Alternative splicing decouples local from global PRC2 activity.

The Polycomb repressive complex 2 (PRC2) mediates epigenetic maintenance of gene silencing in eukaryotes via methylation of histone H3 at lysine 27 (H3K27). Accessory factors define two distinct subtypes, PRC2.1 and PRC2.2, with different actions and chromatin-targeting mechanisms. The mechanisms orchestrating PRC2 assembly are not fully understood. Here, we report that alternative splicing (AS) of PRC2 core component SUZ12 generates an uncharacterized isoform SUZ12-S, which co-exists with the canonical SUZ12-L isoform in virtually all tissues and developmental stages. SUZ12-S drives PRC2.1 formation and favors PRC2 dimerization. While SUZ12-S is necessary and sufficient for the repression of target genes via promoter-proximal H3K27me3 deposition, SUZ12-L maintains global H3K27 methylation levels. Mouse embryonic stem cells (ESCs) lacking either isoform exit pluripotency more slowly and fail to acquire neuronal cell identity. Our findings reveal a physiological mechanism regulating PRC2 assembly and higher-order interactions in eutherians, with impacts on H3K27 methylation and gene repression.

MAF amplification licenses ERα through epigenetic remodelling to drive breast cancer metastasis.

MAF amplification increases the risk of breast cancer (BCa) metastasis through mechanisms that are still poorly understood yet have important clinical implications. Oestrogen-receptor-positive (ER+) BCa requires oestrogen for both growth and metastasis, albeit by ill-known mechanisms. Here we integrate proteomics, transcriptomics, epigenomics, chromatin accessibility and functional assays from human and syngeneic mouse BCa models to show that MAF directly interacts with oestrogen receptor alpha (ERα), thereby promoting a unique chromatin landscape that favours metastatic spread. We identify metastasis-promoting genes that are de novo licensed following oestrogen exposure in a MAF-dependent manner. The histone demethylase KDM1A is key to the epigenomic remodelling that facilitates the expression of the pro-metastatic MAF/oestrogen-driven gene expression program, and loss of KDM1A activity prevents this metastasis. We have thus determined that the molecular basis underlying MAF/oestrogen-mediated metastasis requires genetic, epigenetic and hormone signals from the systemic environment, which influence the ability of BCa cells to metastasize.

Thymine DNA glycosylase regulates cell-cycle-driven p53 transcriptional control in pluripotent cells.

Cell cycle progression is linked to transcriptome dynamics and variations in the response of pluripotent cells to differentiation cues, mostly through unknown determinants. Here, we characterized the cell-cycle-associated transcriptome and proteome of mouse embryonic stem cells (mESCs) in naive ground state. We found that the thymine DNA glycosylase (TDG) is a cell-cycle-regulated co-factor of the tumor suppressor p53. Furthermore, TDG and p53 co-bind ESC-specific cis-regulatory elements and thereby control transcription of p53-dependent genes during self-renewal. We determined that the dynamic expression of TDG is required to promote the cell-cycle-associated transcriptional heterogeneity. Moreover, we demonstrated that transient depletion of TDG influences cell fate decisions during the early differentiation of mESCs. Our findings reveal an unanticipated role of TDG in promoting molecular heterogeneity during the cell cycle and highlight the central role of protein dynamics for the temporal control of cell fate during development.

Quantitative Comparison of Multiple Chromatin Immunoprecipitation-Sequencing (ChIP-seq) Experiments with spikChIP.

The chromatin immunoprecipitation coupled with the next-generation sequencing (ChIP-seq) is a powerful technique that enables to characterize the genomic distribution of chromatin-associated proteins, histone posttranslational modifications, and histone variants. However, in the absence of a reference control for monitoring experimental and biological variations, the standard ChIP-seq scheme is unable to accurately assess changes in the abundance of chromatin targets across different experimental samples. To overcome this limitation, the combination of external spike-in material with the experimental chromatin is offered as an effective solution for quantitative comparison of ChIP-seq data across different conditions. Here, we detail (i) the experimental protocol for preparing quality control spike-in chromatin from Drosophila melanogaster cells and (ii) the computational protocol to compare ChIP-seq samples with spike-in based on the use of the spikChIP software.

In vivo temporal resolution of acute promyelocytic leukemia progression reveals a role of Klf4 in suppressing early leukemic transformation.

Genome organization plays a pivotal role in transcription, but how transcription factors (TFs) rewire the structure of the genome to initiate and maintain the programs that lead to oncogenic transformation remains poorly understood. Acute promyelocytic leukemia (APL) is a fatal subtype of leukemia driven by a chromosomal translocation between the promyelocytic leukemia (PML) and retinoic acid receptor α (RARα) genes. We used primary hematopoietic stem and progenitor cells (HSPCs) and leukemic blasts that express the fusion protein PML-RARα as a paradigm to temporally dissect the dynamic changes in the epigenome, transcriptome, and genome architecture induced during oncogenic transformation. We found that PML-RARα initiates a continuum of topologic alterations, including switches from A to B compartments, transcriptional repression, loss of active histone marks, and gain of repressive histone marks. Our multiomics-integrated analysis identifies Klf4 as an early down-regulated gene in PML-RARα-driven leukemogenesis. Furthermore, we characterized the dynamic alterations in the Klf4 cis-regulatory network during APL progression and demonstrated that ectopic Klf4 overexpression can suppress self-renewal and reverse the differentiation block induced by PML-RARα. Our study provides a comprehensive in vivo temporal dissection of the epigenomic and topological reprogramming induced by an oncogenic TF and illustrates how topological architecture can be used to identify new drivers of malignant transformation.

Differential contribution to gene expression prediction of histone modifications at enhancers or promoters.

The ChIP-seq signal of histone modifications at promoters is a good predictor of gene expression in different cellular contexts, but whether this is also true at enhancers is not clear. To address this issue, we develop quantitative models to characterize the relationship of gene expression with histone modifications at enhancers or promoters. We use embryonic stem cells (ESCs), which contain a full spectrum of active and repressed (poised) enhancers, to train predictive models. As many poised enhancers in ESCs switch towards an active state during differentiation, predictive models can also be trained on poised enhancers throughout differentiation and in development. Remarkably, we determine that histone modifications at enhancers, as well as promoters, are predictive of gene expression in ESCs and throughout differentiation and development. Importantly, we demonstrate that their contribution to the predictive models varies depending on their location in enhancers or promoters. Moreover, we use a local regression (LOESS) to normalize sequencing data from different sources, which allows us to apply predictive models trained in a specific cellular context to a different one. We conclude that the relationship between gene expression and histone modifications at enhancers is universal and different from promoters. Our study provides new insight into how histone modifications relate to gene expression based on their location in enhancers or promoters.

Polycomb Factor PHF19 Controls Cell Growth and Differentiation Toward Erythroid Pathway in Chronic Myeloid Leukemia Cells.

Polycomb group (PcG) of proteins are a group of highly conserved epigenetic regulators involved in many biological functions, such as embryonic development, cell proliferation, and adult stem cell determination. PHD finger protein 19 (PHF19) is an associated factor of Polycomb repressor complex 2 (PRC2), often upregulated in human cancers. In particular, myeloid leukemia cell lines show increased levels of PHF19, yet little is known about its function. Here, we have characterized the role of PHF19 in myeloid leukemia cells. We demonstrated that PHF19 depletion decreases cell proliferation and promotes chronic myeloid leukemia (CML) differentiation. Mechanistically, we have shown how PHF19 regulates the proliferation of CML through a direct regulation of the cell cycle inhibitor p21. Furthermore, we observed that MTF2, a PHF19 homolog, partially compensates for PHF19 depletion in a subset of target genes, instructing specific erythroid differentiation. Taken together, our results show that PHF19 is a key transcriptional regulator for cell fate determination and could be a potential therapeutic target for myeloid leukemia treatment.

RING1B recruits EWSR1-FLI1 and cooperates in the remodeling of chromatin necessary for Ewing sarcoma tumorigenesis.

Ewing sarcoma (EwS) is an aggressive tumor that affects adolescents and young adults. EwS is defined by a chromosomal translocation, EWSR1-FLI1 being the most common, that causes genome reprogramming through remodeling of enhancers. Here, we describe an unexpected function of RING1B, which is highly expressed in EwS. While retaining its repressive activity at Polycomb developmental regulated genes, RING1B colocalizes with EWSR1-FLI1 at active enhancers. We demonstrate that RING1B is necessary for the expression of key EWSR1-FLI1 targets by facilitating oncogene recruitment to their enhancers. Knockdown of RING1B impairs growth of tumor xenografts and expression of genes regulated by EWSR1-FLI1 bound enhancers. Pharmacological inhibition of AURKB with AZD1152 increases H2Aub levels causing down-regulation of RING1B/EWSR1-FLI1 common targets. Our findings demonstrate that RING1B is a critical modulator of EWSR1-FLI1-induced chromatin remodeling, and its inhibition is a potential therapeutic strategy for the treatment of these tumors.

PHF19 mediated regulation of proliferation and invasiveness in prostate cancer cells.

The Polycomb-like protein PHF19/PCL3 associates with PRC2 and mediates its recruitment to chromatin in embryonic stem cells. PHF19 is also overexpressed in many cancers. However, neither PHF19 targets nor misregulated pathways involving PHF19 are known. Here, we investigate the role of PHF19 in prostate cancer cells. We find that PHF19 interacts with PRC2 and binds to PRC2 targets on chromatin. PHF19 target genes are involved in proliferation, differentiation, angiogenesis, and extracellular matrix organization. Depletion of PHF19 triggers an increase in MTF2/PCL2 chromatin recruitment, with a genome-wide gain in PRC2 occupancy and H3K27me3 deposition. Transcriptome analysis shows that PHF19 loss promotes deregulation of key genes involved in growth, metastasis, invasion, and of factors that stimulate blood vessels formation. Consistent with this, PHF19 silencing reduces cell proliferation, while promotes invasive growth and angiogenesis. Our findings reveal a role for PHF19 in controlling the balance between cell proliferation and invasiveness in prostate cancer.

Promoter bivalency favors an open chromatin architecture in embryonic stem cells.

In embryonic stem cells (ESCs), developmental gene promoters are characterized by their bivalent chromatin state, with simultaneous modification by MLL2 and Polycomb complexes. Although essential for embryogenesis, bivalency is functionally not well understood. Here, we show that MLL2 plays a central role in ESC genome organization. We generate a catalog of bona fide bivalent genes in ESCs and demonstrate that loss of MLL2 leads to increased Polycomb occupancy. Consequently, promoters lose accessibility, long-range interactions are redistributed, and ESCs fail to differentiate. We pose that bivalency balances accessibility and long-range connectivity of promoters, allowing developmental gene expression to be properly modulated.

The Dynamic Regulatory Genome of Capsaspora and the Origin of Animal Multicellularity.

The unicellular ancestor of animals had a complex repertoire of genes linked to multicellular processes. This suggests that changes in the regulatory genome, rather than in gene innovation, were key to the origin of animals. Here, we carry out multiple functional genomic assays in Capsaspora owczarzaki, the unicellular relative of animals with the largest known gene repertoire for transcriptional regulation. We show that changing chromatin states, differential lincRNA expression, and dynamic cis-regulatory sites are associated with life cycle transitions in Capsaspora. Moreover, we demonstrate conservation of animal developmental transcription-factor networks and extensive network interconnection in this premetazoan organism. In contrast, however, Capsaspora lacks animal promoter types, and its regulatory sites are small, proximal, and lack signatures of animal enhancers. Overall, our results indicate that the emergence of animal multicellularity was linked to a major shift in genome cis-regulatory complexity, most notably the appearance of distal enhancer regulation.

Role of PRC2-associated factors in stem cells and disease.

The Polycomb group (PcG) of proteins form chromatin-binding complexes with histone-modifying activity. The two main PcG repressive complexes studied (PRC1 and PRC2) are generally associated with chromatin in its repressed state. PRC2 is responsible for methylation of histone H3 at lysine 27 (H3K27me3), an epigenetic mark that is linked with numerous biological processes, including development, adult homeostasis and cancer. The core canonical complex PRC2, which contains the EZH1/2, SUZ12 and EED proteins, may be extended and functionally manipulated through interactions with several other proteins. In this review, we focus on these PRC2-associated proteins. As PRC2 functions are diverse, the variability conferred by these sub-stoichiometrically associated members may help to understand specific changes in PRC2 activity, chromatin recruitment and distribution required for gene repression.

Distinct structural transitions of chromatin topological domains correlate with coordinated hormone-induced gene regulation.

The human genome is segmented into topologically associating domains (TADs), but the role of this conserved organization during transient changes in gene expression is not known. Here we describe the distribution of progestin-induced chromatin modifications and changes in transcriptional activity over TADs in T47D breast cancer cells. Using ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing), Hi-C (chromosome capture followed by high-throughput sequencing), and three-dimensional (3D) modeling techniques, we found that the borders of the ∼ 2000 TADs in these cells are largely maintained after hormone treatment and that up to 20% of the TADs could be considered as discrete regulatory units where the majority of the genes are either transcriptionally activated or repressed in a coordinated fashion. The epigenetic signatures of the TADs are homogeneously modified by hormones in correlation with the transcriptional changes. Hormone-induced changes in gene activity and chromatin remodeling are accompanied by differential structural changes for activated and repressed TADs, as reflected by specific and opposite changes in the strength of intra-TAD interactions within responsive TADs. Indeed, 3D modeling of the Hi-C data suggested that the structure of TADs was modified upon treatment. The differential responses of TADs to progestins and estrogens suggest that TADs could function as "regulons" to enable spatially proximal genes to be coordinately transcribed in response to hormones.

Progesterone receptor interaction with chromatin.

Understanding how eukaryotic gene regulation works implies unraveling the mechanisms used by transcription factors to access DNA information packaged in chromatin. The current view is that different cell types express different parts of the genome because they are equipped with different sets of transcription factors. A few transcription factors are called pioneer factors because they are able to bind to their sites in nucleosomes and to open up chromatin thus enabling access for other transcription factors, which are unable to recognize DNA packaged in nucleosomes. But it is also possible that the way DNA is organized in chromatin differs between cell types and contributes to cell identity by restricting or enhancing access to specific gene cohorts. To unravel these mechanisms we studied the interaction of progesterone receptor with the genome of breast cancer cells and found that it binds preferentially to sites organized in nucleosomes, which contribute to functional interactions leading to gene regulation.

CDC2 mediates progestin initiated endometrial stromal cell proliferation: a PR signaling to gene expression independently of its binding to chromatin.

Although non-genomic steroid receptor pathways have been studied over the past decade, little is known about the direct gene expression changes that take place as a consequence of their activation. Progesterone controls proliferation of rat endometrial stromal cells during the peri-implantation phase of pregnancy. We showed that picomolar concentration of progestin R5020 mimics this control in UIII endometrial stromal cells via ERK1-2 and AKT activation mediated by interaction of Progesterone Receptor (PR) with Estrogen Receptor beta (ERb) and without transcriptional activity of endogenous PR and ER. Here we identify early downstream targets of cytoplasmic PR signaling and their possible role in endometrial stromal cell proliferation. Microarray analysis of global gene expression changes in UIII cells treated for 45 min with progestin identified 97 up- and 341 down-regulated genes. The most over-represented molecular functions were transcription factors and regulatory factors associated with cell proliferation and cell cycle, a large fraction of which were repressors down-regulated by hormone. Further analysis verified that progestins regulate Ccnd1, JunD, Usf1, Gfi1, Cyr61, and Cdkn1b through PR-mediated activation of ligand-free ER, ERK1-2 or AKT, in the absence of genomic PR binding. ChIP experiments show that progestin promoted the interaction of USF1 with the proximal promoter of the Cdc2 gene. Usf1 knockdown abolished Cdc2 progestin-dependent transcriptional regulation and cell proliferation, which also blocked Cdc2 knockdown. We conclude that progestin-induced proliferation of endometrial stromal cells is mediated by ERK1-2 and AKT dependent early regulation of USF1, which directly induces Cdc2. To our knowledge, this is the first description of early target genes of progestin-activated classical PR via crosstalk with protein kinases and independently of hormone receptor binding to the genomic targets.

Genome-wide activity of unliganded estrogen receptor-α in breast cancer cells.

Estrogen receptor-α (ERα) has central role in hormone-dependent breast cancer and its ligand-induced functions have been extensively characterized. However, evidence exists that ERα has functions that are independent of ligands. In the present work, we investigated the binding of ERα to chromatin in the absence of ligands and its functions on gene regulation. We demonstrated that in MCF7 breast cancer cells unliganded ERα binds to more than 4,000 chromatin sites. Unexpectedly, although almost entirely comprised in the larger group of estrogen-induced binding sites, we found that unliganded-ERα binding is specifically linked to genes with developmental functions, compared with estrogen-induced binding. Moreover, we found that siRNA-mediated down-regulation of ERα in absence of estrogen is accompanied by changes in the expression levels of hundreds of coding and noncoding RNAs. Down-regulated mRNAs showed enrichment in genes related to epithelial cell growth and development. Stable ERα down-regulation using shRNA, which caused cell growth arrest, was accompanied by increased H3K27me3 at ERα binding sites. Finally, we found that FOXA1 and AP2γ binding to several sites is decreased upon ERα silencing, suggesting that unliganded ERα participates, together with other factors, in the maintenance of the luminal-specific cistrome in breast cancer cells.

More help than hindrance: nucleosomes aid transcriptional regulation.

A major challenge of modern human biology is to understand how a differentiated somatic cell integrates the response to external signals in the complex context of basic metabolic and tissue-specific gene expression programs. This requires exploring two interconnected basic processes: the signaling network and the global function of the key transcription factors on which signaling acts to modulate gene expression. An apparently simple model to study these questions has been steroid hormones action, since their intracellular receptors both initiate signaling and are the key transcription factors orchestrating the cellular response. We have used progesterone action in breast cancer cells to elucidate the intricacies of progesterone receptor (PR) signaling crosstalk with protein kinases, histone modifying enzymes and ATP-dependent chromatin remodeling complexes. ( 1) Recently we have described the cistrome of PR in these cells at different times after addition of hormone and its relationship to chromatin structure. ( 2) The role of chromatin in transcription factor binding to the genome is still debated, but the dominant view is that factors bind preferentially to nucleosome-depleted regions, usually identified as DNaseI-hypersensitive sites (DHS). In contrast with this vision, we have shown that PR requires nucleosomes for optimal binding and function. In breast cancer cells treated with progestins we identified 25,000 PR binding sites (PRbs), the majority encompassing several copies of the hexanucleotide TGTYCY, highly abundant in the genome. We found that strong functional PRbs accumulate around progesterone-induced genes mainly in enhancers, are enriched in DHS but exhibit high nucleosome occupancy. Progestin stimulation results in remodeling of these nucleosomes with displacement of histones H1 and H2A/H2B dimers. Our results strongly suggest that nucleosomes play crucial role in PR binding and hormonal gene regulation.

Progesterone receptor induces bcl-x expression through intragenic binding sites favoring RNA polymerase II elongation.

Steroid receptors were classically described for regulating transcription by binding to target gene promoters. However, genome-wide studies reveal that steroid receptors-binding sites are mainly located at intragenic regions. To determine the role of these sites, we examined the effect of progestins on the transcription of the bcl-x gene, where only intragenic progesterone receptor-binding sites (PRbs) were identified. We found that in response to hormone treatment, the PR is recruited to these sites along with two histone acetyltransferases CREB-binding protein (CBP) and GCN5, leading to an increase in histone H3 and H4 acetylation and to the binding of the SWI/SNF complex. Concomitant, a more relaxed chromatin was detected along bcl-x gene mainly in the regions surrounding the intragenic PRbs. PR also mediated the recruitment of the positive elongation factor pTEFb, favoring RNA polymerase II (Pol II) elongation activity. Together these events promoted the re-distribution of the active Pol II toward the 3'-end of the gene and a decrease in the ratio between proximal and distal transcription. These results suggest a novel mechanism by which PR regulates gene expression by facilitating the proper passage of the polymerase along hormone-dependent genes.

Nucleosome-driven transcription factor binding and gene regulation.

Elucidating the global function of a transcription factor implies the identification of its target genes and genomic binding sites. The role of chromatin in this context is unclear, but the dominant view is that factors bind preferentially to nucleosome-depleted regions identified as DNaseI-hypersensitive sites (DHS). Here we show by ChIP, MNase, and DNaseI assays followed by deep sequencing that the progesterone receptor (PR) requires nucleosomes for optimal binding and function. In breast cancer cells treated with progestins, we identified 25,000 PR binding sites (PRbs). The majority of these sites encompassed several copies of the hexanucleotide TGTYCY, which is highly abundant in the genome. We found that functional PRbs accumulate around progesterone-induced genes, mainly in enhancers. Most of these sites overlap with DHS but exhibit high nucleosome occupancy. Progestin stimulation results in remodeling of these nucleosomes with displacement of histones H1 and H2A/H2B dimers. Our results strongly suggest that nucleosomes are crucial for PR binding and hormonal gene regulation.

Phf19 links methylated Lys36 of histone H3 to regulation of Polycomb activity.

Polycomb-group proteins are transcriptional repressors with essential roles in embryonic development. Polycomb repressive complex 2 (PRC2) contains the methyltransferase activity for Lys27. However, the role of other histone modifications in regulating PRC2 activity is just beginning to be understood. Here we show that direct recognition of methylated histone H3 Lys36 (H3K36me), a mark associated with activation, by the PRC2 subunit Phf19 is required for the full enzymatic activity of the PRC2 complex. Using NMR spectroscopy, we provide structural evidence for this interaction. Furthermore, we show that Phf19 binds to a subset of PRC2 targets in mouse embryonic stem cells and that this is required for their repression and for H3K27me3 deposition. These findings show that the interaction of Phf19 with H3K36me2 and H3K36me3 is essential for PRC2 complex activity and for proper regulation of gene repression in embryonic stem cells.