PHF2-mediated H3K9me balance orchestrates heterochromatin stability and neural progenitor proliferation.

Heterochromatin stability is crucial for progenitor proliferation during early neurogenesis. It relays on the maintenance of local hubs of H3K9me. However, understanding the formation of efficient localized levels of H3K9me remains limited. To address this question, we used neural stem cells to analyze the function of the H3K9me2 demethylase PHF2, which is crucial for progenitor proliferation. Through mass-spectroscopy and genome-wide assays, we show that PHF2 interacts with heterochromatin components and is enriched at pericentromeric heterochromatin (PcH) boundaries where it maintains transcriptional activity. This binding is essential for silencing the satellite repeats, preventing DNA damage and genome instability. PHF2's depletion increases the transcription of heterochromatic repeats, accompanied by a decrease in H3K9me3 levels and alterations in PcH organization. We further show that PHF2's PHD and catalytic domains are crucial for maintaining PcH stability, thereby safeguarding genome integrity. These results highlight the multifaceted nature of PHF2's functions in maintaining heterochromatin stability and regulating gene expression during neural development. Our study unravels the intricate relationship between heterochromatin stability and progenitor proliferation during mammalian neurogenesis.

LATS1 controls CTCF chromatin occupancy and hormonal response of 3D-grown breast cancer cells.

The cancer epigenome has been studied in cells cultured in two-dimensional (2D) monolayers, but recent studies highlight the impact of the extracellular matrix and the three-dimensional (3D) environment on multiple cellular functions. Here, we report the physical, biochemical, and genomic differences between T47D breast cancer cells cultured in 2D and as 3D spheroids. Cells within 3D spheroids exhibit a rounder nucleus with less accessible, more compacted chromatin, as well as altered expression of ~2000 genes, the majority of which become repressed. Hi-C analysis reveals that cells in 3D are enriched for regions belonging to the B compartment, have decreased chromatin-bound CTCF and increased fusion of topologically associating domains (TADs). Upregulation of the Hippo pathway in 3D spheroids results in the activation of the LATS1 kinase, which promotes phosphorylation and displacement of CTCF from DNA, thereby likely causing the observed TAD fusions. 3D cells show higher chromatin binding of progesterone receptor (PR), leading to an increase in the number of hormone-regulated genes. This effect is in part mediated by LATS1 activation, which favors cytoplasmic retention of YAP and CTCF removal.

Linker histone H1 regulates homeostasis of heterochromatin-associated cRNAs.

Chromatin-associated RNAs (cRNAs) are a poorly characterized fraction of cellular RNAs that co-purify with chromatin. Their full complexity and the mechanisms regulating their packaging and chromatin association remain poorly understood. Here, we address these questions in Drosophila. We find that cRNAs constitute a heterogeneous group of RNA species that is abundant in heterochromatic transcripts. We show that heterochromatic cRNAs interact with the heterogeneous nuclear ribonucleoproteins (hnRNP) hrp36/hrp48 and that depletion of linker histone dH1 impairs this interaction. dH1 depletion induces the accumulation of RNA::DNA hybrids (R-loops) in heterochromatin and, as a consequence, increases retention of heterochromatic cRNAs. These effects correlate with increased RNA polymerase II (RNAPII) occupancy at heterochromatin. Notably, impairing cRNA assembly by depletion of hrp36/hrp48 mimics heterochromatic R-loop accumulation induced by dH1 depletion. We also show that dH1 depletion alters nucleosome organization, increasing accessibility of heterochromatin. Altogether, these perturbations facilitate annealing of cRNAs to the DNA template, enhancing R-loop formation and cRNA retention at heterochromatin.

Choosing the right partner in hormone-dependent gene regulation: Glucocorticoid and progesterone receptors crosstalk in breast cancer cells.

Steroid hormone receptors (SHRs) belong to a large family of ligand-activated nuclear receptors that share certain characteristics and possess others that make them unique. It was thought for many years that the specificity of hormone response lay in the ligand. Although this may be true for pure agonists, the natural ligands as progesterone, corticosterone and cortisol present a broader effect by simultaneous activation of several SHRs. Moreover, SHRs share structural and functional characteristics that range from similarities between ligand-binding pockets to recognition of specific DNA sequences. These properties are clearly evident in progesterone (PR) and glucocorticoid receptors (GR); however, the biological responses triggered by each receptor in the presence of its ligand are different, and in some cases, even opposite. Thus, what confers the specificity of response to a given receptor is a long-standing topic of discussion that has not yet been unveiled. The levels of expression of each receptor, the differential interaction with coregulators, the chromatin accessibility as well as the DNA sequence of the target regions in the genome, are reliable sources of variability in hormone action that could explain the results obtained so far. Yet, to add further complexity to this scenario, it has been described that receptors can form heterocomplexes which can either compromise or potentiate the respective hormone-activated pathways with its possible impact on the pathological condition. In the present review, we summarized the state of the art of the functional cross-talk between PR and GR in breast cancer cells and we also discussed new paradigms of specificity in hormone action.

Chromatin topology defines estradiol-primed progesterone receptor and PAX2 binding in endometrial cancer cells.

Estrogen (E2) and Progesterone (Pg), via their specific receptors (ERalpha and PR), are major determinants in the development and progression of endometrial carcinomas, However, their precise mechanism of action and the role of other transcription factors involved are not entirely clear. Using Ishikawa endometrial cancer cells, we report that E2 treatment exposes a set of progestin-dependent PR binding sites which include both E2 and progestin target genes. ChIP-seq results from hormone-treated cells revealed a non-random distribution of PAX2 binding in the vicinity of these estrogen-promoted PR sites. Altered expression of hormone regulated genes in PAX2 knockdown cells suggests a role for PAX2 in fine-tuning ERalpha and PR interplay in transcriptional regulation. Analysis of long-range interactions by Hi-C coupled with ATAC-seq data showed that these regions, that we call 'progestin control regions' (PgCRs), exhibited an open chromatin state even before hormone exposure and were non-randomly associated with regulated genes. Nearly 20% of genes potentially influenced by PgCRs were found to be altered during progression of endometrial cancer. Our findings suggest that endometrial response to progestins in differentiated endometrial tumor cells results in part from binding of PR together with PAX2 to accessible chromatin regions. What maintains these regions open remains to be studied.

Histone acetylation of bile acid transporter genes plays a critical role in cirrhosis.

BACKGROUND & AIMS: Owing to the lack of genetic animal models that adequately recreate key clinical characteristics of cirrhosis, the molecular pathogenesis of cirrhosis has been poorly characterized, and treatments remain limited. Hence, we aimed to better elucidate the pathological mechanisms of cirrhosis using a novel murine model. METHODS: We report on the first murine genetic model mimicking human cirrhosis induced by hepatocyte-specific elimination of microspherule protein 1 (MCRS1), a member of non-specific lethal (NSL) and INO80 chromatin-modifier complexes. Using this genetic tool with other mouse models, cell culture and human samples, combined with quantitative proteomics, single nuclei/cell RNA sequencing and chromatin immunoprecipitation assays, we investigated mechanisms of cirrhosis. RESULTS: MCRS1 loss in mouse hepatocytes modulates the expression of bile acid (BA) transporters - with a pronounced downregulation of Na+-taurocholate cotransporting polypeptide (NTCP) - concentrating BAs in sinusoids and thereby activating hepatic stellate cells (HSCs) via the farnesoid X receptor (FXR), which is predominantly expressed in human and mouse HSCs. Consistently, re-expression of NTCP in mice reduces cirrhosis, and genetic ablation of FXR in HSCs suppresses fibrotic marks in mice and in vitro cell culture. Mechanistically, deletion of a putative SANT domain from MCRS1 evicts histone deacetylase 1 from its histone H3 anchoring sites, increasing histone acetylation of BA transporter genes, modulating their expression and perturbing BA flow. Accordingly, human cirrhosis displays decreased nuclear MCRS1 and NTCP expression. CONCLUSIONS: Our data reveal a previously unrecognized function of MCRS1 as a critical histone acetylation regulator, maintaining gene expression and liver homeostasis. MCRS1 loss induces acetylation of BA transporter genes, perturbation of BA flow, and consequently, FXR activation in HSCs. This axis represents a central and universal signaling event in cirrhosis, which has significant implications for cirrhosis treatment. LAY SUMMARY: By genetic ablation of MCRS1 in mouse hepatocytes, we generate the first genetic mouse model of cirrhosis that recapitulates human features. Herein, we demonstrate that the activation of the bile acid/FXR axis in liver fibroblasts is key in cirrhosis development.

A set of accessible enhancers enables the initial response of breast cancer cells to physiological progestin concentrations.

Here, we report that in T47D breast cancer cells 50 pM progestin is sufficient to activate cell cycle entry and the progesterone gene expression program. At this concentration, equivalent to the progesterone blood levels found around the menopause, progesterone receptor (PR) binds only to 2800 genomic sites, which are accessible to ATAC cleavage prior to hormone exposure. These highly accessible sites (HAs) are surrounded by well-organized nucleosomes and exhibit breast enhancer features, including estrogen receptor alpha (ERα), higher FOXA1 and BRD4 (bromodomain containing 4) occupancy. Although HAs are enriched in RAD21 and CTCF, PR binding is the driving force for the most robust interactions with hormone-regulated genes. HAs show higher frequency of 3D contacts among themselves than with other PR binding sites, indicating colocalization in similar compartments. Gene regulation via HAs is independent of classical coregulators and ATP-activated remodelers, relying mainly on MAP kinase activation that enables PR nuclear engagement. HAs are also preferentially occupied by PR and ERα in breast cancer xenografts derived from MCF-7 cells as well as from patients, indicating their potential usefulness as targets for therapeutic intervention.

Glucocorticoids uncover a critical role for ASH2L on BCL-X expression regulation in leukemia cells.

Targeting the apoptosis machinery is a promising therapeutic approach in myeloid malignancies. BCL2L1 is a well-known glucocorticoid-responsive gene and a key apoptosis regulator that, when over-expressed, can contribute to tumor development, progression and therapeutic resistance. Moreover, synthetic glucocorticoids, like dexamethasone, are frequently used in the treatment of hematopoietic diseases due to its pro-apoptotic properties. We report here that the trithorax protein ASH2L, considered one of the core subunits of H3K4-specific MLL/SET methyltransferase complexes, contributes to anti-apoptotic BCL-XL over-expression and cell survival in patient-derived myeloid leukemia cells. We find that the unliganded glucocorticoid receptor (uGR) and ASH2L interact in a common protein complex through a chromatin looping determined by uGR and ASH2L binding to BCL2L1 specific +58 HRE and promoter region, respectively. Upon addition of dexamethasone, GR and ASH2L recruitment is reduced, BCL-XL expression diminishes and apoptosis is induced consequently. Overall, our findings indicate that uGR and ASH2L may act as key regulatory players of BCL- XL upregulation in AML cells.

The glucocorticoid receptor interferes with progesterone receptor-dependent genomic regulation in breast cancer cells.

The glucocorticoid and progesterone receptors (GR and PR) are closely related members of the steroid receptor family. Despite sharing similar structural and functional characteristics; the cognate hormones display very distinct physiological responses. In mammary epithelial cells, PR activation is associated with the incidence and progression of breast cancer, whereas the GR is related to growth suppression and differentiation. Despite their pharmacological relevance, only a few studies have compared GR and PR activities in the same system. Using a PR+/GR+ breast cancer cell line, here we report that either glucocorticoid-free or dexamethasone (DEX)-activated GR inhibits progestin-dependent gene expression associated to epithelial-mesenchymal-transition and cell proliferation. When both receptors are activated with their cognate hormones, PR and GR can form part of the same complex according to co-immunoprecipitation, quantitative microscopy and sequential ChIP experiments. Moreover, genome-wide studies in cells treated with either DEX or R5020, revealed the presence of several regions co-bound by both receptors. Surprisingly, GR also binds novel genomic sites in cells treated with R5020 alone. This progestin-induced GR binding was enriched in REL DNA motifs and located close to genes coding for chromatin remodelers. Understanding GR behavior in the context of progestin-dependent breast cancer could provide new targets for tumor therapy.

C/EBPα mediates the growth inhibitory effect of progestins on breast cancer cells.

Steroid hormones are key gene regulators in breast cancer cells. While estrogens stimulate cell proliferation, progestins activate a single cell cycle followed by proliferation arrest. Here, we use biochemical and genome-wide approaches to show that progestins achieve this effect via a functional crosstalk with C/EBPα. Using ChIP-seq, we identify around 1,000 sites where C/EBPα binding precedes and helps binding of progesterone receptor (PR) in response to hormone. These regions exhibit epigenetic marks of active enhancers, and C/EBPα maintains an open chromatin conformation that facilitates loading of ligand-activated PR. Prior to hormone exposure, C/EBPα favors promoter-enhancer contacts that assure hormonal regulation of key genes involved in cell proliferation by facilitating binding of RAD21, YY1, and the Mediator complex. Knockdown of C/EBPα disrupts enhancer-promoter contacts and decreases the presence of these architectural proteins, highlighting its key role in 3D chromatin looping. Thus, C/EBPα fulfills a previously unknown function as a potential growth modulator in hormone-dependent breast cancer.

Hormone-control regions mediate steroid receptor-dependent genome organization.

In breast cancer cells, some topologically associating domains (TADs) behave as hormonal gene regulation units, within which gene transcription is coordinately regulated in response to steroid hormones. Here we further describe that responsive TADs contain 20- to 100-kb-long clusters of intermingled estrogen receptor (ESR1) and progesterone receptor (PGR) binding sites, hereafter called hormone-control regions (HCRs). In T47D cells, we identified more than 200 HCRs, which are frequently bound by unliganded ESR1 and PGR. These HCRs establish steady long-distance inter-TAD interactions between them and organize characteristic looping structures with promoters in their TADs even in the absence of hormones in ESR1+-PGR+ cells. This organization is dependent on the expression of the receptors and is further dynamically modulated in response to steroid hormones. HCRs function as platforms that integrate different signals, resulting in some cases in opposite transcriptional responses to estrogens or progestins. Altogether, these results suggest that steroid hormone receptors act not only as hormone-regulated sequence-specific transcription factors but also as local and global genome organizers.

Steroid hormone receptors silence genes by a chromatin-targeted mechanism similar to those used for gene activation.

How genes are repressed by steroid hormones remains a matter of debate, and several indirect mechanisms have been proposed. We found that the ligand-activated progesterone receptor recruits to the promoter of downregulated genes a repressor complex composed of HP1γ, the lysine demethylase LSD1, histone deacetylases, coREST, the RNA SRA, and the ATPase BRG1. BRG1 is needed for chromatin remodeling and facilitates the deposition of linker histone variant H1.2, which compacts chromatin and hinders RNA polymerase loading and transcription. Thus, steroid hormone receptors can repress genes in ways reminiscent of those used for gene induction, namely by directly targeting factors that remodel chromatin. But while PR-dependent gene induction in T47D cells is mainly achieved by potentiating enhancer activity, repression acts at the level of gene promoters.

Hormone-induced repression of genes requires BRG1-mediated H1.2 deposition at target promoters.

Eukaryotic gene regulation is associated with changes in chromatin compaction that modulate access to DNA regulatory sequences relevant for transcriptional activation or repression. Although much is known about the mechanism of chromatin remodeling in hormonal gene activation, how repression is accomplished is much less understood. Here we report that in breast cancer cells, ligand-activated progesterone receptor (PR) is directly recruited to transcriptionally repressed genes involved in cell proliferation along with the kinases ERK1/2 and MSK1. PR recruits BRG1 associated with the HP1γ-LSD1 complex repressor complex, which is further anchored via binding of HP1γ to the H3K9me3 signal deposited by SUV39H2. In contrast to what is observed during gene activation, only BRG1 and not the BAF complex is recruited to repressed promoters, likely due to local enrichment of the pioneer factor FOXA1. BRG1 participates in gene repression by interacting with H1.2, facilitating its deposition and stabilizing nucleosome positioning around the transcription start site. Our results uncover a mechanism of hormone-dependent transcriptional repression and a novel role for BRG1 in progestin regulation of breast cancer cell growth.

ADP-ribose-derived nuclear ATP synthesis by NUDIX5 is required for chromatin remodeling.

Key nuclear processes in eukaryotes, including DNA replication, repair, and gene regulation, require extensive chromatin remodeling catalyzed by energy-consuming enzymes. It remains unclear how the ATP demands of such processes are met in response to rapid stimuli. We analyzed this question in the context of the massive gene regulation changes induced by progestins in breast cancer cells and found that ATP is generated in the cell nucleus via the hydrolysis of poly(ADP-ribose) to ADP-ribose. In the presence of pyrophosphate, ADP-ribose is used by the pyrophosphatase NUDIX5 to generate nuclear ATP. The nuclear source of ATP is essential for hormone-induced chromatin remodeling, transcriptional regulation, and cell proliferation.

C/EBPα Activates Pre-existing and De Novo Macrophage Enhancers during Induced Pre-B Cell Transdifferentiation and Myelopoiesis.

Transcription-factor-induced somatic cell conversions are highly relevant for both basic and clinical research yet their mechanism is not fully understood and it is unclear whether they reflect normal differentiation processes. Here we show that during pre-B-cell-to-macrophage transdifferentiation, C/EBPα binds to two types of myeloid enhancers in B cells: pre-existing enhancers that are bound by PU.1, providing a platform for incoming C/EBPα; and de novo enhancers that are targeted by C/EBPα, acting as a pioneer factor for subsequent binding by PU.1. The order of factor binding dictates the upregulation kinetics of nearby genes. Pre-existing enhancers are broadly active throughout the hematopoietic lineage tree, including B cells. In contrast, de novo enhancers are silent in most cell types except in myeloid cells where they become activated by C/EBP factors. Our data suggest that C/EBPα recapitulates physiological developmental processes by short-circuiting two macrophage enhancer pathways in pre-B cells.

DNA damage and gene transcription: accident or necessity?

The extent to which DNA repair machinery facilitates gene activation remains poorly appreciated. A new study published in Cell Research reports a novel function of H2AX, a substrate of ATM and known DNA damage marker, in transcriptional initiation.

The chromatin Remodeler CHD8 is required for activation of progesterone receptor-dependent enhancers.

While the importance of gene enhancers in transcriptional regulation is well established, the mechanisms and the protein factors that determine enhancers activity have only recently begun to be unravelled. Recent studies have shown that progesterone receptor (PR) binds regions that display typical features of gene enhancers. Here, we show by ChIP-seq experiments that the chromatin remodeler CHD8 mostly binds promoters under proliferation conditions. However, upon progestin stimulation, CHD8 re-localizes to PR enhancers also enriched in p300 and H3K4me1. Consistently, CHD8 depletion severely impairs progestin-dependent gene regulation. CHD8 binding is PR-dependent but independent of the pioneering factor FOXA1. The SWI/SNF chromatin-remodelling complex is required for PR-dependent gene activation. Interestingly, we show that CHD8 interacts with the SWI/SNF complex and that depletion of BRG1 and BRM, the ATPases of SWI/SNF complex, impairs CHD8 recruitment. We also show that CHD8 is not required for H3K27 acetylation, but contributes to increase accessibility of the enhancer to DNaseI. Furthermore, CHD8 was required for RNAPII recruiting to the enhancers and for transcription of enhancer-derived RNAs (eRNAs). Taken together our data demonstrate that CHD8 is involved in late stages of PR enhancers activation.

Relationship between nucleosome positioning and progesterone-induced alternative splicing in breast cancer cells.

Splicing of mRNA precursors can occur cotranscriptionally and it has been proposed that chromatin structure influences splice site recognition and regulation. Here we have systematically explored potential links between nucleosome positioning and alternative splicing regulation upon progesterone stimulation of breast cancer cells. We confirm preferential nucleosome positioning in exons and report four distinct profiles of nucleosome density around alternatively spliced exons, with RNA polymerase II accumulation closely following nucleosome positioning. Hormone stimulation induces switches between profile classes, correlating with a subset of alternative splicing changes. Hormone-induced exon inclusion often correlates with higher nucleosome occupancy at the exon or the preceding intronic region and with higher RNA polymerase II accumulation. In contrast, exons skipped upon hormone stimulation display low nucleosome densities even before hormone treatment, suggesting that chromatin structure primes alternative splicing regulation. Skipped exons frequently harbor binding sites for hnRNP AB, a hormone-induced splicing regulator whose knock down prevents some hormone-induced skipping events. Collectively, our results argue that a variety of chromatin architecture mechanisms can influence alternative splicing decisions.