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.

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.

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.

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.

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.

The transcription factor GATA6 enables self-renewal of colon adenoma stem cells by repressing BMP gene expression.

Aberrant activation of WNT signalling and loss of BMP signals represent the two main alterations leading to the initiation of colorectal cancer (CRC). Here we screen for genes required for maintaining the tumour stem cell phenotype and identify the zinc-finger transcription factor GATA6 as a key regulator of the WNT and BMP pathways in CRC. GATA6 directly drives the expression of LGR5 in adenoma stem cells whereas it restricts BMP signalling to differentiated tumour cells. Genetic deletion of Gata6 from mouse colon adenomas increases the levels of BMP factors, which signal to block self-renewal of tumour stem cells. In human tumours, GATA6 competes with ß-catenin/TCF4 for binding to a distal regulatory region of the BMP4 locus that has been linked to increased susceptibility to development of CRC. Hence, GATA6 creates an environment permissive for CRC initiation by lowering the threshold of BMP signalling required for tumour stem cell expansion.