Selective coregulator function and restriction of steroid receptor chromatin occupancy by Hic-5.

Steroid receptors (SRs) bind specific DNA regulatory sequences, thereby activating and repressing gene expression. We previously showed that transcriptional coregulator Hic-5 facilitates glucocorticoid regulation of some genes but blocks glucocorticoid regulation of others. Here, in a genome-wide analysis, Hic-5 depletion dramatically increased the global number of sites occupied by glucocorticoid receptor (GR) α (the major GR isoform), and many binding sites blocked by Hic-5 were associated with genes for which Hic-5 also blocked glucocorticoid-regulated expression. Hic-5 had similar effects on GRγ (a splice variant of GRα) and estrogen receptor α (ERα), facilitating hormonal regulation of some genes and blocking hormonal regulation of others. As with GRα, Hic-5 blocking of hormonal gene regulation mediated by GRγ and ERα was associated with blocking of GRγ and ERα occupancy at nearby sites. Hic-5 supported hormonal regulation of many more genes for GRα than for GRγ or ERα and thus exhibited selective coregulator functions for different SRs. In contrast, the number of Hic-5-blocked genes was similar for all 3 SRs. In addition to classic coregulator activity, Hic-5 influences the genomic occupancy of multiple SRs and thereby blocks some aspects of hormonal regulation. Thus, Hic-5, because of its tissue-specific expression, could contribute to tissue-specific genomic occupancy and gene regulation by SRs.

Distinct, genome-wide, gene-specific selectivity patterns of four glucocorticoid receptor coregulators.

Glucocorticoids are a class of steroid hormones that bind to and activate the glucocorticoid receptor (GR), which then positively or negatively regulates transcription of many genes that govern multiple important physiological pathways such as inflammation and metabolism of glucose, fat and bone. The remodeling of chromatin and regulated assembly or disassembly of active transcription complexes by GR and other DNA-binding transcription factors is mediated and modulated by several hundred transcriptional coregulator proteins. Previous studies focusing on single coregulators demonstrated that each coregulator is required for regulation of only a subset of all the genes regulated by a steroid hormone. We hypothesized that the gene-specific patterns of coregulators may correspond to specific physiological pathways such that different coregulators modulate the pathway-specificity of hormone action, thereby providing a mechanism for fine tuning of the hormone response. We tested this by direct comparison of multiple coregulators, using siRNA to deplete the products of four steroid hormone receptor coregulator genes (CCAR1, CCAR2, CALCOCO1 and ZNF282). Global analysis of glucocorticoid-regulated gene expression after siRNA mediated depletion of coregulators confirmed that each coregulator acted in a selective and gene-specific manner and demonstrated both positive and negative effects on glucocorticoid-regulated expression of different genes. We identified several classes of hormone-regulated genes based on the effects of coregulator depletion. Each coregulator supported hormonal regulation of some genes and opposed hormonal regulation of other genes (coregulator-modulated genes), blocked hormonal regulation of a second class of genes (coregulator-blocked genes), and had no effect on hormonal regulation of a third gene class (coregulator-independent genes). In spite of previously demonstrated physical and functional interactions among these four coregulators, the majority of the several hundred modulated and blocked genes for each of the four coregulators tested were unique to that coregulator. Finally, pathway analysis on coregulator-modulated genes supported the hypothesis that individual coregulators may regulate only a subset of the many physiological pathways controlled by glucocorticoids. We conclude that gene-specific actions of coregulators correspond to specific physiological pathways, suggesting that coregulators provide a potential mechanism for physiological fine tuning in vivo and may thus represent attractive targets for therapeutic intervention.

Glucocorticoid receptor binds half sites as a monomer and regulates specific target genes.

BACKGROUND: Glucocorticoid receptor (GR) is a hormone-activated, DNA-binding transcriptional regulatory factor that controls inflammation, metabolism, stress responses, and other physiological processes. In vitro, GR binds as an inverted dimer to a motif consisting of two imperfectly palindromic 6 bp half sites separated by 3 bp spacers. In vivo, GR employs different patterns of functional surfaces of GR to regulate different target genes. The relationships between GR genomic binding and functional surface utilization have not been defined. RESULTS: We find that A477T, a GR mutant that disrupts the dimerization interface, differs from wild-type GRα in binding and regulation of target genes. Genomic regions strongly occupied by A477T are enriched for a novel half site motif. In vitro, GRα binds half sites as a monomer. Through the overlap between GRα- and A477T-bound regions, we identify GRα-bound regions containing only half sites. We further identify GR target genes linked with half sites and not with the full motif. CONCLUSIONS: Genomic regions bound by GR differ in underlying DNA sequence motifs and in the GR functional surfaces employed for regulation. Identification of GR binding regions that selectively utilize particular GR surfaces may discriminate sub-motifs, including the half site motif, that favor those surfaces. This approach may contribute to predictive models for GR activity and therapy.

Hic-5 is a transcription coregulator that acts before and/or after glucocorticoid receptor genome occupancy in a gene-selective manner.

Ligand activation and DNA-binding dictate the outcome of glucocorticoid receptor (GR)-mediated transcriptional regulation by inducing diverse receptor conformations that interact differentially with coregulators. GR recruits many coregulators via the well-characterized AF2 interaction surface in the GR ligand-binding domain, but Lin11, Isl-1, Mec-3 (LIM) domain coregulator Hic-5 (TGFB1I1) binds to the relatively uncharacterized tau2 activation domain in the hinge region of GR. Requirement of hydrogen peroxide-inducible clone-5 (Hic-5) for glucocorticoid-regulated gene expression was defined by Hic-5 depletion and global gene-expression analysis. Hic-5 depletion selectively affected both activation and repression of GR target genes, and Hic-5 served as an on/off switch for glucocorticoid regulation of many genes. For some hormone-induced genes, Hic-5 facilitated recruitment of Mediator complex. In contrast, many genes were not regulated by glucocorticoid until Hic-5 was depleted. On these genes Hic-5 prevented GR occupancy and chromatin remodeling and thereby inhibited their hormone-dependent regulation. Transcription factor binding to genomic sites is highly variable among different cell types; Hic-5 represents an alternative mechanism for regulating transcription factor-binding site selection that could apply both within a given cell type and among different cell types. Thus, Hic-5 is a versatile coregulator that acts by multiple gene-specific mechanisms that influence genomic occupancy of GR as well transcription complex assembly.

G9a functions as a molecular scaffold for assembly of transcriptional coactivators on a subset of glucocorticoid receptor target genes.

Histone H3 lysine-9 methyltransferase G9a/EHMT2/KMT1C is a key corepressor of gene expression. However, activation of a limited number of genes by G9a (independent of its catalytic activity) has also been observed, although the precise molecular mechanisms are unknown. By using RNAi in combination with gene expression microarray analysis, we found that G9a functions as a positive and a negative transcriptional coregulator for discrete subsets of genes that are regulated by the hormone-activated Glucocorticoid Receptor (GR). G9a was recruited to GR-binding sites (but not to the gene body) of its target genes and interacted with GR, suggesting recruitment of G9a by GR. In contrast to its corepressor function, positive regulation of gene expression by G9a involved G9a-mediated enhanced recruitment of coactivators CARM1 and p300 to GR target genes. Further supporting a role for G9a as a molecular scaffold for its coactivator function, the G9a-specific methyltransferase inhibitor UNC0646 did not affect G9a coactivator function but selectively decreased G9a corepressor function for endogenous target genes. Overall, G9a functioned as a coactivator for hormone-activated genes and as a corepressor in support of hormone-induced gene repression, suggesting that the positive or negative actions of G9a are determined by the gene-specific regulatory environment and chromatin architecture. These findings indicate distinct mechanisms of G9a coactivator vs. corepressor functions in transcriptional regulation and provide insight into the molecular mechanisms of G9a coactivator function. Our results also suggest a physiological role of G9a in fine tuning the set of genes that respond to glucocorticoids.

Gene-specific patterns of coregulator requirements by estrogen receptor-α in breast cancer cells.

Progesterone receptor (PgR) controls the menstrual cycle, pregnancy, embryonic development, and homeostasis, and it plays important roles in breast cancer development and progression. However, the requirement of coregulators for estrogen-induced expression of the PgR gene has not been fully explored. Here we used RNA interference to demonstrate dramatic differences in requirements of 10 different coregulators for estrogen-regulated expression of six different genes, including PgR and the well-studied TFF1 (or pS2) gene in MCF-7 breast cancer cells. Full estrogen-induced expression of TFF1 required all ten coregulators, but PgR induction required only four of the 10 coregulators. Chromatin immunoprecipitation studies demonstrated several mechanisms responsible for the differential coregulator requirements. Actin-binding coregulator Flightless-I, required for TFF1 expression and recruited to that gene by estrogen receptor-α (ERα), is not required for PgR expression and not recruited to that gene. Protein acetyltransferase tat-interactive protein 60 and ATP-dependent chromatin remodeler Brahma Related Gene 1 are recruited to both genes but are required only for TFF1 expression. Histone methyltransferase G9a is recruited to both genes and required for estrogen-induced expression of TFF1 but negatively regulates estrogen-induced expression of PgR. In contrast, histone methyltransferase myeloid/lymphoid or mixed-lineage leukemia 1 (MLL1), pioneer factor Forkhead box A1, and p160 coregulator steroid receptor coactivator-3 are required for expression of and are recruited to both genes. Depletion of MLL1 decreased ERα binding to the PgR and TFF1 genes. In contrast, depletion of G9a enhanced ERα binding to the PgR gene but had no effect on ERα binding to the TFF1 gene. These studies suggest that differential promoter architecture is responsible for promoter-specific mechanisms of gene regulation.

Changes in the mouse estrus cycle in response to BRCA1 inactivation suggest a potential link between risk factors for familial and sporadic ovarian cancer.

Menstrual cycle activity is the most important risk factor for sporadic serous ovarian carcinoma, whereas a germ-line mutation in BRCA1 is the most important risk factor for the familial form. Given the rarity of BRCA1 mutations in sporadic ovarian cancers, we hypothesized that BRCA1 influences the menstrual cycle in a way that mimics the factors underlying sporadic ovarian cancer predisposition, making BRCA1 mutations redundant in such cancers. We compared the length of each phase of the estrus cycle (equivalent to the human menstrual cycle) and of circulating levels of estradiol in control mice and in mice carrying a Brca1 mutation in their ovarian granulosa cells, two thirds of which develop ovarian or uterine epithelial tumors. We also compared the length of the different phases of the cycle in mutants that subsequently developed tumors with those in mutants that remained tumor-free. Mutant mice as well as oophorectomized wild-type mice harboring mutant ovarian grafts showed a relative increase in the average length of the proestrus phase of the estrus cycle, which corresponds to the estrogen-dominated follicular phase of the human menstrual cycle. Total circulating levels of estradiol were also increased in mutant mice injected with pregnant mare serum gonadotropins. The relative increase in proestrus length was highest in mutant mice that subsequently developed reproductive epithelial tumors. We conclude that loss of a functional Brca1 increases murine ovarian epithelial tumor predisposition by increasing estrogen stimulation in the absence of progesterone, recapitulating conditions associated with sporadic ovarian cancer predisposition in humans.

Cell-nonautonomous induction of ovarian and uterine serous cystadenomas in mice lacking a functional Brca1 in ovarian granulosa cells.

Women with germline mutations in BRCA1 have a 40% risk of developing ovarian cancer by age 70 and are also predisposed to cancers of the fallopian tubes. Given that ovulatory activity is a strong risk factor for sporadic ovarian cancer, we hypothesized that reduced BRCA1 expression might predispose to gynecological cancers indirectly, by influencing ovarian granulosa cells. These cells secrete sex steroids that control the ovulatory cycle and influence the growth of ovarian epithelial tumors. Granulosa cells also secrete mullerian inhibiting substance (MIS), a hormone that inhibits both the formation of female reproductive organs in male embryos and the proliferation of ovarian epithelial tumor cells. We tested this hypothesis by using the Cre-lox system to inactivate the Brca1 gene in mouse ovarian granulosa cells. A truncated form of the Fsh receptor promoter served as the Cre driver. Here, we show that indeed, inactivation of the Brca1 gene in granulosa cells led to the development of cystic tumors in the ovaries and uterine horns. These tumors carried normal Brca1 alleles, supporting the view that Brca1 may influence tumor development indirectly, possibly through an effector secreted by granulosa cells.

Shift of localized growth zones contributes to skin appendage morphogenesis: role of the Wnt/beta-catenin pathway.

Skin appendage formation represents a process of regulated new growth. Bromodeoxyuridine labeling of developing chicken skin demonstrated the presence of localized growth zones, which first promote appendage formation and then move within each appendage to produce specific shapes. Initially, cells proliferate all over the presumptive skin. During the placode stage they are organized to form periodic rings. At the short feather bud stage, the localized growth zones shifted to the posterior and then the distal bud. During the long bud stage, the localized growth zones descended through the flank region toward the feather collar (equivalent to the hair matrix). During feather branch formation, the localized growth zones were positioned periodically in the basilar layer to enhance branching of barb ridges. Wnts were expressed in a dynamic fashion during feather morphogenesis that coincided with the shifting localized growth zones positions. The expression pattern of Wnt 6 was examined and compared with other members of the Wnt pathway. Early in feather development Wnt 6 expression overlapped with the location of the localized growth zones. Its function was tested through misexpression studies. Ectopic Wnt 6 expression produced abnormal localized outgrowths from the skin appendages at either the base, the shaft, or the tip of the developing feathers. Later in feather filament morphogenesis, several Wnt markers were expressed in regions undergoing rearrangements and differentiation of barb ridge keratinocytes. These data suggest that skin appendages are built to specific shapes by adding new cells from well-positioned and controlled localized growth zones and that Wnt activity is involved in regulating such localized growth zone activity.