries might provide solutions for construction of highly efficient production platforms for biotechnical applications in the future.We show that the formation of the RNAP holoenzyme with the primary σ factor SigA increases in the Δ Recently cyanobacterial research has focused on possibilities to use cyanobacteria as bio-factories to produce valuable compounds. Lack of efficient, easily-controllable promoters has been a bottleneck in the application of cyanobacteria in biotechnology2. Although many E. coli promoters function in cyanobacteria and cyanobacterial promoters in E. coli, none of the commonly used E. coli production systems functions well in cyanobacteria. These experiences suggest that transcriptional regulation differs notably between cyanobacteria and E. coli.Cyanobacteria are eubacteria characterized by oxygenic photosynthesis. They branched early from other eubacteria and are currently found where ever light is available, from oceans to hot springs and fresh water environments to desert crust. These important primary producers are estimated to be responsible for one-third of carbon fixation on Earth3. Only the plastid encoded RNAPs of plants and algae share this arrangement4. As plastids are descendants of cyanobacteria, the splitting of β′ has obviously occurred only once in evolution. The biological reasons and consequences of β′ splitting remain to be solved. The β′ subunit bears a 600 amino acids long cyanobacteria lineage specific insertion5 whose physiological role remains unclear as well. The small ω subunit of RNAP is non-essential in cyanobacteria just like in many other eubacteria6, and it might play a regulatory role in cyanobacteria7.The RNA polymerase (RNAP) has gained specific features in the cyanobacterial lineage. In the vast majority of eubacteria, the RNAP core is composed of two α subunits and a single β, β′ and ω subunit. In the cyanobacterial lineage, the RNAP core has a unique composition with six subunits. The β′ subunit has been split in cyanobacteria, and the N-terminal part is called γ and the C-terminal part retains the name β′8. This competition is affected by numerous factors including the amount of each σ factor, affinity of different σ factors to the RNAP core, amounts and activities of anti-σ factors that prevent recruitment of a particular σ factor, and RNAP modifying factors like the small signaling molecule ppGpp that directly binds to the RNAP core and changes the recruitment efficiency of different σ factors and affect the promoter selectivity of the RNAP holoenzyme10. These processes are not yet well understood in cyanobacteria.For transcription initiation, the RNAP core recruits one of the several σ factors to form a transcription initiation competent RNAP holoenzyme. All σ factors compete for the same RNAP core13. The primary σ factor is assumed to be mainly responsible for transcription of housekeeping genes during growth. In a particular stress condition, the recruitment of one or more of the group 2 σ factors increases. Accordingly cells lacking group 2 σ factors show acclimation defects in high salt15, bright light17 or heat stresses20. In addition to stress responses, the group 2 σ factors control metabolic changes during dark-light transitions24.Cyanobacteria typically encode multiple group 2 σ factors that closely resemble the essential primary σ factor but are non-essential in standard growth conditionsSynechocystis sp. PCC 6803 strain without any functional group 2 σ factors13. The ΔsigBCDE strain is vulnerable to all tested stress conditions but grows well in standard conditions13. However, even in the standard conditions almost 20% of genes are at least two-fold up or down regulated in ΔsigBCDE compared to the control strain13. Interestingly, many genes encoding subunits of the transcriptional or translational machineries are upregulated in the ΔsigBCDE mutant. In the present paper, we further investigated the regulation of transcriptional and translational machineries in cyanobacteria. The results indicate that in the absence of competition between SigA and group 2 σ factors, more RNAP-SigA holoenzyme is formed than in the control strain. Numerous RNAP-SigA holoenzymes enhance the transcription of a particular set of housekeeping genes including those encoding transcription and translation machineries, and ΔsigBCDE produces more RNAPs and ribosomal subunits than the control strain. The extra RNAPs are fully functional, and enhanced transcription leads to a high RNA content per cell whereas extra ribosomal subunits do not form translationally active ribosomes, and ΔsigBCDE cells show similar translation activity and protein content as the control strain. Co-regulation of transcription and translation machineries as well as promoter differences between subgroups of housekeeping genes will be discussed.We have constructed a sigBCDE strain that lacks all functional group 2 σ factors grows well in standard growth conditions13. DNA microarray analysis of the transcriptome of ΔsigBCDE in the standard growth conditions revealed that many genes encoding proteins for transcription and translation machineries were up-regulated in the ΔsigBCDE strain13   Fig. . Similar CS Fig.  althoughsigBCDE than in the control strain while the amount of free SigA protein did not differ between the strains  and the Rpl1  proteins  were only detected in Fraction 19  are also typical. As the amount of translating ribosomes was similar in both strains but many housekeeping genes produce more copies of mRNA in ΔsigBCDE than in CS, the number of ribosomes reading each mRNA molecule must be lower in ΔsigBCDE than in CS.To find reason(s) for the discrepancy between the ribosomal subunit content and translation activity we further analyzed the ribosomes of Δ 19 Fig.  whereas  19 Fig. . AlthougsigBCDE but not in CS. As Fractions 1–11 did not contain full length 16S rRNA, they do not represent functional ribosomal particles. In the lightest fractions, Rps1 might be a free protein while in the other fractions it might form complexes with some other ribosomal proteins and partially degraded 16S rRNA, seen as shorter RNA species. Similarly, the Rpl1 proteins and shorter RNA fragments in Fractions 10–17 detected in the ΔsigBCDE strain might represent functionally incompetent 50S ribosomal particles , secY-adK (TU836), infA (Tu835), rpl36-rpl31 (TU833)26. Our RNA secondary structure analysis revealed possible transcription termination loops downstream of the rpl15 and secY genes. Because combining all these results turns out to be complicated, we next performed a Northern blot analysis.The est Fig. . In addiase Fig. . HoweverrpoA probe. The longest transcripts were circa 16 000 nt and could thus comprise genes from rpl3 to rpl31  were abundant together with 2700 nt, 2200 nt and 1500 nt long transcripts  and rpoB (the β subunit) genes form an operon together with the tatD gene (subunit of the twin-arginine protein export system) and the rps20 gene (a subunit of 30S ribosome), while the rpoC1 gene (the γ subunit) produces a monocistronic transcript . In standard growth conditions the RNAP-SigB holoenzyme content is low13 and thus the regulation of the sigA gene by the SigB factor is important only in special conditions like dark-light transitions. The sigA gene is located just upstream of the rrn operon25 but not co-transcribed with the rrn operon21. Currently three different promoters have been identified for the sigA gene. The sigA gene has suggested to form a transcription unit with a non-coding RNA ncr165027. A typical −10 region is found upstream of ncr1650 but the −35 region is unique . All together, these regulatory mechanisms lower the transcription activity of housekeeping genes in E. coli a lot without large changes in the σ70 content. Unlike in E. coli, the SigA content of cells drops drastically in the stationary phase in Synechocystis, whereas 6S RNA content increases only slightly28, suggesting that the dose of the primary σ factor might play a more direct role in controlling the formation of the RNAP holoenzyme in Synechocystis than in E. coli.In Δ8. In ΔsigBCDE, an elevated dose of SigA factor and a low amount of competitors result in formation of a high number of RNAP-SigA holoenzymes, over expression of many housekeeping genes and accumulation of extra RNAs. However, the growth rate of the ΔsigBCDE is not elevated13, indicating that overall transcriptional enhancement does not induce faster growth and on the other hand that accumulation of extra RNA is not harmful for Synechocystis cells. However, when the growth rate of Synechocystis changes due to changed environmental conditions, the growth rate and formation of RNAP-SigA holoenzymes often correlate. For example, in high CO2 the formation of RNAP-SigA holoenzymes is abundant and cells grow fast7 whereas nitrogen depletion causes cessation of growth and minimal formation of RNAP-SigA holoenzymes32. E. coli cells produce different amounts of RNAP depending on growth medium, and the RNAP level has been shown to be just above the level needed for maximal growth in the particular growth medium33. In E. coli, similarly as in Synechocystis, simultaneous overproduction of RNAP core and the primary σ70 factor enhances transcription of many housekeeping genes but does not enhance growth34.In eubacteria, all σ factors compete for the same RNAP core to form a transcription initiation competent RNAP-holoenzyme, different σ factors having different affinity to the coresigBCDE strain, a high content of RNAP-SigA holoenzyme induces expression of particular housekeeping genes13. We selected a collection of upregulated and non-upregulated housekeeping genes in ΔsigBCDE and compared their promoters. Upregulated Synechocystis promoters often resembled E. coli promoters with an extended −10 region35. The promoters of non-upregulated genes only contain a typical −10 region while the rest of the promoter region varies from gene to gene. Thus promoters recognized by RNAP-SigA holoenzyme in Synechocystis contain a clear −10 region but otherwise those promoters show high variability, suggesting that housekeeping genes are regulated in a complicated manner that requires other factors in addition to the RNAP holoenzyme. Increased availability of RNAP-SigA holoenzymes directly enhanced the transcription efficiency of genes with extended −10 region promoters in Synechocystis, while cyanobacterial-specific promoters of the photosynthetic machinery did not respond to the overdose of RNAP-SigA holoenzyme. Thus, our results suggest that activation of photosynthesis related genes requires more specific regulators than just a high content of RNAP-SigA holoenzyme. Demand for mechanisms strictly regulating expression of photosynthetic genes is obvious, as imbalances in the function of photosynthetic reactions potentially induces production of harmful reactive oxygen species in cyanobacteria36. We have previously shown that in the ΔrpoZ strain, a low RNAP-SigA holoenzyme content lowers especially the transcription of genes encoding the carbon concentrating and carbon fixating machineries while expression of many other housekeeping genes remain at the normal level6. Patterns of gene expression in the ΔsigBCDE and ΔrpoZ strains show that different functional groups of housekeeping genes respond differently to the dose of the RNAP-SigA holoenzyme. This might be a common phenomenon in bacteria, as a recent study showed that the dose sensitivity of the RNAP-σS holoenzyme correlates with the functional category of the σS regulated genes in E. coli37.In the ΔsigBCDE strain produces more rRNAs than CS. In E. coli activity of rRNA operons correlates with growth rate38 and the presence of thirteen highly active rRNA operons in Vibrio natriegens has been use to explain the rapid growth of this bacterium (doubling time of only 10 min)39. High rRNA content but normal growth of ΔsigBCDE shows that growth rate and rRNA content do not directly correlate in Synechocystis.The ΔE. coli40. The rrn operons of E. coli contain two promoters P1 and P2; P2 provides rather constant expression while P1 is highly induced or repressed depending on conditions38. The high transcription efficiency from the P1 promoters has been explained by their unique features that lead to formation of a scrunched open complex, thereby reducing abortive initiation41. The two identical rrn operons of Synechocystis have GTP as an initiator nucleotide and they contain only a single promoter resembling the σ70 consensus promoter with neither an extended −10 region nor a discriminator sequence typical for the P1 promoter  and the amount of the starvation induced ppGpp molecule, set the expression level of the rRNA operons in Synechocystis does contain Nus proteins that regulate Rho-dependent attenuation of the rrn operon in E. coli38, but the Rho factor is missing and thus an E. coli type attenuation mechanism cannot function in cyanobacteria. Nus proteins in Synechocystis might be involved in rRNA processing in the same way as was recently reported in E. coli44. At least rRNAs are efficiently processed in ΔsigBCDE, as only 16S, 23S and 5S rRNAs were detected and according to microarray data13 transcripts of many Nus proteins and rRNA processing enzymes were up-regulated in ΔsigBCDE.45. In accordance with that, the high rRNA content of ΔsigBCDE is accompanied with high ribosomal protein content in the mutant strain. Further studies are required to reveal the actual mechanism regulating ribosome production in Synechocystis.Ribosomal RNA content regulates the production of ribosomal proteins with a feedback mechanism in which ribosomal proteins bind strongly to rRNA and weakly to their own mRNAsigBCDE strain. Interestingly, both the rpoA operon and the rpoB-rpoC2 operon contain ribosomal protein genes in addition to RNAP genes. Actually the long rpoA cluster in Synechocystis comprises three ribosomal protein operons of E. coli -rpoA-rpl17 gene cluster suggests that the rpoA operon might play a central role for simultaneous production of transcriptional and translational machineries. The biogenesis of the RNAP core starts with the formation of the α dimer46 but the actual roles of Rps13, Rps11 and Rpl17 proteins in the formation of functional ribosomes during the complicated assembly of ribosomes (for a review see47) remains to be elucidated.Our results show that the production of both transcription and translation machineries are similarly up-regulated in the ΔsigBCDE, but translation activity and the total protein content of the control and ΔsigBCDE strains are similar. The amount of functional ribosomes in ΔsigBCDE remains similar as in the control strain, pointing to post-translational regulation of the number of active ribosomes. A possibility to engineer a cyanobacterial strain with high transcription and translation capacity without a high growth rate might offer new solutions for the use of cyanobacteria for efficient production of valuable compounds in future.Our findings about cyanobacterial regulation of transcriptional and translational machineries are summarized in Fig. Synechocystis sp. PCC 6803 (CS) and ΔsigBCDE were grown in BG-11 medium buffered with 20 mM Hepes, pH 7.5, in normal air at 32 °C under constant illumination at the photosynthetic photon flux density of 40 µmol m−2 s−1 as described earlier13. The BG-11 agar plates of ΔsigBCDE were supplemented with appropriate antibiotics but for the experiments, liquid cultures were grown without antibiotics.The glucose-tolerant strain of 730, dense cultures were diluted to OD730 = 0.4 before measurements. To determine the cell number of the CS and ΔsigBCDE cultures, the OD730 was set to 0.6 and cells were calculated with a flow cytometer BD LSRRortessa (BD Biosciences).The cell content was detected by measuring OD730 was 1.0) grown in standard conditions was harvested at 4 °C by centrifugation at 4500 g for 6 min. Then total proteins were isolated as described earlier12 and protein concentration was measured with Bio-Rad DC protein assay kit. Then 15 µg of proteins were solubilized and separated with 10% NEXT GEL® SDS-PAGE (Amresco), transferred to Immobilon-P membrane (Millipore) with Trans-Blot® (BioRad) according to the manufacturer’s instructions. The RNAP core proteins and the primary σ factor SigA were detected by Western blotting as described13. Immunoblots were quantified using a FluorChem image analyzer (Alpha Innotech Corp). As a loading control, ATPase β subunit was detected with a specific antibody (Agrisera). Three independent biological replicates were analyzed.To analyze the amounts of the RNAP subunits, 30 mL of culture ). Then 180 µL of BN isolation buffer and the pellet volume of acid-washed 150–212 µm glass beads (Sigma) were added, and cells were broken by vortexing 8 × 1 min at 4 °C. Cell debris and membranes were removed by two centrifugation steps, first at 10 000 g for 5 min and then 18 000 g for 15 min.−1, 2% glycerol, 90 mM sucrose, 2.5 mM BisTris) for 5 min on ice, and separated using 5–12% gradient gels (acrylamide:bis-acrylamide 32:1) with 4% of stacking gel using Mini protean gel system (BioRad) at 4 °C. The anode buffer was 50 mM BisTris pH 7.0 and the cathode buffer 50 mM Tricine, 15 mM BisTris pH 7.0 supplemented with 0.01% Serva G; the voltage was increased after 30 min of run from 75 V to 100 V and again after 30 min to 150 V. Then after 30 min, the cathode buffer was changed to a similar buffer without Serva G and the run was continued at 200 V until the blue color ran out of the gel. The gel was soaked in a transfer buffer  for 30 min and thereafter protein transfer and immunodetection was performed as described above. Two independent biological replicates were analyzed.To separate protein complexes, 60 μg of soluble proteins were solubilized with BN solubilization buffer (Serva G Blue 5 mg mL730 was 1) with the hot phenol method as described earlier48, and any DNA contamination was removed with TURBO DNA-freeTM kit (Ambion). The RNA content was measured with a spectrophotometer at 260 nm (Biodrop). To visualize RNA, a sample of isolated RNA from the same amount of the cells was separated on 1.2% agarose gel and stained with ethidium bromide. The DNA microarray data13 is available in GEO accession GSE69981.Total RNAs was isolated from 1-ml cell culture (OD6. Reverse transcription (RT) was performed for 900 ng RNA using SuperScript III kit (Invitrogen) according to the manufacturer’s instructions. Then 2 µL of RT reaction was used in sequent PCR amplification with Phusion HeatShock II (Thermo Scientific). The PCR primers are listed in Supplemental Table DNA-free RNA was extracted from CS as described earlier730 was 1) was supplemented with rifampicin (500 μg mL−1) and cells were collected by centrifugation after 0, 5 or 15 min of incubation in the standard growth conditions. RNA was isolated with the hot-phenol method48. Samples containing 8 μg of total RNA were denatured with the glyoxal system, separated on 1.2% agarose gel in phosphate buffer and transferred to a Hybond-N membrane48. The rpoA gene was amplified from genomic DNA using specific primers (Supplementary Table 32P-dCTP 10 mCi mL−1 (Perkin Elmer) with the Prime-a-gen labelling system (Promega) according to the manufacturer’s instructions. After prehybridization in 6xSSC, 1xDenhardt’s, 0.1% SDS, 100 μg mL−1 Herring sperm DNAS for 1 h at 67 °C, denatured probe was added and hybridized overnight at 67 °C. Then membrane was washed once with 2xSCC, 0.1% SDS at 60 °C for 15 min and once with 1xSCC, 0.1% SDS at 60 °C for 15 min and autoradiographed.The cell culture  were collected from standard conditions by centrifugation and ribosomes were isolated at 4 °C. The cell pellet was rapidly washed with 20 mM Na-EDTA, 20 mM Tris-HCl pH 8 and then with isolation buffer ). The pellet was resuspended in 2 mL of isolation buffer and the cells were broken using a French press (Constant Systems Ltd) treating the samples twice at 2.7 kPa. Cell debris was removed by centrifugation at 10 000 g for 10 min, and the supernatant was centrifuged again at 30 000 g for 30 min. The supernatant was collected and protein concentration was determined. Two mg of proteins were loaded onto the top of 5% to 45% linear sucrose density gradient in isolation buffer and the samples were centrifuged in TH-641 rotor  at 107000 g for 18 h at 4 °C. After centrifugation, 19 fractions, 620 μL of each, were collected. Three independent biological replicates were analyzed.Cells . Proteins were transfer to the membrane and immunodetected as described above. The Rps1 (AS08309) and Rpl1 (AS111738) antibodies were purchased from Agrisera.Ribosomal proteins were analyzed by western blotting. A 24-μL sample of each fraction was solubilized and proteins were separated on 12% SDS-PAGE gels with 4% stacking gel (acrylamide: bis-acrylamide 37.5:1) using ProteanTotal RNAs were isolated from each fraction. An aliquot of 300 μL of a fraction was mixed with 200 μL of RNA isolation buffer  and treated with one unit of DNaseI (Life Technologies) for 5 min and then extracted once with phenol:chlororoform (1:1) and once with chloroform. RNAs were precipitated at −20 °C overnight after addition of LiCl to a final concentration of 0.725 M and 2.5 volumes of ethanol. RNAs were collected by centrifugation at 18 000 g for 15 min and the RNA pellet was washed once with 70% ethanol and dissolved into 20 μL of water. Fifteen μL samples were separated on 1.2% agarose gels in TAE buffer and stained with ethidium bromide.35S-methionine  was added to 20 mL of cell culture (OD730 was 1) and cells were labelled for 10 or 30 min under the standard growth conditions. Cold L-Met was added to a final concentration 0.4 mg mL−1, samples were rapidly cooled and cells were harvested by centrifugation at 4500 g for 6 min at 4 °C and total proteins were isolated as described15. Proteins (60 μg) were separated by 10% NEXT GEL SDS-PAGE, transferred onto membrane and autoradiographed. Two independent biological replicates were performed.Cells were grown in standard growth conditions. Five μL of radioactive Supplementary informationDataset 1


Schizosaccharomyces pombe, meiotic genes are tightly repressed during vegetative growth. Despite being embedded in heterochromatin these genes are transcribed and believed to be repressed primarily at the level of RNA. However, the mechanism of facultative heterochromatin formation and the interplay with transcription regulation is not understood. We show genome-wide that HDAC-dependent histone deacetylation is a major determinant in transcriptional silencing of facultative heterochromatin domains. Indeed, mutation of class I/II HDACs leads to increased transcription of meiotic genes and accumulation of their mRNAs. Mechanistic dissection of the pho1 gene where, in response to phosphate, transient facultative heterochromatin is established by overlapping lncRNA transcription shows that the Clr3 HDAC contributes to silencing independently of SHREC, but in an lncRNA-dependent manner. We propose that HDACs promote facultative heterochromatin by establishing alternative transcriptional silencing.It is important to accurately regulate the expression of genes involved in development and environmental response. In the fission yeast  The me+-dependent, ‘sirtuins’) histone deacetylase complexes (HDACs). Hypoacetylation of histones often depends on HDAC recruitment via low level non-coding (nc) transcription occurring within these regions. Heterochromatic ncRNAs are converted into dsRNAs through the action of the RNA-directed RNA polymerase complex (RDRC) and subsequently cleaved by the ribonuclease III Dicer (Dcr1) to produce small interfering RNAs (siRNAs) of 21–23 nucleotides (Snf2/Hdac [histone deacetylase]-containing Repressor Complex) that deacetylates H3K14. In addition to Clr3, SHREC is composed of three other factors—Clr1, Clr2 and the ATPase and chromatin-remodelling subunit Mit1  that methylates Lys9 of histone H3 (H3K9me), as well as the class I Clr6 and Hos2), class II (Clr3) and class III . prt is produced in the presence of phosphate from a promoter located upstream of pho1  pho1 transcription is fully activated. Surprisingly though, in contrast to the complete loss of silencing observed upon deletion of the prt non-coding promoter, deletion of Clr4 had a minor effect on pho1 expression and instead affected only induction kinetics  sequences on RNA ) and Zin of pho1 ,15–17. USaccharomyces cerevisiae it has been shown that meiotic genes are repressed at the level of transcription by HDAC activity . This seems to be due to the action of Clr3 as its deletion has the same effect on pho1 levels. Strikingly, Clr3-dependent repression of pho1 is mediated via the lncRNA prt, which is needed for Clr3 recruitment to the loci. Interestingly, loss of Set1 and Set2 methyltransferases also leads to loss of transcriptional silencing, with the Set1 deletion having the most striking effect, suggesting that the role of Set1 in facilitating recruitment of HDACs via non-coding transcription is conserved in budding and fission yeast. In contrast to constitutive heterochromatin  and hybridized at 42 °C overnight. After repeated washes in 2× SSC, 0.1% SDS, blots were exposed on Amersham Hyperfilm MP .Northern blot experiments were essentially performed as described . Gene-spChIP was performed as previously described . ImmunopRNase ChIP was performed as described above with the following exceptions: (i) formaldehyde cross-linking time was reduced from 20 to 5 min (ii) Chromatin was prepared in FA lysis buffer with 0.05% SDS and (iii) cross-linked chromatin was treated with either 7.5 U of RNase A (Thermo Scientific) and 300 U of RNase T1 (Thermo Scientific) or an equivalent volume of RNase storage buffer (50 mM Tris–HCl (pH 7.4) and 50% (v/v) glycerol). After incubation at room temperature for 30 min, immunoprecipitations were performed as usual.Chromatin was prepared as above. After washing and eluting bound material from the beads, protein was removed by incubation with 0.4 mg pronase for 1 h at 42°C, followed by overnight incubation at 65°C. RNA was degraded by incubating samples with 0.02 mg RNase A (Roche) for 1 h at 37°C. DNA was purified using ChIP DNA Clean & Concentrator kit  according to the manufacturer's instructions. A sequencing library was constructed using NEBNext Fast DNA Library Prep Set for Ion Torrent™ Kit . Libraries with different barcodes were pooled together and loaded onto the Ion PI™ Chip v3 using the Ion Chef™ Instrument . Library sequencing was carried out on the Ion Torrent Proton. The resulting sequences were trimmed to remove low quality reads (less than Phred score 20) and reads shorter than 20 nt using Trimmomatic (version 0.36)  was added to 1 μg of total RNA, then rRNAs were depleted using the RiboMinus Eukaryote System v2 (Life Technologies). Strand-specific total RNA-Seq libraries were prepared using the TruSeq Stranded Total RNA Sample Preparation Kit (Illumina). Paired-end sequencing (2 × 50 nt) of the libraries was performed on a HiSeq 2500 sequencer.For identification of CUTs, total RNA was extracted from biological duplicates of WT and S. pombe cells were treated for 2.5 h with 20 μg/ml of the HDAC inhibitor trichostatin A (TSA). After lysis, each S. pombe lysate was mixed with an aliquot of a lysate of Rpb3-Flag S. cerevisiae cells (spike), in a 50:1 ratio.Biological duplicates of Rpb3-Flag Libraries were constructed according to the previously published protocol , with miS. pombe and S. cerevisiae reference genomes using version 2.2.5 of Bowtie, with a tolerance of one mismatch. Bioinformatics analyses used uniquely mapped reads. Tags densities were normalized either on the total number of uniquely mapped reads (IP & Input samples), or the signal for S. cerevisiae ribosomal proteins-coding genes.After removal of the 5′-adapter sequence using cutadapt, reads were uniquely mapped to the S. pombe genome (ASM294v2.28) and the S. cerevisiae genome (sacCer3) separately either with TopHat  (http://broadinstitute.github.io/picard) to keep only reads unique for one of the genomes. All remaining S. cerevisiae reads were counted and used for the normalization of S. pombe samples. In the case of ChIP-Seq, differences in the input was also taken into account when calculating normalization values and H3K14ac was normalized to H3 density as determined by H3 ChIP-Seq. For NET-Seq, reads were trimmed to leave only the 3′ nucleotide corresponding to the nucleotide in the active centre of Pol II at the time of the experiment. For RNA-Seq, only properly aligned pairs were kept and all other reads were removed using samtools   in the c_013035)  in the csamtools . Spearmasamtools . For difsamtools . The anasamtools  using mapackages ,47.S. pombe reference genome using version 2.0.6 of TopHat, with a tolerance of three mismatches and a maximum size for introns of 5 kb. Tags densities were normalized on the ERCC Spike-In signals. Segmentation was performed using the ZINAR algorithm  <0.05 upon differential expression analysis using DESeq . Strikingly, a substantial increase in pho1 mRNA is observed by Northern blot when cells are transiently treated with TSA  due to increased expression of prt  followed by qPCR was performed to determine whether Clr3 is recruited to the locus. This revealed that Clr3 indeed localizes to the gene, particularly at the non-coding region  levels at the R Figure ; similarR Figure . Concomipho1 locus. In order to address this, we generated the double mutant clr3Δclr4Δ. Remarkably, this mutant showed an additive accumulation of pho1 mRNA levels   , Clr3 red Figure . H3K14acd Figure , similarΔ Figure . Moreovet Figure . This inprt ncRNA other than its Mmi1 binding site are involved in pho1 silencing, a series of five different 189 bp deletions within the 5′ proximal region of prt were generated (prt-1Δ (–1197 to –1008), prt-2Δ (–1008 to –819), prt-3Δ (–819 to –630), prt-4Δ (–441 to –252), and prt-5Δ (–252 to –63) (pho1 ATG = 1)) . We next wanted to examine whether inhibition of Clr3 HDAC activity by TSA has any additive effect on pho1 levels in the prt mutants. In prt mutants prt-1Δ, prt-2Δ, prt-4Δ, and prt-5Δ, pho1 levels were identical to the wild-type upon TSA treatment  were noticeably increased in TSA-treated compared to untreated cells  of the ) Figure . We were mapped  or deletion of the Seb1 binding site (prt-bsΔ) . Similar to pho1, tgp1 is repressed in response to inorganic phosphate, via non-coding transcription  than increased (289)  Figure . Northers Figure  upon TSAs Figure ,64. IndeIn order to test whether the observed changes in transcript level are due to changed transcription or post-transcriptional RNA stability, as is the case in mammals , we perfTo determine the direct effect of TSA treatment on gene expression and histone acetylation levels, we performed ChIP-seq experiments to measure histone H3K14ac and histone H3 levels genome-wide. A Spearman correlation matrix was generated from all biological replicates of all strains used in this experiment . SpearmaSPAC2E1P3.05c and ctr4 can be seen upon mutation of Clr6 . Ou. Oupho1,nt rrp6Δ  the densncreased . By clusncreased  we find ncreased . One suc11c loci . Interes11c loci . These tci .The ability of cells to invoke rapid changes in gene expression is critical for the execution of developmental programs, adaptation to environmental stress, and during the fate decision of stem cells . Many melability . Despitelability ,5,16. InHow HDACs control transcriptional repression of only specific regions of the genome is not fully understood. Recent studies have proposed that the pattern of histone methylation at promoters of protein-coding genes could provide the specificity. Histone H3 methylation by Set1 at promoter proximal regions controls gene expression either positively or negatively depending on the H3K4me2/me3 ratio ,24. A chtgp1 and pho1 , at facand Pcr1 . It has and Pcr1 . While oIt has previously been proposed that SHREC promotes H3K9me . The mecThis study significantly advances our understanding of the molecular mechanisms underlying repression of meiotic genes. However, many questions are still left unanswered. It remains to be seen how HDACs are targeted to regions of facultative heterochromatin and why it is that many genes are down-regulated upon loss of HDAC activity. Future studies addressing some of these unresolved questions will provide further insight into the function of HDACs in the regulation of transcription.http://www.ncbi.nlm.nih.gov/geo, accession number GSE104713 (RNA-seq), GSE104712 (NET-seq), GSE108668 (ChIP-seq and RNA-seq).Raw (fastq) and processed sequencing data can be downloaded from the NCBI Gene Expression Omnibus repository (Supplementary DataClick here for additional data file.


AMH was high homology in geese with other species. Several regulatory elements were identified and transcriptional factors were predicted in the AMH promoter sequence. Through a double-luciferase reporter assay, potential regulatory relationship spanning from −637 to −87 bp were identified. In addition, the mRNA expression of AMH gradually decreased during the development of follicles in geese. In the Chinese hamster ovary (CHO) cell line, the luciferase activity significantly increased by co-expression of AMH and GATA-4. However, when the binding sites of GATA-4 to the promoter of AMH were mutated, the luciferase activity significantly decreased. These results indicated that the transcription of AMH was activated by GATA-4 to inhibit the development of follicles in geese.Anti-Müllerian hormone (AMH) plays a vital role in the development of follicles. We found that the cloning nucleotide sequence of AMH gene remains poorly understood. In the present study, both the full-length coding sequence (CDS) and promoter sequence of goose AMH have been cloned. Its CDS consisted of 2013 nucleotides encoding 670 amino acids and the amino acid sequence contained two structural domain: AMH-N and transforming growth factor beta (TGF-β) domain. The obtained promoter sequence spanned from the −2386 bp to its transcription start site (ATG). Core promoter regions and regulatory elements were identified as well as transcription factors were predicted in its promoter sequence. The luciferase activity was the highest spanning from the −331 to −1 bp by constructing deletion promoter reporter vectors. In CHO cells, the luciferase activity significantly increased by co-expression of AMH and GATA binding protein 4 (GATA-4), while that significantly decreased by mutating the binding sites of GATA-4 located in the −778 and −1477 bp. Results from quantitative real-time polymerase chain reaction (qPCR) indicated that levels of AMH mRNA in geese granulosa layers decreased gradually with the increasing follicular diameter. Taken together, it could be concluded that the transcriptional activity of AMH was activated by GATA-4 to inhibit the development of small follicles in goose.Anti-Müllerian hormone (AMH) is recognized as a reliable marker of ovarian reserve. However, the regulatory mechanism of goose  AMH and its receptor across a range of species, accumulating evidence has shown that AMH plays a key role in the control of mammalian and avian gonadal functions, such as spermatogenesis and ovarian follicle development [AMH negatively regulated the recruitment of primordial follicles to avoid their excessive consumption during the reproductive cycle [AMH resulted in a significant increase in the number of all growing follicles compared to the control group [AMH inhibited the growth of follicles by decreasing their sensitivity to follicle-stimulating hormone (FSH) [FSHR) decreased after treatment with AMH [AMH might inhibit the growth of follicles via the FSH/FSHR system in mammals. In avian, the mRNA expression pattern of AMH in GCs during follicle development was similar to that observed in mammals, with higher levels in small follicles [AMH mRNA significantly declined with the increasing follicular diameter [AMH suppressed the FSHR signaling to prevent premature GCs differentiation, however, FSH signaling suppressed AMH expression to initiate GCs differentiation [AMH played a key role during the development of small follicles in avian. The follicles of the avian ovary were arranged in a strict hierarchy, which were generally classified into the pre-hierarchical and hierarchical follicles [AMH in geese ovarian follicles still remain unknown.Anti-Müllerian hormone (AMH) is known as a member of the transforming growth factor-β (TGF-β) superfamily and exerts its biological actions by interacting with its specific type I and II receptors (AMHR I and II) . With thelopment ,3,4,5. Ive cycle ,7. In mool group . It was ne (FSH) ,9,10. Inwith AMH . These rollicles . In hensdiameter . In cultntiation . These rollicles . The uniollicles . HoweverAMH has been cloned in humans and mice [AMH gene expression. For example, steroidogenic factor 1 (SF-1) directly activated the transcription of AMH by binding to its proximal promoter in both embryonic and postnatal gonads [AMH expression. Barbara et al. showed that the SRY-box transcription factor 9 (SOX9), as an interaction partner of SF-1, could be related to the Sertoli cell-specific expression of AMH during embryogenesis [AMH transactivation [AMH regulation via protein-protein interactions during the development of human ovarian follicle [AMH transcription in a number of species, little is known about such mechanisms in geese.Promoter is usually located in the 5′ proximal region of one gene and plays a pivotal role in gene expression. The promoter of and mice ,18. At pl gonads . In GCs l gonads . Besidesogenesis . In moustivation . In addifollicle . AlthougAMH gene, to determine the relative levels of AMH mRNA in GCs during geese follicular development and to reveal the regulatory mechanisms underlying AMH transcription. These results will help broaden our understanding of the role of AMH in the goose ovary as well as its transcriptional mechanism during follicle development.Therefore, the aim of the present study was to amplify the complete coding sequence (CDS) and the 5′-flanking region of goose AMH mRNA and six geese were used for collection of GCs for in vitro culture. Different classified follicles were collected at 7–9 h before oviposition. The granulosa layers from different diameters  and different size ) follicles were separated as the methods of Gilbert and Deng [The healthy female maternal line of Tianfu meat geese (Anser cygnoide) were approved by the waterfowl breeding of Sichuan Agricultural University. The name of the ethics committee is the Institutional Animal Care and Use Committee (IACUC) of Sichuan Agricultural University, the number/ID of the approval is DKY-B20141401.The geese had free access to water and food with natural lighting and temperature conditions. Geese aged 34–36 weeks during a regular period of egg laying were selected. Three geese were used for determining the relative abundance of and Deng ,25. All AMH gene (GenBank accession No. XM_013196337.1) and the goose genomic DNA sequence (GenBank accession No. NW_013185857.1) were obtained from NCBI (https://www.ncbi.nlm.nih.gov/gene/?term=AMH+Anser). The primers used for amplifying both the coding sequence and the 5′-flanking region of AMH were designed by the Primer Premier 5.0. The CDS sequence and 5′-flanking sequence were amplified by segmenting amplification and spliced using the SeqMan Pro. The fragments were overlapping with each other. Primers used for cloning CDS were listed in E. coli DH5α competent cells. Positive clones that contained the expected-size fragments were screened by using colony PCR and were then sequenced by using the Qinke procedure .The predicted sequence of https://blast.ncbi.nlm.nih.gov/Blast.cgi) and 5′-flanking sequence of goose AMH gene was confirmed on genomic DNA of geese (https://www.ncbi.nlm.nih.gov/assembly/GCF_000971095.1/). Homology analyses of nucleotide and amino acid sequences were performed using the DNAMAN software. Multiple alignments of AMH gene amino acid sequence were performed with the ExPASy ProtParam System (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). The structure domains of amino acid sequence were analyzed using the Conserved Domains Database . A phylogenetic tree was calculated using the MEGA 7.0 software by neighbor-joining method and bootstrap replicates with 1000 times. The GenBank accession numbers of AMH sequences among various species used for sequence characterization, homology and phylogenetic analysis were listed in Accuracy of cloning sequences was detected by the BlASTn program (http://www.fruitfly.org/seq_tools/promoter.html). The Promoter Typical Components were Analyzed by the Plant Care (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The Crucial Transcription Factor Binding Sites were Predicted by the TF SEARCH (http://diyhpl.us/~bryan/irc/protocol-online/protocol cache/TFSEARCH.html). Possible CpG Islands were Searched for Using DataBase of CpG Islands & Analytical Tools (http://dbcat.cgm.ntu.edu.tw/).The Putative Transcription Start Sites of the Goose AMH Gene were Predicted Using Neural Network Promoter Prediction (AMH mRNA were measured in granulosa layers of different sized follicles by quantitative polymerase chain reaction (q-PCR). Q-PCR reactions were performed in a CFX96TM Real-Time System  using SYBR PrimerScriptTM RT-PCR kit . The procedures included 95 ℃ for 30 s, followed by 40 cycles of 95 °C for 5 s imposing the annealing temperature for 30 s and at 72 °C for 10 min. An 80-cycle melting curve was performed, starting at a temperature of 65 °C and increasing by 0.5 °C every 10 s to determine primer specificity. Each sample was in triplicate and the expression profile of AMH mRNA was evaluated by 2−ΔΔCt method [GAPDH and β-actin. Primers designed for qPCR were listed in The expression profiles of t method  by normaAMH promoter reporter vectors were constructed by PCR cloning. Seven specific primers containing Kpn I and Xho I restriction enzyme cleavage sites were designed to amplify the desired promoter fragments of the geese AMH gene and the PCR products were cloned into a Kpn I- and Xho I-digested pGL4.10-Basic vector . The primers including restriction enzyme sites were listed in The goose AMH promoter were generated using the Fast Site-Directed Mutagenesis Kit . Mutagenic primers were listed in AMH could not be bound. These mutation vectors were named pGL4.10-AMH2-GATA4-778, 1399, 1477 , respectively. All the plasmids were confirmed by DNA sequencing.Mutation vectors of three binding sites for GATA4  of the geese 2-Humidified Atmosphere to Reach 50–60% Confluence for Transfection. CHO Cells were Seeded into 96-well Plates at a Density of 1 × 104/Well.Chinese Hamster Ovary (CHO) Cells Line was Obtained from Kunming Cell Bank, Chinese Academy of Sciences and was Incubated for Dual-Luciferase Reporter Assay. The Culture Medium Included High Glucose Dulbecco’s Modified Eagle’s Medium , 10% Fetal Bovine Serum  and 1% Streptomycin and Penicillin Mixture . Cells were Incubated at 37 °C in 5% CO® 3000 Transfection Reagent  according to the manufacture’s protocol. For dual-luciferase reporter assays, CHO cells were transfected with 100 ng/well of reporter gene plasmids (PGL4.10-AMH), pRL-TK renilla luciferase plasmids  and expression plasmids  or pAc5.1-GFP plasmids (as control) in 96-well plate. After transfection for 24 h, the cells were collected. Luciferase activities were measured using the Dual-Luciferase® Reporter Assay System  and promoter activity was calculated as firefly-luciferase activity/sea pansy-luciferase activity. All transfections were performed in triplicate.Subsequently, plasmids were transfected into CHO cells using Lipofectaminep < 0.05 was considered statistically significant.All data were presented as the mean ± SD. Data were analyzed using the GraphPad Prism 5.0 Software . The normal distribution and homogeneity of variances of all data were tested with a Shapiro-Wilk test  and LeveAMH was obtained (AMH (GenBank accession No. MK061535) contained 2013 nucleotides encoding 670 amino acids. Homology analysis of goose AMH showed that there was a high sequence homology with other avian species such as duck (95.0%) and chicken (85.1%)  at the N-terminus and transforming growth factor-β (TGF-β)-like domain (TGFB) at the C-terminus  . The conterminus B. HomoloB domain . In addiB domain , which iAMH was obtained , AP-1 binding site (AP-1), AP-2 binding site (AP-2), SP1, forkhead box M1 (FOXM1) and GATA-4  of the goose obtained . Analysiectively . Among tectively . FurtherAMH gene A. Severad GATA-4 B. In addpromoter C. The leAMH mRNA in granulosa layers gradually decreased during the development of follicles. The highest mRNA level of AMH was detected in the 4–8 mm follicles (p < 0.05). Besides, the mRNA levels of AMH in the 4–10 mm follicular granulosa layers were significantly higher than that in the F5–F1 follicles (p < 0.05) and there was no evident difference in the F5–F1 follicular granulosa layers (p > 0.05).As depicted in AMH gene, seven deletion promoter reporter vectors (pGL4.10-AMH 1 ~ 7) were constructed successfully . However, further deletion of −331 to −1 bp significantly increased the promoter activity (p < 0.05), while deletion of −87 to −1 bp significantly decreased the promoter activity (p < 0.05), suggesting that there was a negative regulatory element in the region within −637 to −331 bp and a positive regulatory element in the region within −331 to −87 bp.To characterize the promoter activities of the 5′-flanking sequence and identify the putative essential regulatory elements for transcription of the goose essfully  and the AMH gene. Three putative binding sites of GATA-4 were predicted at −778 bp, −1399 bp and −1477 bp, respectively  (p < 0.05), which excluded the possibility of endogenous expression of GATA-4 in CHO cells. To analyze whether GATA-4 could regulate the transcription of AMH, a reporter gene assay was developed in CHO cells by overexpressing GATA-4. Results showed that the relative luciferase activity significantly increased in co-expression of AMH and GATA-4 (p < 0.05). When the putative binding sites of GATA-4 were mutated in the 5′-flanking sequence of AMH, the relative luciferase activity significantly decreased in both −778 and −1477 bp binding sites (p < 0.05). The luciferase activity was the lowest in −1477 bp binding site (p < 0.05), indicating the importance of the −1477 bp binding site of GATA-4 for the transcription of AMH promoter.As depicted in ectively . After tectively  and greeectively . The expAMH is an important factor regulating sex differentiation and is also produced by GCs from the ovaries [AMH gene has been cloned in various species such as mouse, rat, pig and chicken [AMH gene had a higher homology with that of chicken and duck and the phylogenetic analysis indicated that goose AMH was clustered with chicken and duck into the same clade. These data suggested that AMH gene might be highly conserved among goose, chicken and duck. Further analysis of the amino acid sequence showed that goose AMH contained an AMH-N domain at the N terminus and a TGF-β domain at the C terminus. The TGF-β domain shared a high homology by alignment with the counterparts of other species, which was consistent with the result of a previous study where Hu et al. showed that a high homology of the AMH amino acid at the C terminus was due to the conservatism of the TGF-β family among different species [AMH amino acid was longer and diverged from the counterpart in mammals, the divergence resulted in low homology [AMH might be similar to other species and the TGF-β domain might be a crucial region for its functions. ovaries ,29,30. T chicken ,32,33,34 species . While thomology . TherefoAMH decreased gradually with the increasing follicular diameter, which was in line with the previous studies in chicken [AMH was related to the growth of small follicles [AMH inhibited the activation of primordial follicles [AMH in the selection and growth of small follicles. It was also found that the expression level of AMH in GCs of the 4–10 mm follicles was higher than that in those of the F5–F1 follicles, suggesting that AMH might play a negative role in geese follicle recruitment and selection. Recently, AMH has been suggested to be a prognostic biomarker for small yellow follicles (SYFs) and identified as a reliable marker of ovarian reserve [AMH during the early development of follicles.We also found that the relative expression of goose  chicken ,36. In collicles . Severalollicles , attenuaollicles  and inhiollicles , suggest reserve ,42, indiAMH, its 5′-flanking sequence was cloned in the present study. Subsequent analysis predicted several core promoter elements in its 5′-flanking sequence as well as potential transcription factors. The promoters of rodent and human AMH shared common transcription factors that were tightly clustered in their 5′ proximal sequences, including the SOX9, NR5A1 and GATA-4 . Resollicles , which wollicles . The rolles [AMH ,48,49. Ocription  but GATApromoter . Studiesand SF-1 , GATAs aAMH gene was obtained, which encoded for 670 amino acids consisting of an AMH-N domain at the N terminus and a TGF-β family at the C terminus. The promoter sequence of AMH contained five core transcription regions and there were essential regulatory elements spanning from the −637 to −87 bp. The relative expression of AMH in granulosa layers gradually decreased during the development of follicles. The luciferase activity significantly increased in co-expression of AMH and GATA-4. However, when the binding sites of GATA-4 at the −778 bp and −1477 bp were mutated respectively, the luciferase activities significantly decreased. These results suggested that the transcriptional activity of AMH was activated by GATA-4 to inhibit the development of small follicles in goose.In conclusion, the intact CDS of goose