Given that lncRNAs (long noncoding RNAs) are transcribed from the fbp1 upstream region and are important for fbp1 gene expression, we hypothesized that local changes in DNA topological state caused by topoisomerase activity could alter lncRNA and fbp1 transcription. In support of this, we found that topoisomerase overexpression caused destabilization of positioned nucleosomes within the fbp1 promoter region, which was accompanied by aberrant fbp1 transcription. Similarly, the direct recruitment of topoisomerase, but not a catalytically inactive form, to the promoter region of fbp1 caused local changes in nucleosome positioning that was also accompanied by altered fbp1 transcription. These data indicate that changes in DNA topological state induced by topoisomerase activity could lead to altered fbp1 transcription through modulating nucleosome positioning.Chromatin structure, including nucleosome positioning, has a fundamental role in transcriptional regulation through influencing protein-DNA interactions. DNA topology is known to influence chromatin structure, and in doing so, can also alter transcription. However, detailed mechanism(s) linking transcriptional regulation events to chromatin structure that is regulated by changes in DNA topology remain to be well defined. Here we demonstrate that nucleosome positioning and transcriptional output from the fission yeast  DNA topology, defined by the degree of interwinding of two complementary strands, is an important factor to consider in various DNA-related biological processes, such as DNA repair, replication, and transcription . PositivIn eukaryotic cells, DNA is packed in chromatin, which composes of an array of histone–DNA complexes called nucleosomes. The position of nucleosomes plays critical roles in the regulation of gene expression and most DNA-related biological processes through limiting the accessibility of DNA binding proteins to DNA , 11. Forin vitro reconstitution assay [via the topological state of DNA has not been well defined.Nucleosome formation is further impacted by the DNA topological state, as evidenced by the fact that the efficiency of nucleosome formation on topologically constrained DNA is lower than that on relaxed DNA in an on assay . Moreoveon assay , 17. Topon assay , highligfbp1 gene, encoding fructose-1,6-bisphosphatase, is one of many genes upregulated during glucose starvation stress, which is known to involve regulation of chromatin structure [fbp1 gene promoter region, a lncRNA (long noncoding RNA) referred to as mlonRNA (metabolic stress-induced long noncoding RNA) is also weakly transcribed from a region upstream of the two TF binding sites in the presence of glucose (referred to as mlonRNA-a). Upon glucose starvation, the transcription initiation site shifts towards the TF binding sites, which is preceded by two distinct lncRNA species (mlonRNAs-b and -c) that are transcribed prior to the strong induction of fbp1 mRNA [fbp1 upstream region converts to an open configuration, with eviction of nucleosomes positioned at UAS1 and UAS2, and TF binding occurs [The fission yeast tructure –22. In gtructure . In the bp1 mRNA –26. Coupg occurs , 27, 28.fbp1 upstream region impact chromatin state and transcriptional output. In this study, we have used topoisomerase overexpression and the direct recruitment of topoisomerase to the fbp1 gene to begin investigating these potential links. We find that topoisomerase activity impacts the maintenance of positioned nucleosomes upstream of fbp1, which is important for proper transcriptional control. These findings indicate an importance of DNA supercoiling in the maintenance of positioned nucleosomes that may alter gene expression regulation, including gene loci regulated by lncRNAs.lncRNA-coupled chromatin modulation raises the possibility that DNA negative supercoils caused by lncRNA-transcription at the Schizosaccharomyces pombe strains used in this study are listed in nmt1 promoter, respectively as described previously [nmt1 promoter were pre-cultured in SD medium. After cells had been washed twice with sterile water, they were transferred to MM containing 6% glucose and cultured for 16 h (glucose-rich conditions). Then, cells were transferred to MM containing 0.1% glucose and 3% glycerol (glucose starvation condition). Cells without the expression vector were cultured as described previously [The eviously . This previously .The primer sequences used in this study are listed in top1 and reported top2 coding regions were cloned in the pREP1 vector [top1 gene, a 0.4-kb 5′-fragment flanked with a SalI site and a 2-kb 3′-fragment flanked with a SalI site were amplified from cDNA and genomic DNA, respectively. The primer sets p1/p2 and p3/p4 were used for amplification of the 5′ and 3′ fragments, respectively. The full-length top1 sequence was generated by PCR using these fragments as templates and primers p1 and p4, and was then cloned into the SalI site of the pREP1 vector. For the cloning of the top2 gene, the top2 sequence flanked with SalI sites was amplified from genomic DNA using primers p5 and p6 and cloned into the SalI site of the pREP1 vector.Annotated 1 vector . For theGAL4-bd gene encoding N-terminus Gal4 DNA binding domain of S. cerevisiae (1–93 amino acid) at NotI-BglII restriction site in the pRGT1 vector [nmt1 promoter, which strongly induces expression in the absence of thiamine and weakly (~1% of w/o thiamine condition) express in the presence of thiamine [NotI site, generating pREP1-Gal4-BD vector. For construction of pREP1-top2-Gal4-BD vector, top2 sequence flanked with SalI sites was amplified from genomic DNA using primer set p5/p91 and cloned into the SalI site in pREP1-Gal4-BD vector. For construction of pREP1-top2Y781F-Gal4-BD vector, top2 gene was amplified using mutated primer sets p5/p93 and p92/p91. The resultant PCR products were purified using QIAquick gel extraction kit (Qiagen), then these products were combined by PCR using primer p5/p91 and cloned into SalI site in pREP1-Gal4-BD vector. The adjacent UAS2 sequence (-594 to -540) was replaced by the 3xGal4 binding.For the expression of the Gal4-BD fusion protein, we replaced the GFP tag gene with the 1 vector , which i1 vector . This vethiamine . Ser-Glyprp3 and Southern blotting for prp3 were amplified by PCR using the primer sets p9/p10 and p11/p12, respectively.Northern blot and chromatin analyses by MNase partial digestion were performed as described previously . The procam1 promoter region, respectively. The cam1 promoter region was analyzed for normalization.ChIP was performed as described previously . For the7/mL and 50 mL of cell culture was crosslinked with 1% formaldehyde for 20 min at room temperature. The reaction was terminated by the addition of glycine to 0.125 M. After centrifugation at 3,500 rpm and 4°C for 1 min, the cells were washed in 1 mL of water, resuspended in 8 mL of pre-incubation solution  and incubated at 30°C for 10 min. After centrifugation at 3,500 rpm and 4°C for 5 min, the cells were resuspended with 4 mL of Sorbitol/Tris buffer  containing 2 mg/mL Zymolyase 100T and 10 mM 2-mercaptoethanol  and incubated at 30°C for 30 min. Then, the cells were washed in 16 mL of cold Sorbitol/Tris buffer without Zymolyase and 2-mercaptoethanol. After washing, the cells were resuspended in 1.5 mL of cold NP-buffer  containing 1 mM 2-mercaptoethanol and protease inhibitor cocktail (Roche). The 500-μL aliquots were treated with 100 U/mL MNase for 30 min at 30°C. In this condition, approximately 70% of whole genome DNA was digested as mono-nucleosomal DNA . Quantification of the expression level of top1 and top2 was carried out by qPCR using primer sets: p94/p95 for top1, p96/p97 for top2 and p98/p99 for 18S-rRNA as internal control.Reverse transcription PCR (RT-PCR) was carried out using SuperScriptnmt1 promoter , which can be conditionally induced by the depletion of thiamine from the medium [top1 (~4 fold) or top2 (~6 fold) mRNA as compared to control cells in the presence of thiamine (repressive condition), which increased in levels to ~100 fold upon thiamine removal . Top1 and Top2 resolve both positive and negative supercoils and have overlapping roles in fission yeast . To overe medium . Using q removal . Cell prnditions , but uponditions . These rfbp1 transcriptional activation profile in Top1 or Top2 overexpressing cells. Given that topoisomerase overexpression strongly inhibits growth, experiments were performed 16 h after Top1 or Top2 induction, which was the earliest timepoint where cultures showed growth differences as compared to control strains  partial digestion of chromatin DNA followed by indirect end-labeling to map nucleosome-free MNase-hypersensitive sites. In control cells under repressive conditions (e.g. time 0 min), chromatin at UAS1 was protected from MNase digestion, while multiple sensitive sites were present around UAS1  to induce fbp1, two new peaks were detected (referred to here as −1 and −2), while peak +7 remained  to directly tether Top2 to a region upstream of UAS2 in fbp1 fbp1 locufbp1 upstream region were unaffected (p<0.01). This small change is in line with our expectations of this assay, as the topoisomerase is expressed at low levels, it may not always be bound to the promoter (e.g. displaced by RNA polymerase II), and would need to continually resolve supercoils generated by ongoing mlonRNA transcription. Importantly, cells carrying the Top2-Gal4-BD fusions exhibited no obvious changes in nucleosomes positioning at the prp3 locus , this strain exhibited six peaks by mono-nucleosome profiling, in which peaks +2 and +3 in  were fusaffected . This loaffected , p<0.01.p3 locus . During -Gal4-BD , but it p2-Y781F . Taken tfbp1 gene promoter region and are involved in regulating chromatin remodeling in the fbp1 upstream region. The production of mlonRNAs is required for the efficient induction of fbp1 [fbp1 upstream region that modulate chromatin structure at this locus. In this study, we begin to address the possible role of DNA supercoils in the regulation of fbp1 by assaying the impact of topoisomerase activity on fbp1 transcriptional control. We found that over expression of either Top1 or Top2 altered positioned nucleosome in the fbp1 upstream region in either the repressed or active state  in fission yeast. Top1 and Top2 localize at intergenic regions in a similar pattern and have roles in the regulation of transcription through facilitating nucleosome disassembly in fission yeast [top3- cells was completely rescued by the loss of Rqh1 [The link between topoisomerase activity and altered nucleosome positioning in on yeast . Througham1 gene . It is pon yeast , suggeston yeast . Moreoveon yeast , 46, as  of Rqh1 . As suchfbp1 in fission yeast, it is known that all transcription from this region is immediately repressed when cells are returned from glucose starvation to glucose-rich conditions [fbp1 upstream region, leading to the destabilization and rephasing of nucleosomes to reconstitute a repressive chromatin state. In other words, rapid transcriptional shutoff could directly serve as a signal for the reconstitution to repressive chromatin via the alterations this causes at the level of DNA topology. More broadly, recent studies have revealed that negative supercoils accumulate around TSSs and that the level of DNA supercoils correlates with transcriptional activity in human and fly cells [top1Δ/top2ts double mutants, with loss of PHO5 transcription in this mutant attributed to a nucleosome-remodeling defect [Stress induced transcription programs must also be effectively repressed once cells adapt to a new environmental condition or the stress is removed. In the case of nditions . One outly cells , 9. Sincly cells . Similarg defect . These rin vivo that is linked to pervasive transcription and gene expression regulation. We expect that further studies to understand how topoisomerase activity is linked to DNA topology in gene promoter regions, and how this influences lncRNA-transcription, will provide important insights into pervasive transcription and associated biological processes.In noncoding regions, such as intergenic and promoter regions, pervasive transcription is commonly observed and in some instances a role of pervasive transcriptions in the regulation of chromosome functions has been highlighted –52. HoweS1 Fig(A) Representative image showing a DNA sample partially digested with MNase. Partial digestion of nucleosomal DNA with MNase was performed as indicated in Materials and Methods. The sample was run on 1.5% agarose gel at 100 V. (B) Intensities of each band corresponding to mono-, di- and tri-nucleosome were quantified using ImageJ (Plot Profile command). (C) Histogram shows the quantification of intensities of each band corresponding to mono-, di- and tri-nucleosome. Error bars show the standard deviation from three independent experiments.(PDF)Click here for additional data file.S2 Figtop1 and top2 in cells carrying pREP1-top1 or pREP1-top2 in indicated condition was examined by RT-PCR. Level of 18S-rRNA was measured as internal control and used for normalization. Error bars indicate standard deviation in three biological replicates.mRNA expression level of (PDF)Click here for additional data file.S3 Figfbp1 transcript levels in (A) top1-overexpressing cells and (B) top2-overexpressing cells as compared to control. Images presented are of a longer exposure of the same northern blot images from Representative image of a northern blot showing (PDF)Click here for additional data file.S4 Fig(A) Representative image showing a nucleosomal DNA sample partially digested with MNase. Partial digestion of nucleosomal DNA with MNase was performed as indicated in Materials and Methods. The sample was run on 1% agarose gel at 100 V.(PDF)Click here for additional data file.S5 Figcam1 locus was analyzed by a MNase partial digestion assay in glucose-rich conditions in wild-type and topoisomerase overexpressing cells. The MNase-digested DNA samples used in Chromatin state at the (PDF)Click here for additional data file.S6 Figtop2-Gal4-BD, and pREP1-top2-Y781F-Gal4-BD carrying cells were streaked to thiamine-containing SD medium or thiamine-free MM and incubated for 3 days at 30°C.Single colonies of pREP1, pREP1-(PDF)Click here for additional data file.S7 Figfbp1 locus were analyzed as in prp3 promoter region. Error bars indicate standard deviation in at least two biological replicates.Mono-nucleosome positionings in the cells tethering Top2-Gal4-BD or Top2-Y781F-Gal4-BD to (PDF)Click here for additional data file.S8 Figfbp1, prp3 and cam1 upstream region was presented. Total genome DNA (WCE) was analyzed as a control.Chromatin immunoprecipitation (ChIP)-seq data of Top2 in the previous study  was anal(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


I am Kazuko Okamoto working as a researcher in RIKEN-Hiroshima University Collaboration Research Facility, which was opened in 2018. In the past 4 years, I have been working for single molecular imaging of transcription factors in living nuclei in order to understand the relationships between chromatin architecture and molecular dynamics of transcription factors. I have been fascinated by microscopy technology developing in biophysics field, including single molecular imaging; therefore, I started to study about the dynamics of transcription factors using such microscopes. Then, I joined The Biophysical Society of Japan (BSJ) 3 years ago, and they gave me a chance to write this commentary here as a young BSJ member.I studied developmental biology in the early days of my scientific career; thus, I was estranged in the techniques and studies in the biophysics field. I have never thought that it is now possible to observe nucleoproteins including transcription factors at single molecular level and obtain spatio-temporal information inside living nuclei in the past.The first trigger to get curious about the biological meaning of the relationship between chromatin architecture and transcription regulation was a CCCTC-binding factor (CTCF) protein which plays a role in the regulation of 3D architecture of chromatin and facilitates transcriptional insulation activity (Arzate-Mejía et al. More than 10 years have passed since then; sequencing technology has greatly developed to detect the 3D chromatin architectures (Ohno et al. My question is very simple. Chromatin architectures control the transcription process by the binding of core transcriptional proteins, and then cellular states change. However, it is poorly understood how the chromatin architecture controls the binding of core transcriptional proteins. Now, I am enthusiastically involved in the observation of single molecular behavior of transcriptional proteins Fig. .Fig. 1ExIn my case, advanced microscopic technology encouraged me to join biophysics field, and I am trying to understand the mechanisms of the interplay between chromatin architecture and transcription regulation again. The studies in transcription process will gain more attention in biophysical studies in Japan, and I am willing to contribute to the understanding of the transcription machinery by observing molecular behaviors of transcriptional proteins one by one. Finally, I would like to thank the BSJ for giving me the Early Career Presentation Award at the 57th Annual Meeting of BSJ in Miyazaki.


Stx1a) gene, which is involved in synaptic transmission and neurodevelopmental disorders, is a TATA-less gene with several transcription start sites. It is activated by the binding of Sp1 and acetylated histone H3 to the −204 to +2 core promoter region (CPR) in neuronal cell/tissue. Furthermore, it is depressed by the association of class 1 histone deacetylases (HDACs) to Stx1a–CPR in non-neuronal cell/tissue. To further clarify the factors characterizing Stx1a gene silencing in non-neuronal cell/tissue not expressing Stx1a, we attempted to identify the promoter region forming DNA–protein complex only in non-neuronal cells. Electrophoresis mobility shift assays (EMSA) demonstrated that the −183 to −137 OL2 promoter region forms DNA–protein complex only in non-neuronal fetal rat skin keratinocyte (FRSK) cells which do not express Stx1a. Furthermore, the Yin-Yang 1 (YY1) transcription factor binds to the −183 to −137 promoter region of Stx1a in FRSK cells, as shown by competitive EMSA and supershift assay. Chromatin immunoprecipitation assay revealed that YY1 in vivo associates to Stx1a–CPR in cell/tissue not expressing Stx1a and that trichostatin A treatment in FRSK cells decreases the high-level association of YY1 to Stx1a-CPR in default. Reporter assay indicated that YY1 negatively regulates Stx1a transcription. Finally, mass spectrometry analysis showed that gene silencing factors, including HDAC1, associate onto the −183 to −137 promoter region together with YY1. The current study is the first to report that Stx1a transcription is negatively regulated in a cell/tissue-specific manner by YY1 transcription factor, which binds to the −183 to −137 promoter region together with gene silencing factors, including HDAC.The HPC-1/syntaxin 1A ( Stx1a) was first discovered as a gene exhibiting neuron-specific expression and is a key acceptor molecule of a complex called soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE), which is involved in presynaptic vesicle exocytosis in the central nervous system (CNS) [Stx1a gene is a TATA-less gene with multiple transcription start sites and is activated by the binding of Sp1 and acetylated histone H3 (AcH3) to the −204 to +2 core promoter region (CPR) in neuronal cell and tissue [Stx1a–CPR in non-neuronal cell and tissue [Stx1a gene silencing is unclear.HPC-1/syntaxin 1A (em (CNS) ,3,4. Theem (CNS) ,6,7. We d tissue . We alsod tissue ,9. HowevStx1a gene silencing in non-neuronal cell and tissue, in the present study, we first attempted to identify the promoter region forming DNA–protein complex only in non-neuronal cells by electrophoresis mobility shift assays (EMSA). EMSA demonstrated that DNA–protein complex is formed on the −183 to −137 OL2 region of Stx1a–CPR only in non-neuronal cells such as fetal rat skin keratinocyte (FRSK) and fibroblast 3Y1 not expressing Stx1a. Competitive EMSA and supershift assay also revealed that the DNA–protein complex formed on the −183 to −137 region consists of the Yin-Yang 1 (YY1) transcription factor in FRSK cells. Furthermore, the association of YY1 to the Stx1a–CPR in vivo was examined by chromatin immunoprecipitation (ChIP) assay. Results showed that YY1 associates to Stx1a–CPR in cell/tissue not expressing Stx1a and that trichostatin A (TSA) treatment in FRSK cells decreases the high-level association of YY1 to the Stx1a–CPR in default. YY1 belongs to the GLI-Kruppel class of zinc finger transcription factors and is a ubiquitously distributed transcription factor that positively or negatively regulates a diverse number of gene promoters. YY1 is implicated in histone modification via interaction with various proteins, including histone acetyltransferases and HDACs, to activate or repress gene promoters [Stx1a transcription activity by conducting a luciferase reporter assay. Results showed that YY1 negatively regulates Stx1a transcription. Finally, we identified proteins that bind to the −183 to −137 promoter region by mass spectrometry analysis. Results suggest that gene silencing factors, including HDAC1, associate to the −183 to −137 promoter region together with YY1. In the present study, we demonstrated that Stx1a gene transcription is negatively regulated in a cell-/tissue-specific manner by the YY1 transcription factor and that YY1 binds to the −183 to −137 promoter region together with factors participating in gene silencing, such as HDACs.To further clarify the factors characterizing romoters ,11,12,13Drosophila melanogaster SL2 cell line was purchased from ATCC . Cells were maintained in RPMI medium or Schneider’s drosophila medium supplemented with 10% (v/v) fetal calf serum (FCS), streptomycin and penicillin.Cell lines of fetal rat skin keratinocyte (FRSK), rat fibroblast 3Y1, rat adrenal pheochromocytoma PC12 and human neuroblast IMR32 were purchased from HSRRB . 2, 0.1 mM EDTA, 0.5 mM DTT, 2 mM spermidine, 0.1 mg/mL bovine serum albumin and 10% glycerol). For competition assays, 0–125 pmol of cold oligonucleotides was added to the reaction mixtures and incubated before probe addition. For supershift assay, polyclonal antibodies to SP1 , SP3 and SP4  and monoclonal antibodies to YY1 (H10) and NFκB (F-6) (Santa Cruz) were used. Antibody (4 µg) was incubated with nuclear extract in EMSA buffer at 4 °C for 16 h before the addition of the OL2 probe. EMSA reaction mixtures were loaded and separated on a native 3–5% polyacrylamide gels containing 0.5× Tris borate–EDTA buffer. Dried gels were exposed to BASS-2000 imaging plate (IP) film . Preparation of nuclear extracts, EMSA and supershift assays were performed as previously described . The seq3 and 1% sodium dodecyl sulfate (SDS)). The supernatant from the reaction lacking a primary antibody was saved as total input chromatin and processed alongside the eluted immunoprecipitates, beginning at the cross-link reversal step. After the cross-links were reversed with NaCl and RNaseA and incubated with proteinase K, the samples were precipitated and analyzed by PCR. PCR was carried out with primers rStx1ag F−284 to −255 (5′-CTG GGG CAG AGG AGG CAG CAG GGT CTC TGG-3′) and rStx1a R+30 to +11 (5′-CAT ACA AAA CTC CTA AAT TC-3′). Cycling conditions were as follows: 95 °C for 3 min, followed by 35 cycles of 95.5 °C for 30 s, 50 °C for 30 s and 72 °C for 40 s. PCR products were run on a 1.5% agarose gel and visualized by SYBR Green staining.ChIP assay was adapted from reference  with modDrosophila melanogaster SL2 cells, pPac-mock, pPac-YY1, and/or pPac-Sp1 vectors were co-transfected with phRG-TK and pGL3-CPR (−204 to +2) reporter vectors into cells by using FuGENE 6 . For reporter assays in PC12 cells, pcDNA3-mock or pcDNA3-YY1 vector was co-transfected with phRG-TK and pGL3-CPR (−204 to +2) reporter vectors. At 48 h after transfection, cells were lysed in reporter passive lysis buffer . Luciferase activity in cell lysates was measured by a luminometer . Relative luciferase units were evaluated after normalization against the phRG activity of each sample. Fold change was expressed as relative luciferase unit with pPac or pcDNA expression plasmid/relative luciferase unit with a mock vector.Luciferase reporter assay was carried out in accordance with our previous report . For rep2, 1 mM EDTA, 5 mM DTT, 1 mM spermidine, 0.1 µg/µl poly(dI-dC), 0.01% BSA, 10% glycerol and 0.1% PI cocktail). The reaction beads were washed with binding buffer and eluted by boiling in Laemmli sample buffer. Eluted samples were subjected to 5% SDS–PAGE gel electrophoresis. Gels were stained with Coomassie blue and then prepared for mass spectrometry analyses.The isolation of DNA–protein complexes based on streptavidin and biotin interaction was adapted from reference ,15 with All protein bands were excised from a Coomassie blue-stained SDS-PAGE gel and treated with trypsin for 16 h at 37 °C. Digested proteins were desalted and concentrated with StageTips [t-test or one-way analysis of variance. Differences at p values less than or equal to 0.05 were considered significant.Data were expressed as means ± SE and analyzed using Stx1a −204 to +2 CPR (accession no. D10392) promotes Stx1a transcription in neuronal cell/tissue; furthermore, we showed that the association of class 1 HDACs, such as HDAC1, HDAC2 and HDAC8, with Stx1a–CPR negatively regulates Stx1a transcription in non-neuronal cell/tissue [Stx1a–CPR includes two SP/GC sites  is suppressed by co-transfection of pPac-YY1 (p < 0.01 compared with pPac-Sp1) (Stx1a transcription. To study the effect of YY1 on the g of Sp1 . The reppPac-YY1 B. On thePac-Sp1) B. These Stx1a–CPR in non-neuronal cell/tissue not expressing Stx1a [To further identify OL2 DNA–protein complexes, we carried out mass spectrometry analysis by using proteins that were affinity purified from FRSK nuclear extracts by biotinylated OL2 DNA see . In geneng Stx1a . FurtherStx1a–CPR forms DNA–protein complex only in the non-neuronal FRSK cells which do not express Stx1a. Furthermore, Stx1a transcription is negatively regulated via the binding of YY1 transcription factor to the −183 to −137 region of Stx1a–CPR in non-neuronal cell/tissue not expressing Stx1a. Moreover, the high level association of YY1 to the Stx1a–CPR in default in non-neuronal FRSK cells is decreased by the treatment of the HDAC inhibitor TSA. Mass spectrometry analysis also showed that YY1 binds to the −183 to −137 promoter region together with factors involved in gene silencing, such as non-POU domain-containing octamer-binding protein [Stx1a–CPR only in non-neuronal cell/tissue not expressing Stx1a [Stx1a. Given that the YY1 transcription factor is well known to interact with proteins, including HDACs [Stx1a via complex formation with the YY1 transcription factor. In the present study, we found that the −183 to −137 OL2 region of  protein , HDAC1 [ protein  and hete protein . Interesng HDACs ,12,13,22Stx1a gene transcription is negatively regulated in a cell-/tissue-specific manner by the YY1 transcription factor, which may form the complex with gene silencing factors, including HDACs, on the −183 to −137 promoter region of Stx1a. In addition, a genetic deficiency of YY1 involved in Stx1a transcription has been reported to cause an autosomal dominant neurodevelopmental disorder, Gabriele–DeVries syndrome; this disorder is characterized by neuronal symptoms, such as intellectual disability, variable cognitive impairment and behavioral problems [Stx1a gene regulated by YY1 is known to be implicated in neuronal transmission [Stx1a expression will aid in understanding how YY1-regulated genes are controlled in the CNS and the role of the Stx1a promoter region in pathogenesis.Taken together, these findings suggest that problems . Given tsmission , further


Squamous cell carcinoma (SCC) is an aggressive malignancy that can originate from various organs. TP63 is a master regulator that plays an essential role in epidermal differentiation. It is also a lineage-dependent oncogene in SCC. ΔNp63α is the prominent isoform of TP63 expressed in epidermal cells and SCC, and overexpression promotes SCC development through a variety of mechanisms. Recently, ΔNp63α was highlighted to act as an epidermal-specific pioneer factor that binds closed chromatin and enhances chromatin accessibility at epidermal enhancers. ΔNp63α coordinates chromatin-remodeling enzymes to orchestrate the tissue-specific enhancer landscape and three-dimensional high-order architecture of chromatin. Moreover, ΔNp63α establishes squamous-like enhancer landscapes to drive oncogenic target expression during SCC development. Importantly, ΔNp63α acts as an upstream regulator of super enhancers to activate a number of oncogenic transcripts linked to poor prognosis in SCC. Mechanistically, ΔNp63α activates genes transcription through physically interacting with a number of epigenetic modulators to establish enhancers and enhance chromatin accessibility. In contrast, ΔNp63α also represses gene transcription via interacting with repressive epigenetic regulators. ΔNp63α expression is regulated at multiple levels, including transcriptional, post-transcriptional, and post-translational levels. In this review, we summarize recent advances of p63 in epigenomic and transcriptional control, as well as the mechanistic regulation of p63. Squamous cell carcinomas (SCCs) are a series of aggressive malignancies from various tissue origins, including the skin, head and neck, lung, and esophagus , 2. SquaLineage dependency occurs where cancer cells depend on survival and self-renewal mechanisms co-opted from the original healthy tissues from where they arose . TranscrThe TAp63 and ∆Np63 isoforms regulate distinct target gene sets and often exert opposing regulatory functions , 16. HetTP63 plays essential roles in lineage commitment during epidermal development through a variety of epigenetic mechanisms Table . Mutatiolama5. The chromatin accessibility of the pioneered p63-binding sites is significantly reduced in tp63−/− mutants. Consequently, the levels of expression of these epidermal genes are significantly decreased in the tp63−/− mutant, such as col18a1a [It has been speculated that TP53 family members may exert intrinsic pioneer factor activity to specify epithelial lineage-specific chromatin landscape , and thicol18a1a .Loss of p63 reduces the chromatin accessibility of keratinocyte-specific genes, such as KRT5. Furthermore, depletion of p63 reduces the chromatin accessibility and occupancy probability of other maturation-associated TFs, including p53, RFX, and Kruppel-like factor 4 (KLF4) , indicatITGA6, KRT5, and KRT14, are dramatically decommissioned as epidermal stem cells (EpSCs) differentiate to keratinocytes, whereas enhancers driving the expression of epidermal differentiation genes such as IVL and SPRR1A are activated de novo, which is accompanied by a shift of Dnmt3a genomic binding from the former enhancers cluster to the latter [Enhancers are cis-regulatory elements defined by H3K4me1 modification. Enhancers can be categorized as poised or active depending on the co-occupancy of H3K27me3 or H3K27ac, respectively , 71. Thee latter .Chromatin immunoprecipitation-sequencing (ChIP-seq) has revealed that, while the majority of p63-bound regulatory elements and p63-bound open chromatin regions in epidermal keratinocytes are active enhancers, and only a small number of p63-bound regions belong to the promoter , 76, sugIt has been demonstrated that embryonic epithelia from p63-null mice display compromised epithelial fate, concomitant with the activation of mesodermal-specific genes , suggestFAT2 and NECTIN1 is significantly reduced upon depletion of ΔNp63α in squamous-like PC cells, as well as downregulation of FAT2 and NECTIN1 [Chromatin states are closely associated with tumorigenesis and tumor phenotypes in SCCs . There a NECTIN1 . This in NECTIN1 , 87. ΔNp NECTIN1 , 88, 89. NECTIN1 , 91. Ove NECTIN1 . In addi NECTIN1  and LINC NECTIN1 , through NECTIN1 –94. New  NECTIN1 , 96. It  NECTIN1 . SEs are NECTIN1 . It is p NECTIN1 . Thus, eIt has been demonstrated that ΔNp63α physically interacts with SOX2 in SCC cells , which iThere are multiple epigenetic modulators implicated in regulating enhancer and spatiotemporal transcription of lineage-specific genes, including enzymatic regulators histone methylases, histone acetyltransferases, histone deacetylases, polycomb repressive complex (PRC), DNA methylase and a number of chromatin remodelers. The p63 activates enhancers and increases chromatin accessibility through interaction with these various epigenetic modulators, which will be discussed in the section below.COMPASS is a multiple-protein complex, which exerts histone H3K4 methylase activity. In mammalian cells, incorporation of H3K4me1 at enhancers is mainly accomplished by histone lysine methyltransferase 2C (KMT2C/MLL3) and 2D (KMT2D/MLL4) COMPASS-related complexes . UTX is S35-induced skin tumorigenesis [Deposition of H3K27ac at the H3K4me1-defined enhancer is proposed to be catalyzed by the cAMP response element-binding protein (CBP) or p300 histone acetyltransferases . Transcrigenesis . Thus, iigenesis . It has igenesis , 120. Fuigenesis . This su−/− mice and is accompanied by insufficient expression of EDC genes in epidermal progenitor cells [The mammalian SWI/SNF complex is an ATP-dependent chromatin remodeling and histone acetylation complex highly expressed in pluripotent stem cells . The mSWor cells . The speor cells . Thus, tor cells  and Satbor cells , respectIn addition to histone modifications, the status of DNA methylation also influences the activity of enhancers. Early studies suggest that patterns of 5-methylcytosine can mark enhancers. It is widely appreciated that lineage-specific enhancer regions are usually hypomethylated –131. It Not only does ΔNp63α act as a dominant negative competitor to repress p53-mediated transcription , but it By using a tandem affinity purification strategy, Ramsey et al. observed that ΔNp63α interacts with histone deacetylase (HDAC) 1 and HDAC2 via its transactivation inhibitory domain in SCC cells. ΔNp63α recruits HDAC1 binding to promoter region of PUMA and represses its transcription. Treatment with the HDAC inhibitor, trichostatin A (TSA), alleviates repression of PUMA transcription by ΔNp63α and induces substantial apoptotic cell death in SCC cells. TSA treatment increases H4 acetylation levels at the p63-binding site within the PUMA promoter, whereas silencing ΔNp63α promotes deposition of H4 acetylation level at PUMA promoter and induces PUMA expression, indicating that ΔNp63α represses gene transcription through HDAC1-mediated histone deacetylation  is the major chromatin-remodeling complex responsible for catalyzing the incorporation of H2AZ-H2B dimers into nucleosomes , 163. StR279H mutant causing EEC syndrome, is sufficient to bypass OIS in Ras-expressing primary keratinocytes, indicating downregulation of ΔNp63α is required for OIS. Mechanistically, ΔNp63α cooperates with Ras to promote proliferation and survival of keratin 15-expressing stem cells. Lsh was identified as a direct target of ΔNp63α, which initiates senescence bypass, as silencing of the gene leads to a stronger senescent-like morphology [Lsh is an ATPase that belongs to the SNF2/helicases family involved in chromatin remodeling . Bypassirphology . Both p6rphology . In addirphology , 170, Lsrphology , 172. Lsrphology –175. It trans to cis catalyzed by Pin1, a peptidyl-prolyl isomerase, prevents the binding of p63 with Itch, thus stabilizing p63 protein [cis/trans conformation switch, since E3 ligases favor the trans conformation [The N6-methyladenosine (m6A) modification, which is added by methyltransferase-like 3 (METTL3), promotes the increased expression of ΔNp63α mRNA and accelerates proliferation of cutaneous SCC . There a protein . These sormation . Stxbp4 ormation . This suThe WW domain containing E3 ubiquitin protein ligase 1 (WWP1) is a member of HECT type E3 family. WWP1 physically associates with both TAp63α and ΔNp63α via PY/WW motif interaction, leading to ubiquitination and degradation of TAp63α and ΔNp63α proteins . PhosphoRACK1 is a WD-40 repeat-containing scaffold protein binding to p63α , 191. UpSquamous cell carcinoma remains a great challenge to human health due to its highly aggressive and limited therapeutic options. SCC from various organs is highly dependent on ΔNp63α, which is recently highlighted for its pioneer activity and ability to establishing active enhancers through interaction with epigenetic regulators. The existence of p63 in chromatin-remodeling complexes provides an opportunity to target p63-positive SCCs via epigenetic therapy. However, many of the interaction partners of p63 in keratinocytes, including KMT2D and SWI/SNF components, are frequently mutated in SCC, implying that the p63-mediated differentiation transcriptional program is disturbed during SCC. It remains unclear how ΔNp63α cooperates with epigenetic regulators to activate enhancer activity of oncogenes in SCC. Additional efforts are necessary to fully understand the complexity of the interactions between ΔNp63α and epigenetic regulators in the control of SCC development. Characterization of ΔNp63α-binding partners in SCCs may enable the development novel strategies to indirectly interfere with ΔNp63α activity. Disruption of the interaction between ΔNp63α and diverse coactivators in SCC cells will broadly affect ΔNp63α-dependent transcription and may provide a novel therapeutic approach for SCC. ΔNp63α expression is sufficient to establish a squamous subtype-specific SE to drive oncogene expression in SCC. Thus, another area worthy of attention is the mechanisms underlying phase separation of ΔNp63α-dependent SE in SCC cells. It has been shown that ΔNp63α interacts with the c-Rel subunit of nuclear factor-kappa B (NF-κB) in keratinocytes and HNSCC cells . Given t


Telomeres are organized into a heterochromatin structure and maintenance of silent heterochromatin is required for chromosome stability. How telomere heterochromatin is dynamically regulated in response to stimuli remains unknown. Pyruvate kinase Pyk1 forms a complex named SESAME (Serine-responsive SAM-containing Metabolic Enzyme complex) to regulate gene expression by phosphorylating histone H3T11 (H3pT11). Here, we identify a function of SESAME in regulating telomere heterochromatin structure. SESAME phosphorylates H3T11 at telomeres, which maintains SIR (silent information regulator) complex occupancy at telomeres and protects Sir2 from degradation by autophagy. Moreover, SESAME-catalyzed H3pT11 directly represses autophagy-related gene expression to further prevent autophagy-mediated Sir2 degradation. By promoting H3pT11, serine increases Sir2 protein levels and enhances telomere silencing. Loss of H3pT11 leads to reduced Sir2 and compromised telomere silencing during chronological aging. Together, our study provides insights into dynamic regulation of silent heterochromatin by histone modifications and autophagy in response to cell metabolism and aging. Pyruvate kinase phosphorylates histone H3T11 (H3pT11) and represses gene expression by forming a large complex SESAME (Serine-responsive SAM-containing Metabolic Enzyme). Here the authors show that SESAME-catalyzed H3pT11 regulates telomere silencing by promoting Sir2 binding at telomeres and preventing autophagy-mediated Sir2 degradation. In budding yeast, heterochromatin is localized at three major sites, including silent mating-type loci (HML and HMR), telomeres, and ribosomal DNA (rDNA). Genes near these regions are subject to transcriptional repression, also known as position-effect variegation, which is highly conserved in higher organisms4. The proper organization of this transcriptionally silent heterochromatin is important to maintain telomere length, genome integrity, and chromosome segregation fidelity6. Disruption or loss of silent chromatin may lead to chromosomal instability, a hallmark and causal factor of tumorigenesis and cell aging10.The eukaryotic genome is organized into distinct euchromatin and heterochromatin domains, which contain the bulk of actively transcribed regions and transcriptionally silent regions, respectively. Maintaining the integrity of euchromatin and heterochromatin within the nucleus is required for cell development and differentiation13. During this process, Sir4 is recruited by Rap1 and the yeast Ku complex to cis-acting silencer elements15. Sir4 then recruits Sir2, a highly conserved NAD+-dependent histone deacetylase (HDAC) to deacetylate H4K1616. Deacetylation of H4K16 generates a high-affinity binding site for Sir3, which then recruits more Sir4 and Sir217. The cycles of Sir2-mediated H4K16 deacetylation and Sir3 recruitment allow the formation of SIR complex and its propagation along the telomere regions, which shields the telomere-proximal DNA from the transcription machinery, leading to transcription repression18. The spread of SIR complex to transcriptionally active chromatin is antagonized by H4K16ac, which acts as the heterochromatin–euchromatin boundary19. In addition to H4K16ac, Dot1-catalyzed H3K79me3 and Set1-catalyzed H3K4me3 have been shown to inhibit the spread of SIR complex into euchromatin regions21. These modifications occur predominantly in euchromatin regions and thus affect heterochromatin structure in an indirect manner22.The silent heterochromatin at yeast telomeres is formed by assembly of Silent Information Regulator (SIR) complex (Sir2/Sir3/Sir4) and their spread along chromatin in a stepwise manner2. During the aging process, the global loss of heterochromatin was observed in Drosophila, Caenorhabditis elegans, and mammalian stem cells6. The telomere silencing is enhanced by increasing the temperature within the range of 23–37 °C in budding yeast23. Telomere silencing is also affected by cell metabolic states. During the aging process when carbon source is exhausted, the long-lived quiescent cells group their telomeres into a unique focus or hypercluster localized in the center of the nucleus, which correlates with more stable telomere silencing25. Changing carbon source from glucose to galactose induces telomere silencing defects, whereas switching from glucose to glycerol enhances telomere silencing by affecting the redox status27. Calorie restriction (CR) by glucose depletion has been proposed to increase NAD+/NADH and enhance Sir2 activity, which promotes telomere silencing28. However, glucose deficiency has also been shown to have no significant effect on Sir2 activity and telomere silencing30. These results suggest the relationship between glucose metabolism and telomere silencing is complex.The silent heterochromatin is dynamically regulated in response to external and internal stimuli+ levels, suggesting that it is nuclear NAD+ instead of global NAD+ changes that regulate telomere silencing31. The glutamate dehydrogenase 1 (Gdh1) can translocate into the nucleus and maintain telomere silencing likely by reducing nuclear α-ketoglutarate levels32. All these results emphasize the importance of examining the nuclear functions of metabolic enzymes in modulating telomere silencing.Some metabolic enzymes have been reported to translocate into the nucleus, where they regulate telomere silencing. For example, the glyceraldehyde 3-phosphate dehydrogenase (Tdh3) enhances telomere silencing by maintaining the nuclear NAD33. Glycolysis and serine metabolism stimulate SESAME to phosphorylate H3T11 and confer cell resistance to oxidative stress34. In this study, by examining the genome-wide binding profiles for SESAME and H3pT11, we unexpectedly discovered that SESAME binds at telomeres to phosphorylate H3T11. SESAME-catalyzed H3pT11 maintains telomere silencing, promotes SIR complex binding at telomeres, and prevents autophagy-mediated Sir2 degradation. Our study also reveals a dynamic regulation of silent heterochromatin assembly at telomeres by SESAME-phosphorylated H3T11 in response to cell metabolism and aging.We have previously found that the glycolytic enzyme, pyruvate kinase (Pyk1), can translocate into the nucleus, where it exerts non-metabolic functions. Pyk1 phosphorylates histone H3T11 (H3pT11) to repress the expression of a subset of genes as a part of a 400 kDa complex, Serine-responsive SAM-containing Metabolic Enzyme (SESAME), which contains serine metabolic enzymes, SAM synthetases, and an acetyl-CoA synthetasePYK1 gene promoter33. We also classified RNA polymerase II-regulated genes into five groups based on their transcription levels and then compared H3pT11 levels among these groups. The H3pT11 signals were positively correlated with gene transcription levels , we performed chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) of H3pT11 in yeast cells grown in glucose-containing rich media and compared it with the positions of H3K4me3, a histone marker associated with active gene transcription. Figure els Fig. . Like thr33 Fig. . Kyoto Er33 Fig. .Fig. 1SEYJR011C, there is a 2.01-, 2.16-, 2.95-fold enrichment of Pyk1, Ser33, and Sam1 at the non-telomeric PYK1 gene  Fig. . We alsoene Fig. . Compareons Fig. , suggestons Fig. . The H3p11C Fig. . The H3p11C Fig. . The varnts Fig. . The losPYK1 and PMA133. We then examined the effect of Set1 and H3K4me3 on the occupancy of SESAME and H3pT11 at telomeres. In set1Δ and H3K4A mutants, H3pT11 occupancy at the PYK1 gene was significantly reduced but its occupancy at telomeres was unaffected  , a suicide substrate for cells expressing URA3. The growth of H3T11A mutant on 5-FOA plate was reduced when compared to its wild-type counterpart cells  . Cells with normal silenced ADE2 are red after grown for 4–6 days but will become white once ADE2 is derepressed39. In agreement with the results with the URA3 reporter assay, we observed that H3T11A mutant cells became white  Fig. 39. In thlls Fig. . Similarm2Δ Fig. . The redCR) Fig. , indicatYFR057W, which is located near the right telomere of chromosome VI (Tel VI-R) and subject to transcriptional silencing in a SIR-dependent manner  complex Fig. , suggestHML and rDNA loci . The transcription of telomere proximity genes was significantly increased in H3T11A mutant; however, overexpression of Sir2 abrogated the increased transcription of these genes in H3T11A mutant  responsible for Sir2 degradation in H3T11A mutant, we treated WT and H3T11A cells with the proteasome inhibitor, MG132 and found that the reduced Sir2 in H3T11A mutant was unaffected by MG132 treatment  proteins that coordinately function at different stages47. To test whether autophagy regulates Sir2 degradation, we treated cells with Phenylmethanesulfonyl fluoride (PMSF), an inhibitor for lysosomal proteases48. The reduced Sir2 proteins in H3T11A mutant were restored by PMSF treatment  autophagy process. As heterochromatin is gradually lost during agingess Fig. , suggest49, while the autophagy is a cytoplasmic process. To be degraded in the vacuole, Sir2 needs to be transported from the nucleus into the cytoplasm. We thus examined the localization of Sir2 in WT and the H3T11A mutant expressing a Sir2-GFP fusion protein. Monitoring GFP by fluorescence microscopy showed that Sir2 was localized primarily in the nucleus in WT cells with only 25.08% cells having cytoplasmic-localized Sir2  treatment -rich nuclear export sequence (NES), LPEDLNSLYI, for the exportin chromosomal region maintenance 1 (Crm1). We thus examined Sir2 localization and protein levels in ir2 Fig. , indicatWe treated WT and H3T11A mutant with rapamycin and found that rapamycin treatment resulted in less Sir2 protein remaining in H3T11A compared to WT cells Fig. . To showPEP4, which encodes a major vacuolar protease, in the H3T11A mutant. Loss of Pep4 restored Sir2 levels in the H3T11A mutant  liberation assay, which detects free GFP that is liberated upon the delivery of endogenous promoter-driven Atg8 with an N-terminal GFP tag (GFP-Atg8) to the vacuole and subsequent proteolysis of the Atg8 portion of the fusion proteinlls Fig. . Moreovells Fig. . Consistlls Fig. . We alsoant Fig. . To strents Fig. . These dATG genes52. Analysis of our ChIP-seq data showed that H3pT11 was enriched at ATG genes, especially ATG31, ATG23, ATG8, ATG3, and ATG5 . The transcription of ATG8, ATG5, ATG23, ATG12, and ATG31 was significantly increased in H3T11A but not in H3T11D mutants , H4K20 monomethylation (H4K20me1) and H4K12ac58. However, their functions at telomeres have not been explored extensively. Here, we identified H3pT11 as a histone marker that is enriched in telomere regions and promotes SIR complex occupancy at telomeres. The function of SESAME and H3pT11 at telomere heterochromatin regions is independent on Set1-catalyzed H3K4me3. As SESAME-catalyzed H3pT11 regulates telomere silencing but has no effect on silencing at mating-type loci, it is possible that H3pT11 could indirectly regulate telomere silencing. Our ChIP-seq data also showed that at some silent loci, the localization of H3pT11 is immediately adjacent to SIR binding sites, suggesting that H3pT11 may function as a boundary like other histone modifications, i.e., H3K4me3 and H3K79me3 to block the spread of SIR complex. Moreover, H3pT11 is highly enriched at the transcription start site (TSS) of highly transcribed genes and Gcn5 has a preference towards H3T11 phosphorylation59. It is likely that H3T11A mutant strongly affects highly active promoters across the genome, which then indirectly lead to shifting of SIR complex to other sites across the genome and altered telomere structure. In addition, some factors have been reported to indirectly regulate the binding of active histone modifiers at heterochromatin. For example, loss of H3K79 methylase Dot1 leads to rearrangement of heterochromatin and one plausible mechanism is promoting the binding of histone acetyltransferase Gcn5 at heterochromatin60. Moreover, MRG1, which a factor exclusively bound at euchromatin in Caenorhabditis elegans, promotes the anchoring of heterochromatin at the nuclear periphery61. Loss of MRG1 increases the occupancy of histone acetyltransferase CBP-1 and H3K27ac at heterochromatin61. Hence, it is possible that SESAME and H3pT11 may prevent the mislocalization of euchromatic factors and/or histone modifications at telomere heterochromatin.Histone modifications play important roles in regulation of telomere silencing by restricting the spread of SIR complex into euchromatic regions in budding yeast, including Sas2-catalyzed H4K16ac+, and telomere silencing is complex, recent studies emphasize the importance of examining the nuclear functions of metabolic enzymes in modulating telomere silencing. For example, the glyceraldehyde 3-phosphate dehydrogenase (Tdh3) and glutamate dehydrogenase 1 (Gdh1) regulate the activity of Sir2 and telomere silencing by affecting the nuclear NAD+ and α-ketoglutarate levels, respectively32. In addition, Gas1, a β-1,3-glucanosyltransferase, has been shown to regulate telomere silencing by interacting with Sir2 and perhaps adding β-1,3-glucan to Sir262. Unlike these enzymes, pyruvate kinase-containing SESAME complex epigenetically regulates telomere silencing independent of NAD+ and α-ketoglutarate. SESAME directly binds to telomeres, phosphorylates H3T11, and maintains telomere silencing by promoting SIR complex occupancy and preventing autophagy-mediated Sir2 degradation. The homolog of pyruvate kinase in mammals, PKM2 has been shown as a telomere-binding protein by tandem affinity purification coupled with the label-free comparative mass spectrometry despite little being known about its effect on telomere structure63. Given the high similarity between PKM2 and Pyk1, it is possible that PKM2 may regulate telomere silencing in mammals like Pyk1. Moreover, we found a function of serine and serine metabolic enzymes (Ser33 and Shm2) in regulating telomere silencing by stimulating Pyk1-catalyzed H3pT11 and preventing Sir2 degradation. Hence, serine metabolism enhances telomere silencing by inducing SESAME-catalyzed H3pT11 and maintaining Sir2 homeostasis.Telomere silencing is regulated by cell metabolism. Although the relationship between glucose deficiency, NAD24. The telomere hyperclusters correlate with more stable telomere silencing25. Although we observed that H3pT11 prevents the nuclear export of Sir2 during the aging process , H3pT11 has no clear effect on telomere hyperclustering . These data suggest that H3pT11 does not maintain telomere silencing by promoting the formation of telomere hyperclusters. This could be explained by the fact that telomere hyperclustering in quiescent cells is not driven by an increase of Sir3 spreading and gene silencing is not necessary for telomere hyperclustering25.During carbon source exhaustion in the aging process, the long-lived quiescent cells group their telomeres into a unique focus or hypercluster localized in the center of the nucleus64; however, little is known about the effect of autophagy on SIRT1 (Sir2) protein level. We find that Sir2 protein stability is controlled by autophagy, which interestingly is regulated by H3pT11. Our findings thus establish autophagy as an important regulator of telomere silencing. Notably, autophagy is responsible for the loss of Sir2 and transcriptional silencing during chronological aging. By focusing on SESAME and H3pT11, we not only link autophagy and telomere silencing but also show how they are dynamically controlled: during the aging process, H3pT11 is reduced, which leads to reduced Sir2 binding and accelerated nuclear export of Sir2; meanwhile, reduced H3pT11 induces autophagy to degrade Sir2. By analyzing the global genetic interaction network generated by Costanzo et al.65, we find a negative genetic interaction between PYK1 and autophagy-related genes , consistent with our findings that Pyk1 represses autophagy. Knockdown of pyruvate kinase PKM2 has been shown to induce autophagy in mammalian cells66. As PKM2 has also been shown to phosphorylate H3T1167, it is possible that PKM2-phosphorylated H3T11 may prevent autophagy-degradation of Sir2 homologs in mammals. In agreement with the role of Pyk1 and H3pT11 in autophagy, the SESAME subunit, Acs2 has also been reported to repress autophagy-related gene transcription52, suggesting the concerted function of SESAME subunits in regulation of autophagy.Autophagy is an evolutionarily conserved cellular process that primarily participates in lysosome-mediated protein degradation. It has been reported that SIRT1, the Sir2 homolog in mammals, promotes autophagyATG gene expression. Autophagy has been reported to be regulated by few histone modifications. Under starvation conditions, the reduced H2B monoubiquitination (H2Bub1) results in the activation of autophagy by controlling the transcription of autophagy regulatory genes68. Rpd3-mediated histone deacetylation represses autophagy69. The acetyltransferase hMOF catalyzed H4K16ac is involved in autophagy regulation70. Here, we show that H3pT11 inhibits autophagy by directly repressing the transcription of autophagy genes. It remains unclear how H3pT11 represses the transcription of autophagy genes.We identified a histone modification that prevents autophagy flux by repressing Together, our study reveals a pathway by which metabolism can influence SIR-mediated silencing through the SESAME complex. We identify SESAME-catalyzed H3pT11 as an epigenetic regulator of telomere heterochromatin structure. Our results also reveal functions of autophagy in regulating telomere silencing.71. Fusion of the tags  to individual SESAME subunits and Sir2 does not interfere with the functions of the proteins studied as no defects in cell growth, histone modifications, and telomere silencing were observed. All yeast strains were verified by colony PCR, DNA sequencing, qRT-PCR, and/or Western blots before used for experiments. The sequence of primers used for qPCR is listed in Supplementary Table All yeast strains used in this study are described in Supplementary Table pTEFpro-SIR2) was constructed as follows: SIR2 was amplified by primers  or selective medium as indicated until OD600 of 0.7–1.0. Cells were harvested and lysed in alkaline lysis buffer  β-mercaptoethanol) on ice for 15 min. After centrifugation, the protein pellet was resuspended in 150 μl 2×SDS-sample buffer. Protein samples were separated by 8–15% SDS-PAGE and transferred to Immobilon-P PVDF membrane. The blots were probed with primary antibodies followed by incubation with horseradish peroxidase-labeled IgG secondary antibodies. The protein bands were visualized using the ECL Chemiluminescence Detection Kit  and quantified with Image J software (v.1.8.0) (https://imagej.en.softonic.com/). All Western blots were performed at least three times and one typical example was shown.Proteins were extracted from exponentially growing yeast cells600 of ~0.7–1.0. The crosslinking was performed in 1% formaldehyde and quenched by adding 10 ml of 2.5 M glycine. Harvested cells were washed once with TBS + PMSF pre-chilled to 4 °C, lysed with glass beads in FA-SDS lysis buffer . DNA was sonicated to an average size of 500 bp and subjected to immunoprecipitation with anti-H3 , anti-H3pT11 , anti-FLAG antibody , anti-H4K16ac (2 μl) pre-bound to Protein G Dynabeads (Invitrogen) at 4 °C overnight. The beads were subsequently washed with FA lysis buffer, FA buffer + 1 M NaCl, FA buffer + 0.5 M NaCl, TEL buffer , and TE . The eluted DNA/protein complexes were treated with 20 µg Proteinase K at 55 °C for 1 h and then treated at 65 °C overnight. The DNA was digested by RNase (Roche), purified with ethanol precipitation and quantitated by qPCR with primers listed in Supplementary Table Yeast cells were grown in 200 ml YPD media at 30 °C until OD33. Library was constructed and sequenced on an Illumina platform33. Data was aligned to yeast genome sacCer3 from UCSC using bowtie2 version 2.1.0 with parameter -k 1. As the telomere repeated sequences cannot be mapped to specific telomeres, they were excluded for further analysis. Data was read into R (3.1.0) for further analysis. Peaks were called using MACS2  with parameter -t -c -g 1.2e7 -n -B -q 0.01–nomodel. Peaks annotation was performed on a website service (https://manticore.niehs.nih.gov/pavis2/). Tracks was smoothed by deepTools2 (v.2.0) and visualized by IGV software (v.2.0) with a reference genome of S. cerevisiae (sacCer3). The control tracks were generated using untagged BY4741 strain with anti-FLAG antibody. The raw ChIP-seq dataset for Sam1 and Pyk1 is derived from GSE72972. The raw ChIP-seq dataset for Sir2, Sir3, and Sir4 is derived from SRP030670. Raw data were downloaded by SRA toolkit (v.2.9.2). Files were unzipped and filtrated by Fastqc (v.0.11.9) and Trim Galore (v.2.11).ChIP-seq was done for four biological replicates with anti-H3pT11 and anti-H3 antibodies72. qPCR was carried out on a Bio-Rad real-time PCR machine with iTaq™ Universal SYBR® Green Supermix . Primers used for qRT-PCR are described in Supplementary Table (-ΔΔCt) to determine the quantity of relative transcription level. The mRNA level of the gene of interest was normalized to that of beta-actin.Total RNA was isolated from exponentially growing yeast cells by standard phenol-chloroform extraction procedures. The purified RNA was digested with DNase I (RNase-free)  at room temperature for 30 min and quantified by Nanodrop 2000 (Thermo scientific). The integrity of RNA was assessed by agarose gel electrophoresis. 500 ng total RNA was used for reverse transcription PCR (RT-PCR) in a 10 μl reaction volume with Reverse Transcriptase Kit (M-MLV) (ZOMANBIO)600 of 1.0. After washing with cold phosphate buffered saline once, the cells were resuspended in phosphate buffered saline. Cells were fixed with 4% formaldehyde in phosphate buffered saline for 30 min and treated with DAPI for 15 min at room temperature. Cells were then washed with cold phosphate buffered saline. The cell morphology was visualized using a ZEISS LSM710 microscope (Germany) with a 100× oil immersion objective by fluorescent microscopy. Images were acquired using ZEN Imaging Software ZEN 2.1 (ZEISS). DAPI was used to indicate the nucleus. The merged color Images were generated by Image J.Yeast cells were cultured in YPD or selective medium until ODAll antibodies used in this study were listed in Supplementary Table t test in Microsoft Excel  was used for comparison between two groups and a p-value <0.05 was considered statistically significant. *p < 0.05; **p < 0.01; ***p < 0.001; n.s. no significance. For all error bars, data are mean ± standard error (SE).Representative results of at least three biological independent experiments were performed in all of the figure panels. The two-sided Student’s Further information on research design is available in the Supplementary InformationReporting summary


Precise control of gene expression is fundamental to cell function and development. Although ultimately gene expression relies on DNA-binding transcription factors to guide the activity of the transcription machinery to genes, it has also become clear that chromatin and histone post-translational modification have fundamental roles in gene regulation. Polycomb repressive complexes represent a paradigm of chromatin-based gene regulation in animals. The Polycomb repressive system comprises two central protein complexes, Polycomb repressive complex 1 (PRC1) and PRC2, which are essential for normal gene regulation and development. Our early understanding of Polycomb function relied on studies in simple model organisms, but more recently it has become apparent that this system has expanded and diverged in mammals. Detailed studies are now uncovering the molecular mechanisms that enable mammalian PRC1 and PRC2 to identify their target sites in the genome, communicate through feedback mechanisms to create Polycomb chromatin domains, and control transcription to regulate gene expression. In this Review, we discuss and contextualise the emerging principles that define how this fascinating chromatin-based system regulates gene expression in mammals. However, through studying these mechanisms it has become apparent that chromatin, and histone post-translational modification, can also profoundly influence transcription and gene expression.The development of multicellular organisms relies on cells and tissues establishing unique gene expression programmes. To achieve this, signalling pathways converge on DNA-binding transcription factors, which guide the binding and activity of RNA polymerase II (Pol II) at gene promotersDrosophila melanogaster, where they are required for repression of homeotic (Hox) genes and thus for body plan specification2. Polycomb genes were subsequently shown to have mammalian orthologues that have important roles in controlling gene expression throughout development3.The importance of chromatin modifying complexes in controlling development is exemplified by the Polycomb repressive system. Polycomb genes were initially discovered in 5, whereas PRC2 mono-, di- and tri-methylates histone H3 at Lys27 9. Importantly, PRC1 and PRC2 tend to spatially converge on the same sites in the genome to form Polycomb chromatin domains, which are uniquely enriched in H2AK119ub1, H3K27me3 and Polycomb complexes14. Polycomb chromatin domains are then thought to counteract transcription, though the mechanisms that enable this remain incompletely defined.Since these initial discoveries, biochemical analyses have shown that Polycomb proteins typically assemble into one of two large multi-protein complexes that post-translationally modify histones. Polycomb repressive complex 1 (PRC1) mono-ubiquitylates histone H2A at Lys119 (H2AK119ub1)D. melanogaster, detailed studies over the past decade have revealed that the Polycomb system has expanded in mammals and that some key molecular principles that define its function have diverged from D. melanogaster. Based on these discoveries, we have gained a new understanding of how mammalian Polycomb complexes identify their target sites to form Polycomb chromatin domains and ultimately control gene expression. Several Reviews have recently discussed the general features and functions of Polycomb systems across phyla, and in mammalian development and disease18. In this Review we discuss and contextualise our emerging understanding of the molecular principles that enable the functions of mammalian Polycomb complexes in gene regulation. We first introduce the diverse complement of mammalian Polycomb repressive complexes and describe their enzymatic activities. We examine how Polycomb repressive complexes identify their target sites in the genome and communicate with each other to initiate the formation of Polycomb chromatin domains. We then discuss the regulation of Polycomb chromatin domain formation and ultimately how this is integrated with and regulates gene expression. Finally, we conclude by considering future avenues of research that will provide further mechanistic understanding of the principles of Polycomb biology in health and disease.Although early genetic and biochemical investigations of Polycomb proteins were focussed on The rapid progress in understanding mammalian Polycomb biology has relied on detailed and systematic biochemical interrogation of Polycomb repressive complexes and their histone modifying activities. This work has shown that Polycomb complexes comprise catalytic cores, which bind auxiliary proteins to create distinct PRC1 and PRC2 assemblies. In this section we introduce the composition, structure and enzymatic activity of the different Polycomb complexes, as a prerequisite for examining how these complexes underpin the functions of mammalian Polycomb complexes in gene regulation.19. RING1 and PCGF proteins have similar domain architecture, including an N-terminal RING domain and C-terminal RING-finger and WD40-associated ubiquitin-like (RAWUL) domain. The RING1 protein and the PCGF protein dimerise through their RING domains, which facilitates their interaction with an E2 conjugating enzyme to enable histone ubiquitylation22. Their RAWUL domains bind to a range of auxiliary subunits, which regulate the catalytic activity of PRC1 and target it to specific sites in the genome24. Importantly, the PCGF components dictate which auxiliary subunits are incorporated into specific PRC1 complexes. These complexes are generally categorised as either canonical PRC1 (cPRC1) or variant PRC1  and a Polyhomeotic (PHC) subunit . By contrast, vPRC1 complexes, which were identified later27, can assemble around any of the six PCGF proteins (PCGF1–6) and include RING1 and YY1-binding protein (RYBP), or its paralogue YAF2, and various additional subunits depending on the PCGF component present in the complex.The catalytic core of PRC1 is composed of RING1B or its paralogue RING1A, and one of six Polycomb group ring finger (PCGF) proteins  domain of SUZ12 interact with EZH1 or EZH2, causing its SET (Su(var)3-9, Enhancer-of-zeste and Trithorax) domain to transition from an autoinhibitory conformation into an active one that supports methyltransferase activity41. Also important for the catalytic activity of PRC2 is the CXC domain of EZH2, which engages with nucleosomal DNA, and a patch of residues on the surface of EZH2, which interacts with the N-terminal portion of the H3 tail43. Together, these interactions position the N-terminal tail of H3 such that Lys27 is adjacent to the active site of PRC2, to enable its methylation.PRC2 complexes form around a tetrameric core complex consisting of EZH2 or its paralogue EZH1, EED, SUZ12, and RBBP4 or RBBP7 . Biochem44, giving rise to PRC2 assemblies with distinct biochemical properties , was recently shown to interact with the core PRC2 complex and inhibit its methyltransferase activity while also blocking interactions with PRC2.1-specific and PRC2.2-specific subunits54. This function suggests that context-specific regulators of PRC2 activity have important roles during development.The targeting and regulatory lobe of PRC2 includes the N-terminal portion of SUZ12, which binds to RBBP4 or RBBP7 and interacts with various additional auxiliary factors in a mutually exclusive manneroperties . PRC2.1 63, indicating that more specific mechanisms must dictate the observed binding patterns. In the following sections, we examine emerging evidence that primary mechanisms of Polycomb complex targeting in mammals rely on a complement of sequence-specific DNA-binding factors, RNA-dependent mechanisms and binding to CpG islands (CGIs).Despite an intrinsic capacity to engage with nucleosomes, genome-wide studies have shown that Polycomb complexes are enriched at gene promoters and other gene regulatory elementsD. Melanogaster, targeting of Polycomb complexes is thought to rely primarily on sequence-specific DNA-binding factors. However, many of these factors lack clear mammalian orthologues, or if they are present in mammals, do not target Polycomb complexes to chromatin66. This difference has brought into question whether these mechanisms are also important for mammalian Polycomb target site identification. In beginning to address this important question, it has recently been shown that the PCGF6-vPRC1 complex, which is absent in D. Melanogaster, stably incorporates auxiliary subunits that function as sequence-specific DNA-binding domains  contains bHLH and T-box DNA-binding domains70. Studying these domains has revealed that MGA dimerises with MYC-associated factor X (MAX) to bind E-box motifs (5’-CACGTG-3’), and that it can also use its T-box domain to recognise longer T-box motifs (5’-TCACACCT-3’)73. These domains are important for PCGF6-vPRC1 occupancy at a subset of its target sites, which in somatic cells includes a number of germline-specific genes75. In addition, E2F6–DP-1 (or DP-2) heterodimers also associate with PCGF6-vPRC1 and provide the complex with distinct target-site specificity in other contexts72. Together, these findings demonstrate that PCGF6-vPRC1 utilises sequence-specific DNA-binding activities to identify a subset of its target sites in the genome  by interacting with the transcription factors upstream stimulatory factor 1 (USF1) and USF280. Mammalian X chromosome inactivation (XCI), which is mediated by the lncRNA X inactive specific transcript (XIST), involves Polycomb repressive complexes. During XCI, XIST is expressed from one of the two female X chromosomes and spreads in cis to drive mono-allelic chromosomal gene silencing. Detailed characterization of this process has revealed that the PCGF3/5-vPRC1 complex interacts with the RNA-binding protein heterogeneous nuclear ribonucleoprotein K (hnRNPK), which recognises XIST, leading to PCGF3/5-vPRC1 enrichment on the inactive X chromosome  that associate with defined sites in chromatinromosome 81-84. HoXIST studies, numerous other lncRNAs have been proposed to target Polycomb complexes to sites in autosomes. Notably, the lncRNA HOX transcript antisense RNA (HOTAIR) was reported to function as a trans-acting repressor of the HOXD locus by physically interacting with and recruiting PRC287. However, recent work has demonstrated that transcription repression by HOTAIR is independent of PRC2, and that PRC2 recruitment to the HOXD locus occurs primarily in response to gene silencing, not through direct targeting by HOTAIR88. These findings highlight the need for caution when interpreting how lncRNAs influence gene expression and the recruitment of Polycomb complexes90. However, consistent with a potential role for lncRNAs in targeting the Polycomb complexes to chromatin, the lncRNAs Airn and Kcnq1ot1 promote mono-allelic expression of autosomal genes in mouse trophoblast stem cells through a mechanism that appears to be highly analogous to XCI  regions of CpG-rich DNA that are associated with approximately 70% of mammalian gene promoters99. In CGIs, CpG dinucleotides remain free of DNA methylation, whereas elsewhere in the genome they are heavily methylated and function to maintain heterochromatin and repress the expression of parasitic DNA elements101. CGIs are absent in non-vertebrate model organisms, including D. melanogaster, which lacks DNA methylation. This led to speculation that CGIs may be used by Polycomb complexes to identify target gene promoters and other regulatory elements in mammals102.Polycomb complexes appear to recognise some of their target sites using sequence-specific DNA-binding factors or through associating with lncRNAs. However, these mechanisms alone cannot explain all Polycomb complex binding observed in mammalian genomes, suggesting that additional targeting mechanisms exist. Early genome-wide studies revealed that Polycomb complexes associate primarily with CGIs104. KDM2B contains a zinc finger-CxxC domain that specifically binds to non-methylated CpG dinucleotides106. Consistent with this specificity, KDM2B can target the PCGF1-vPRC1 complex to CGIs  to CGIs 107-110. s of DNA 111,112. Once Polycomb complexes engage with target sites, this primary targeting must then be converted into the formation of repressive Polycomb chromatin domains, which can extend up to tens of kilobases and have extremely high levels of H2AK119ub1, H3K27me3, and Polycomb complex occupancy. In the following sections, we describe an intricate series of feedback and communication mechanisms that link the activity of Polycomb complexes on chromatin to enable the formation and maintenance of Polycomb chromatin domains.de novo enrichment of PRC2, accumulation of H3K27me3 and the formation of a Polycomb chromatin domain that spread several kilobases from the primary engagement site115. Interestingly, this activity was exclusive to vPRC1 complexes and relied on their capacity to ubiquitylate H2AK119 (Ref.30)  . Subsequ PRC2117 . The str126. This interaction forms the basis of a feedback mechanism that supports spreading of H2AK119ub1 onto neighbouring nucleosomes in a manner that appears to be aided by histone H1 (Ref.32). Together, these data demonstrate that once Polycomb target sites are identified through the primary targeting of vPRC1, feedback mechanisms inherent to vPRC1 complexes and H2AK119ub1  to polymerise in a head-to-tail orientation and form long filaments150  or through the pre-mRNA splicing factor RBFOX2 (Ref.195), supports PRC2 occupancy at target sites.The realisation that gene activity appears to be incompatible with Polycomb chromatin domain formation has led to a concerted effort to understand the mechanisms that regulate this process. Although some lncRNAs appear to target Polycomb complexes to specific sites, an overwhelming body of evidence now also indicates that PRC2 binds promiscuously to nascent RNA194. In this model, nascent transcripts from very lowly transcribed genes help bring PRC2 into proximity with promoter elements to support chromatin binding and tri-methylation of H3K27. However, at more highly transcribed genes, elevated levels of nascent transcripts would compete with PRC2 for chromatin binding and decrease H3K27 tri-methylation. Although the molecular details of this model remain to be fully tested, it appears that PRC2 is highly attuned to sensing transcript levels and using this information to regulate the formation of Polycomb chromatin domains.To reconcile these seemingly contradictory effects, an ‘RNA-bridging model’ has been proposed196. Importantly, in vitro studies have demonstrated that the methyltransferase activity of PRC2 is inhibited by the presence of H3K4me3, H3K36me2 or H3K36me3 (Ref.202), and disruption of H3K4 methyltransferase activity in vivo can lead to enhanced Polycomb chromatin domain formation203. In addition, histone demethylases act as part of transcription co-activator complexes to remove H3K27 methylation209, and transcription-associated H3K27, H4K16 and H4K20 acetylation counteracts PRC2 activity215. Similarly, the H2AK119ub1-specific deubiquitylases USP16 and 2A-DUB  have been shown to associate with promoters of actively transcribed genes and counteract H2AK119 ubiquitylation217.In addition to producing nascent RNA, transcription also profoundly influences the chromatin environment at transcribed genes. For example, during transcription initiation, histone methyltransferases tri-methylate H3K4 at gene promoters, whereas elongation is associated with H3K36 tri-methylation in gene bodies220. This activity was reported to target RYBP-containing vPRC1 complexes, suggesting that promoter-associated chromatin remodelling activities can displace vPRC1 complexes and limit H2AK119 ubiquitylation221. Together, these findings suggest that although primary targeting activities may enable Polycomb complexes to engage with a wide range of gene promoters, transcription-associated histone modifications and chromatin remodelling activities at regulatory elements can limit the catalysis and feedback mechanisms required for efficient formation of Polycomb chromatin domains.In addition to histone modification-dependent effects, the chromatin remodelling ATPase BRG1 can actively evict PRC1 from promoters of transcribed target genes222  by which H2AK119ub1 affects transcription remain to be determined . Recently, an evolutionarily-conserved bromo adjacent homology (BAH) domain in BAHD1 and BAHCC1 was shown to specifically recognise H3K27me3 and contribute to gene repression, possibly by recruiting histone deacetylases, which are known to counteract gene expression255 . This suggests that pervasive H2AK119 ubiquitylation may function like a rheostat to control the transcriptional potential of the genome.Although H2AK119ub1 and H3K27me3 levels are highly elevated in Polycomb chromatin domains, they are also found elsewhere in the genome, and there is mounting evidence that these alternative pools of H2AK119ub1 and H3K27me can affect gene expression in various contexts. The PCGF3/5-vPRC1 complexes deposit a ‘blanket’ of H2AK119ub1 across the genome, with approximately 10% of H2A molecules being modified 275. Although the mechanisms that control H3K27 methylation outside of Polycomb chromatin domains remain poorly understood, this pervasive activity primarily relies on the core complex and not on PRC2.1-specific and PRC2.2-specific subunits274. Importantly, there is evidence that these alternative pools of H3K27me can broadly antagonise transcription, possibly by counteracting H3K27 acetylation and limiting the activity of enhancers276. Therefore, it is clear that Polycomb complexes can also influence gene expression by functioning outside of Polycomb chromatin domains and it has recently been suggested that these activities may be particularly relevant in germ cells and in very early developmental stages-278.PRC2 also acts pervasively throughout the genome: approximately 70% of H3K27 residues undergo mono-methylation or di-methylation and up to 15% undergo tri-methylation282. For example, a PCGF5-vPRC1 complex that contains AUTS2 and casein kinase 2 and inefficiently ubiquitylates H2AK119, was shown to potentiate transcription by recruiting the histone acetyltransferase P300 (Ref.279). Alternatively, the H2AK119ub1-specific reader ZRF1  was linked to activation of Polycomb-repressed genes during differentiation283. These effects are in agreement with earlier reports of PRC1 supporting gene expression in resting B cells and T cells280.Although Polycomb complexes appear to primarily support transcription repression, there is increasing evidence that in some contexts, Polycomb complexes can potentiate gene expression286. Specifically, it has been proposed that, by supporting long-range chromatin interactions, cPRC1 can bring into close proximity enhancers that are poised for activation and their target promoters, thereby enabling rapid induction of transcription in response to activation signals during cell differentiation285 –MLL2 (KMT2D) and SETD1A–SETD1B290. Interestingly, some Trithorax group complexes can also sample CGIs through zinc finger-CxxC DNA binding domains, but in contrast to the Polycomb complexes, they initiate the formation and spreading of transcription-permissive, H3K4me3-containing Trithorax chromatin domains through communication and feedback mechanisms linked to active transcription296. Antagonism between the Polycomb and Trithorax complexes helps to ensure that at most gene promoters, either a transcriptionally repressive or a transcriptionally permissive chromatin state predominates.During cellular differentiation, Polycomb target genes can be activated to support new cellular functions300 (301 (Arabidopsis thaliana303, and there is some evidence that this gene expression switch also occurs in mammals304. We envisage that switch-like transitions in the expression of master regulators of cell fate could help to ensure decisive cell fate decisions, and Polycomb and Trithorax complexes are known to have important roles in regulating these types of genes during mammalian development3. Furthermore, once individual genes acquire defined chromatin states, this could provide hysteresis of their current transcription status to help maintain cell identify in the face of inherently stochastic and pulsatile transcription signals.Based on these feedback mechanisms and antagonism between the Polycomb and Trithorax systems, CGIs have been proposed to support the formation of bistable chromatin states at gene promoters to regulate gene expression300 . In defi300 (301 . By cont300 (301 . In supp303. Importantly, interrogation of these models also suggests that bivalent chromatin states, which are characterised by the combined presence of both H3K4me3 and H3K27me3 and previously proposed to define an alternative poised transcriptional state305, are unlikely to be stable and may instead correspond to the transition between Polycomb and Trithorax chromatin states. Although many of the underlying biochemical activities of Polycomb and Trithorax systems are consistent with the possibility that bistable chromatin states could be created at their target sites, a major challenge for our research field will be to directly test these models and their relevance to the kinetics of gene expression during cell differentiation.Recent mathematical models incorporating the known activities of Polycomb and Trithorax systems have provided theoretical evidence to support the existence of bistable chromatin states at gene regulatory elementsin vivo, we anticipate that addressing this important question will require a reductionist approach leveraging in vitro transcription reactions on reconstituted chromatin templates, and single-molecule approaches that can capture the behaviour of the transcription machinery307.Detailed biochemical and structural characterisation has revealed that the Polycomb system comprises a diverse array of multi-protein complexes that are highly specialised in how they engage with nucleosomes, catalyse histone modifications and function through communication and feedback mechanisms to form Polycomb chromatin domains. Our understanding of the mechanics that underpin these processes has advanced rapidly in recent years, but it still remains poorly understood at the mechanistic level how, once formed, Polycomb chromatin domains influence the transcription machinery to enable gene repression. This represents a central unresolved question in our field. Given the complexity of gene regulatory elements in vivo has indicated they are more dynamic than originally anticipated and, somewhat counterintuitively, appear to respond to transcription in forming Polycomb chromatin domains. This is forcing us to rethink how the Polycomb system both influences, and is related to, gene transcription. In this Review, we have discussed some emerging molecular principles that could explain these dynamic behaviours. However, the static and ensemble experimental approaches that our field has relied on until now are largely unsuited to exploring the relevance of these concepts. Instead, this will require live, single-cell, and quantitative measurements. These approaches will be of particular relevance in determining whether the Polycomb system enables chromatin bistablity, how it shapes the kinetics of gene-expression transitions during cell differentiation and development, and how the functions of Polycomb complexes are disrupted in human disease.Building on these biochemical underpinnings, the molecular study of how Polycomb systems function Finally, much of our understanding of the mammalian Polycomb system has come from studies in stem cells and in early developmental contexts. Although the ideas and models discussed in this Review primarily draw on this body of work, it is becoming increasingly clear that the mechanisms that guide Polycomb function in specific tissues and at later developmental stages are likely to vary considerably. Therefore, moving forward, a central objective remains to uncover the complement of mechanisms and principles that define mammalian Polycomb-mediated gene regulation in diverse cellular and developmental contexts.As our field ventures into testing the relevance of new and emerging concepts in Polycomb biology, the coming years will inevitably be a fast-paced and exciting period in the ongoing quest to uncover the molecular principles that define gene regulation by Polycomb repressive complexes.