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.