Daniel Soronellas

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.

CDK2-dependent activation of PARP-1 is required for hormonal gene regulation in breast cancer cells

Eukaryotic gene regulation implies that transcription factors gain access to genomic information via poorly understood processes involving activation and targeting of kinases, histone-modifying enzymes, and chromatin remodelers to chromatin. Here we report that progestin gene regulation in breast cancer cells requires a rapid and transient increase in poly-(ADP)-ribose (PAR), accompanied by a dramatic decrease of cellular NAD that could have broad implications in cell physiology. This rapid increase in nuclear PARylation is mediated by activation of PAR polymerase PARP-1 as a result of phosphorylation by cyclin-dependent kinase CDK2. Hormone-dependent phosphorylation of PARP-1 by CDK2, within the catalytic domain, enhances its enzymatic capabilities. Activated PARP-1 contributes to the displacement of histone H1 and is essential for regulation of the majority of hormone-responsive genes and for the effect of progestins on cell cycle progression. Both global chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) and gene expression analysis show a strong overlap between PARP-1 and CDK2. Thus, progestin gene regulation involves a novel signaling pathway that connects CDK2-dependent activation of PARP-1 with histone H1 displacement. Given the multiplicity of PARP targets, this new pathway could be used for the pharmacological management of breast cancer.

Unliganded progesterone receptor-mediated targeting of an RNA-containing repressive complex silences a subset of hormone-inducible genes

A close chromatin conformation precludes gene expression in eukaryotic cells. Genes activated by external cues have to overcome this repressive state by locally changing chromatin structure to a more open state. Although much is known about hormonal gene activation, how basal repression of regulated genes is targeted to the correct sites throughout the genome is not well understood. Here we report that in breast cancer cells, the unliganded progesterone receptor (PR) binds genomic sites and targets a repressive complex containing HP1γ (heterochromatin protein 1γ), LSD1 (lysine-specific demethylase 1), HDAC1/2, CoREST (corepressor for REST [RE1 {neuronal repressor element 1} silencing transcription factor]), KDM5B, and the RNA SRA (steroid receptor RNA activator) to 20% of hormone-inducible genes, keeping these genes silenced prior to hormone treatment. The complex is anchored via binding of HP1γ to H3K9me3 (histone H3 tails trimethylated on Lys 9). SRA interacts with PR, HP1γ, and LSD1, and its depletion compromises the loading of the repressive complex to target chromatin-promoting aberrant gene derepression. Upon hormonal treatment, the HP1γ-LSD1 complex is displaced from these constitutively poorly expressed genes as a result of rapid phosphorylation of histone H3 at Ser 10 mediated by MSK1, which is recruited to the target sites by the activated PR. Displacement of the repressive complex enables the loading of coactivators needed for chromatin remodeling and activation of this set of genes, including genes involved in apoptosis and cell proliferation. These results highlight the importance of the unliganded PR in hormonal regulation of breast cancer cells.

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

A nuclear power source in the cell DNA is packaged onto nucleosomes, the principal component of chromatin. This chromatin must be remodeled to allow gene transcription, DNA replication, and DNA repair machineries access to the enclosed DNA. Chromatin-remodeling complexes require high levels of cellular energy to do their job. Wright et al. show that the energy needed to remodel chromatin can be derived from a source, poly-ADP-ribose, in the cell nucleus, rather than by diffusion of ATP from mitochondria in the cytoplasm, the usual powerhouse of the cell. Poly-ADP-ribose is converted to ADP-ribose and then to ATP, which can be used to fuel chromatin remodeling within the nucleus. Science, this issue p. 1221 Energy needed to remodel chromatin to make DNA accessible can be generated in situ in the nucleus from ADP-ribose. 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.

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.

Activation of mitogen- and stress-activated kinase 1 is required for proliferation of breast cancer cells in response to estrogens or progestins.

Growth of breast cancers is often dependent on ovarian steroid hormones making the tumors responsive to antagonists of hormone receptors. However, eventually the tumors become hormone independent, raising the need to identify downstream targets for the inhibition of tumor growth. One possibility is to focus on the signaling mechanisms used by ovarian steroid hormones to induce breast cancer cell proliferation. Here we report that the mitogen- and stress-activated kinase 1 (MSK1) could be a potential druggable target. Using the breast cancer cell line T47D, we show that estrogens (E2) and progestins activate MSK1, which forms a complex with the corresponding hormone receptor. Inhibition of MSK1 activity with H89 or its depletion by MSK1 short hairpin RNAs (shRNAs) specifically abrogates cell proliferation in response to E2 or progestins without affecting serum-induced cell proliferation. MSK1 activity is required for the transition from the G1- to the S-phase of the cell cycle and inhibition of MSK1 compromises both estradiol- and progestin-dependent induction of cell cycle genes. ChIP-seq experiments identified binding of MSK1 to progesterone receptor-binding sites associated with hormone-responsive genes. MSK1 recruitment to epigenetically defined enhancer regions supports the need of MSK1 as a chromatin remodeler in hormone-dependent regulation of gene transcription. In agreement with this interpretation, expression of a histone H3 mutated at S10 eliminates the hormonal effect on cell proliferation and on induction of relevant target genes. Finally, we show that E2- or progestin-dependent growth of T47D cells xenografted in immunodefficient mice is inhibited by depletion of MSK1, indicating that our findings are not restricted to cultured cells, and that MSK1 plays an important role for hormone-dependent breast cancer growth in a more physiological context.

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.

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.

ADP-ribose derived Nuclear ATP is Required for Chromatin Remodeling and Hormonal Gene Regulation

Highlights – Hormonal gene regulation requires synthesis of PAR and its degradation to ADP-ribose by PARG – ADP-ribose is converted to ATP in the cell nuclei by hormone-activated NUDIX5/NUDT5 – Blocking nuclear ATP formation precludes hormone-induced chromatin remodeling, gene regulation and cell proliferation Summary Key nuclear processes in eukaryotes including DNA replication or repair and gene regulation require extensive chromatin remodeling catalyzed by energy consuming enzymes. How the energetic demands of such processes are ensured in response to rapid stimuli remains unclear. We have 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. Nuclear ATP synthesis requires the combined enzymatic activities of PARP1, PARG and NUDIX5/NUDT5. Although initiated via mitochondrial derived ATP, the nuclear source of ATP is essential for hormone induced chromatin remodeling, gene regulation and cell proliferation and may also participate in DNA repair. This novel pathway reveals exciting avenues of research for drug development.

Distinct structural transitions of chromatin topological domains coordinate 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 described the distribution of Progestin-induced chromatin modifications and changes in transcriptional activity over TADs in T47D breast cancer cells. Using ChIP-Seq, Hi-C and 3D modelling techniques, we found that the borders of the ∼2,000 TADs in these cells are largely maintained after hormone treatment but that some TADs operate as discrete regulatory units in which the majority of the genes are either transcriptionally activated or repressed upon hormone stimulus. The epigenetic signatures of the TADs are coordinately modified by hormone 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. In response to hormone activated TADs exhibit higher density of internal contacts, while repressed TADs show less intra-TAD contacts. Integrative 3D modelling revealed that TADs structurally expanded if activated and compacted when repressed, and that this is accompanied by differential changes in their global accessibility. We thus propose that TADs function as “regulons” to enable spatially proximal genes to be coordinately transcribed in response to hormones.