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.

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 HP1g (heterochromatin protein 1g), 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 HP1g to H3K9me3 (histone H3 tails trimethylated on Lys 9). SRA interacts with PR, HP1g, and LSD1, and its depletion compromises the loading of the repressive complex to target chromatinpromoting aberrant gene derepression. Upon hormonal treatment, the HP1g-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.

Yeast SWI/SNF is a multisubunit, 1.14-MDa ATP-dependent chromatin-remodeling enzyme required for transcription of a subset of inducible genes. Biochemical studies have demonstrated that SWI/SNF uses the energy from ATP hydrolysis to generate superhelical torsion, mobilize mononucleosomes, enhance the accessibility of nucleosomal DNA and remove H2A-H2B dimers from mononucleosomes. Here we describe the ATP-dependent activities of a SWI/SNF sub complex that is composed of only three subunits, Swi2p, Arp7p and Arp9p. Whereas this sub complex is fully functional in most remodeling assays, Swi2p-Arp7p-Arp9p is defective for ATP-dependent removal of H2A-H2B dimers. We identify the acidic N terminus of the Swi3p subunit as a novel H2A-H2B-binding domain required for ATP-dependent dimer loss. Our data indicate that H2A-H2B dimer loss is not an obligatory consequence of ATP-dependent DNA translocation, and furthermore they suggest that SWI/SNF is composed of at least four interdependent modules.

Steroid hormones regulate gene expression by interaction of their receptors with hormone responsive elements (HREs) and recruitment of kinases, chromatin remodeling complexes, and coregulators to their target promoters. Here we show that in breast cancer cells the BAF, but not the closely related PBAF complex, is required for progesterone induction of several target genes including MMTV, where it catalyzes localized displacement of histones H2A and H2B and subsequent NF1 binding. PCAF is also needed for induction of progesterone target genes and acetylates histone H3 at K14, an epigenetic mark that interacts with the BAF subunits by anchoring the complex to chromatin. In the absence of PCAF, full loading of target promoters with hormone receptors and BAF is precluded, and induction is compromised. Thus, activation of hormone-responsive promoters requires cooperation of at least two chromatin remodeling activities, BAF and PCAF.

Steroid hormones regulate gene expression by interaction of their receptors with hormone-responsive elements on DNA or with other transcription factors, but they can also activate cytoplasmic signaling cascades. Rapid activation of Erk by progestins via an interaction of the progesterone receptor (PR) with the estrogen receptor is critical for transcriptional activation of the mouse mammary tumor virus (MMTV) promoter and other progesterone target genes. Erk activation leads to the phosphorylation of PR, activation of mitogen- and stress-activated protein kinase 1, and the recruitment of a complex of the three activated proteins and of P300/CBP-associated factor (PCAF) to a single nucleosome, resulting in the phosphoacetylation of histone H3 and the displacement of heterochromatin protein 1γ. Hormone-dependent gene expression requires ATP-dependent chromatin remodeling complexes. Two switch/sucrose nonfermentable-like complexes, Brahma-related gene 1-associated factor (BAF) and polybromo-BAF are present in breast cancer cells, but only BAF is recruited to the MMTV promoter and cooperates with PCAF during activation of hormone-responsive promoters. PCAF acetylates histone H3 at K14, an epigenetic mark recognized by BAF subunits, thus anchoring the complex to chromatin. BAF catalyzes localized displacement of histones H2A and H2B, facilitating access of nuclear factor 1 and additional PR complexes to the hidden hormone-responsive elements on the MMTV promoter. The linker histone H1 is a structural component of chromatin generally regarded as a general repressor of transcription. However, it contributes to a better regulation of the MMTV promoter by favoring a more homogeneous nucleosome positioning, thus reducing basal transcription and actually enhancing hormone induced transcription. During transcriptional activation, H1 is phosphorylated and displaced from the promoter. The kinase cyclin-dependent kinase 2 is activated after progesterone treatment and could catalyze progesterone-induced phosphorylation of histone H1 by chromatin remodeling complexes. The initial steps of gene induction by progestins involve changes in the chromatin organization of target promoters that require the activation of several kinase signaling pathways initiated by membrane anchored PR. Because these pathways also respond to other external signals, they serve to integrate the hormonal response in the global context of the cellular environment.

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 HP1c-LSD1 complex repressor complex, which is further anchored via binding of HP1c 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.

Transcription from the mouse mammary tumor virus (MMTV) promoter can be induced by glucocorticoids or progestins. Progesterone treatment of cultured cells carrying an integrated single copy of an MMTV transgene leads to recruitment of progesterone receptor (PR), SWI/SNF, and SNF2h-related complexes to MMTV promoter. Recruitment is accompanied by selective displacement of histones H2A and H2B from the nucleosome B. In nucleosomes assembled on promoter sequences, SWI/SNF displaces histones H2A and H2B from MMTV nucleosome B, but not from other MMTV nucleosomes or from an rDNA promoter nucleosome. Thus, the outcome of nucleosome remodeling by purified SWI/SNF depends on the DNA sequence. On the other hand, 5 min after hormone treatment, the cytoplasmic signaling cascade Src/Ras/Erk is activated via an interaction of PR with the estrogen receptor, which activates Src. As a consequence of Erk activation PR is phosphorylated, Msk1 is activated, and a ternary complex PR-Erk-Msk1 is recruited to MMTV nucleosome B. Msk1 phosphorylates H3 at serine 10, which is followed by acetylation at lysine 14, displacement of HP1gamma, and recruitment of Brg1, PCAF, and RNA polymerase II. Blocking Erk activation or Msk1 activity prevents induction of the MMTV transgene. Thus, the rapid nongenomic effects of progestins are essential for their transcriptional effects on certain progestin target genes. In rat endometrial stromal cells, picomolar concentrations of progestins trigger the cross talk of PR with ERbeta that activates the Erk and Akt kinase pathways leading to cell proliferation in the absence of direct transcriptional effects of the ligand-activated PR. Thus, depending on the cellular context rapid kinase activation and transcriptional effect play different roles in the physiological response to progestins.

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.