Transcriptional coactivation by EHMT2 restricts glucocorticoid-induced insulin resistance in a study with male mice.

The classical dogma of glucocorticoid-induced insulin resistance is that it is caused by the transcriptional activation of hepatic gluconeogenic and insulin resistance genes by the glucocorticoid receptor (GR). Here, we find that glucocorticoids also stimulate the expression of insulin-sensitizing genes, such as Irs2. The transcriptional coregulator EHMT2 can serve as a transcriptional coactivator or a corepressor. Using male mice that have a defective EHMT2 coactivation function specifically, we show that glucocorticoid-induced Irs2 transcription is dependent on liver EHMT2's coactivation function and that IRS2 play a key role in mediating the limitation of glucocorticoid-induced insulin resistance by EHMT2's coactivation. Overall, we propose a model in which glucocorticoid-regulated insulin sensitivity is determined by the balance between glucocorticoid-modulated insulin resistance and insulin sensitizing genes, in which EHMT2 coactivation is specifically involved in the latter process.

How Protein Methylation Regulates Steroid Receptor Function.

Steroid receptors (SRs) are members of the nuclear hormonal receptor family, many of which are transcription factors regulated by ligand binding. SRs regulate various human physiological functions essential for maintenance of vital biological pathways, including development, reproduction, and metabolic homeostasis. In addition, aberrant expression of SRs or dysregulation of their signaling has been observed in a wide variety of pathologies. SR activity is tightly and finely controlled by post-translational modifications (PTMs) targeting the receptors and/or their coregulators. Whereas major attention has been focused on phosphorylation, growing evidence shows that methylation is also an important regulator of SRs. Interestingly, the protein methyltransferases depositing methyl marks are involved in many functions, from development to adult life. They have also been associated with pathologies such as inflammation, as well as cardiovascular and neuronal disorders, and cancer. This article provides an overview of SR methylation/demethylation events, along with their functional effects and biological consequences. An in-depth understanding of the landscape of these methylation events could provide new information on SR regulation in physiology, as well as promising perspectives for the development of new therapeutic strategies, illustrated by the specific inhibitors of protein methyltransferases that are currently available.

Gene-Specific Actions of Transcriptional Coregulators Facilitate Physiological Plasticity: Evidence for a Physiological Coregulator Code.

The actions of transcriptional coregulators are highly gene-specific, that is, each coregulator is required only for a subset of the genes regulated by a specific transcription factor. These coregulator-specific gene subsets often represent selected physiological responses among multiple pathways targeted by a transcription factor. Regulating the activity of a coregulator via post-translational modifications would thus affect only a subset of the transcription factor's physiological actions. Using the context of transcriptional regulation by steroid hormone receptors, this review focuses on gene-specific actions of coregulators and evidence linking individual coregulators with specific physiological pathways. Such evidence suggests that there is a 'physiological coregulator code', which represents a fertile area for future research with important clinical implications.

Relapse-associated AURKB blunts the glucocorticoid sensitivity of B cell acute lymphoblastic leukemia.

Glucocorticoids (GCs) are used in combination chemotherapies as front-line treatment for B cell acute lymphoblastic leukemia (B-ALL). Although effective, many patients relapse and become resistant to chemotherapy and GCs in particular. Why these patients relapse is not clear. We took a comprehensive, functional genomics approach to identify sources of GC resistance. A genome-wide shRNA screen identified the transcriptional coactivators EHMT2, EHMT1, and CBX3 as important contributors to GC-induced cell death. This complex selectively supports GC-induced expression of genes contributing to cell death. A metaanalysis of gene expression data from B-ALL patient specimens revealed that Aurora kinase B (AURKB), which restrains GC signaling by phosphorylating EHMT1-2, is overexpressed in relapsed B-ALL, suggesting it as a potential contributor to relapse. Inhibition of AURKB enhanced GC-induced expression of cell death genes, resulting in potentiation of GC cytotoxicity in cell lines and relapsed B-ALL patient samples. This function for AURKB is distinct from its canonical role in the cell cycle. These results show the utility of functional genomics in understanding mechanisms of resistance and rapidly identifying combination chemotherapeutics.

Increasing G9a automethylation sensitizes B acute lymphoblastic leukemia cells to glucocorticoid-induced death.

Synthetic glucocorticoids (GCs) are used to treat lymphoid cancers, but many patients develop resistance to treatment, especially to GC. By identifying genes that influence sensitivity to GC-induced cell death, we found that histone methyltransferases G9a and G9a-like protein (GLP), two glucocorticoid receptor (GR) coactivators, are required for GC-induced cell death in acute lymphoblastic leukemia (B-ALL) cell line Nalm6. We previously established in a few selected genes that automethylated G9a and GLP recruit heterochromatin protein 1γ (HP1γ) as another required coactivator. Here, we used a genome-wide analysis to show that HP1γ is selectively required for GC-regulated expression of the great majority of GR target genes that require G9a and GLP. To further address the importance of G9a and GLP methylation in this process and in cell physiology, we found that JIB-04, a selective JmjC family lysine demethylase inhibitor, increased G9a methylation and thereby increased G9a binding to HP1γ. This led to increased expression of GR target genes regulated by G9a, GLP and HP1γ and enhanced Nalm6 cell death. Finally, the KDM4 lysine demethylase subfamily demethylates G9a in vitro, in contrast to other KDM enzymes tested. Thus, inhibiting G9a/GLP demethylation potentially represents a novel method to restore sensitivity of treatment-resistant B-ALL tumors to GC-induced cell death.

Aberrant expression of SETD1A promotes survival and migration of estrogen receptor α-positive breast cancer cells.

The histone H3 lysine 4-specific methyltransferase SETD1A is associated with transcription activation and is considered a key epigenetic regulator that modulates the cell cycle and metastasis in triple-negative breast cancer cells. However, the clinical role of SETD1A in estrogen receptor (ER)-positive breast cancer cells remains unclear. Here, we examined whether SETD1A is a potential target for ERα-positive breast cancer therapy. SETD1A expression was upregulated in breast tumor tissue compared to that in normal breast tissue. Moreover, ER-target genes regulated by SETD1A were particularly enriched in cell cycle and cancer pathways. SETD1A is involved in histone H3K4 methylation, subsequent recruitment of ERα, and the establishment of accessible chromatin structure at the enhancer region of ERα target genes. In addition to ERα target genes, other cell survival genes were also downregulated by SETD1A depletion in MCF-7 cells, leading to significant decrease in cell proliferation and migration, and spontaneous induction of apoptosis. We also found that miR-1915-3p functioned as a novel regulator of SETD1A expression in breast cells. Importantly, the growth of tamoxifen-resistant MCF-7 cells was effectively repressed by SETD1A knockdown. These results indicate that SETD1A may serve as a molecular target and prognostic indicator in ERα-positive breast cancer.

Different chromatin and DNA sequence characteristics define glucocorticoid receptor binding sites that are blocked or not blocked by coregulator Hic-5.

The glucocorticoid receptor (GR) regulates genes in many physiological pathways by binding to enhancer and silencer elements of target genes, where it recruits coregulator proteins that remodel chromatin and regulate the assembly of transcription complexes. The coregulator Hydrogen peroxide-inducible clone 5 (Hic-5) is necessary for glucocorticoid (GC) regulation of one group of GR target genes, is irrelevant for a second group, and inhibits GR binding to a third gene set, thereby blocking their regulation by GC. Gene-specific characteristics that distinguish GR binding regions (GBR) at Hic-5 blocked genes from GBR at other GC-regulated genes are unknown. Here we show genome-wide that blocked GBR generally require CHD9 and BRM for GR occupancy in contrast to GBR that are not blocked by Hic-5. Hic-5 blocked GBR are enriched near Hic-5 blocked GR target genes but not near GR target genes that are not blocked by Hic-5. Furthermore blocked GBR are in a closed conformation prior to Hic-5 depletion, and require Hic-5 depletion and glucocorticoid treatment to create an open conformation necessary for GR occupancy. A transcription factor binding motif characteristic of the ETS family was enriched near blocked GBR and blocked genes but not near non-blocked GBR or non-blocked GR target genes. Thus, we identify specific differences in chromatin conformation, chromatin remodeler requirements, and local DNA sequence motifs that contribute to gene-specific actions of transcription factors and coregulators. These findings shed light on mechanisms that contribute to binding site selection by transcription factors, which vary in a cell type-specific manner.

A post-translational modification switch controls coactivator function of histone methyltransferases G9a and GLP.

Like many transcription regulators, histone methyltransferases G9a and G9a-like protein (GLP) can act gene-specifically as coregulators, but mechanisms controlling this specificity are mostly unknown. We show that adjacent post-translational methylation and phosphorylation regulate binding of G9a and GLP to heterochromatin protein 1 gamma (HP1γ), formation of a ternary complex with the glucocorticoid receptor (GR) on chromatin, and function of G9a and GLP as coactivators for a subset of GR target genes. HP1γ is recruited by G9a and GLP to GR binding sites associated with genes that require G9a, GLP, and HP1γ for glucocorticoid-stimulated transcription. At the physiological level, G9a and GLP coactivator function is required for glucocorticoid activation of genes that repress cell migration in A549 lung cancer cells. Thus, regulated methylation and phosphorylation serve as a switch controlling G9a and GLP coactivator function, suggesting that this mechanism may be a general paradigm for directing specific transcription factor and coregulator actions on different genes.

Glucocorticoid receptor binding to chromatin is selectively controlled by the coregulator Hic-5 and chromatin remodeling enzymes.

The steroid hormone-activated glucocorticoid receptor (GR) regulates cellular stress pathways by binding to genomic regulatory elements of target genes and recruiting coregulator proteins to remodel chromatin and regulate transcription complex assembly. The coregulator hydrogen peroxide-inducible clone 5 (Hic-5) is required for glucocorticoid (GC) regulation of some genes but not others and blocks the regulation of a third gene set by inhibiting GR binding. How Hic-5 exerts these gene-specific effects and specifically how it blocks GR binding to some genes but not others is unclear. Here we show that site-specific blocking of GR binding is due to gene-specific requirements for ATP-dependent chromatin remodeling enzymes. By depletion of 11 different chromatin remodelers, we found that ATPases chromodomain helicase DNA-binding protein 9 (CHD9) and Brahma homologue (BRM, a product of the SMARCA2 gene) are required for GC-regulated expression of the blocked genes but not for other GC-regulated genes. Furthermore, CHD9 and BRM were required for GR occupancy and chromatin remodeling at GR-binding regions associated with blocked genes but not at GR-binding regions associated with other GC-regulated genes. Hic-5 selectively inhibits GR interaction with CHD9 and BRM, thereby blocking chromatin remodeling and robust GR binding at GR-binding sites associated with blocked genes. Thus, Hic-5 regulates GR binding site selection by a novel mechanism, exploiting gene-specific requirements for chromatin remodeling enzymes to selectively influence DNA occupancy and gene regulation by a transcription factor.

Identifying differential transcription factor binding in ChIP-seq.

ChIP seq is a widely used assay to measure genome-wide protein binding. The decrease in costs associated with sequencing has led to a rise in the number of studies that investigate protein binding across treatment conditions or cell lines. In addition to the identification of binding sites, new studies evaluate the variation in protein binding between conditions. A number of approaches to study differential transcription factor binding have recently been developed. Several of these methods build upon established methods from RNA-seq to quantify differences in read counts. We compare how these new approaches perform on different data sets from the ENCODE project to illustrate the impact of data processing pipelines under different study designs. The performance of normalization methods for differential ChIP-seq depends strongly on the variation in total amount of protein bound between conditions, with total read count outperforming effective library size, or variants thereof, when a large variation in binding was studied. Use of input subtraction to correct for non-specific binding showed a relatively modest impact on the number of differential peaks found and the fold change accuracy to biological validation, however a larger impact might be expected for samples with more extreme copy number variations between them. Still, it did identify a small subset of novel differential regions while excluding some differential peaks in regions with high background signal. These results highlight proper scaling for between-sample data normalization as critical for differential transcription factor binding analysis and suggest bioinformaticians need to know about the variation in level of total protein binding between conditions to select the best analysis method. At the same time, validation using fold-change estimates from qRT-PCR suggests there is still room for further method improvement.

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.

Role of distinct surfaces of the G9a ankyrin repeat domain in histone and DNA methylation during embryonic stem cell self-renewal and differentiation.

BACKGROUND: Epigenetic modifications such as histone and DNA methylation are essential for silencing pluripotency genes during embryonic stem cell (ESC) differentiation. G9a is the major histone H3 Lys9 (H3K9) methyltransferase in euchromatin and is required for the de novo DNA methylation of the key regulator of pluripotency Oct3/4 during ESC differentiation. Surprisingly, the catalytic activity of G9a is not required for its role in de novo DNA methylation and the precise molecular mechanisms of G9a in this process are poorly understood. It has been suggested that the G9a ankyrin repeat domain, which can interact with both H3K9me2 and the DNA methyltransferase DNMT3A, could facilitate de novo DNA methylation by bridging the interaction between DNMT3A and H3K9me2-marked chromatin. RESULTS: Here, we demonstrate that the G9a ankyrin domain H3K9me2-binding function is not required for the de novo DNA methylation of Oct3/4 during ESC differentiation. Moreover, we show that the interaction between the G9a ankyrin domain and DNMT3A is not sufficient to ensure efficient de novo DNA methylation. More importantly, we characterize a specific residue of the G9a ankyrin domain (Asp905) that is critical for both maintaining cellular H3K9me2 levels in undifferentiated ESCs and for the establishment of de novo DNA methylation during differentiation. CONCLUSIONS: These results represent an exciting breakthrough, which reveals 1) an unexpected critical biological function of the G9a ankyrin domain in global histone H3K9 methylation and 2) valuable insights into the molecular mechanisms and interaction surfaces through which G9a regulates de novo DNA methylation of Oct3/4 during ESC differentiation.

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.

Coregulator cell cycle and apoptosis regulator 1 (CCAR1) positively regulates adipocyte differentiation through the glucocorticoid signaling pathway.

Glucocorticoids contribute to adipocyte differentiation by cooperating with transcription factors, such as CCAAT/enhancer-binding protein ß (C/EBPß), to stimulate transcription of the gene encoding peroxisome proliferator-activated receptor (PPARγ), a master regulator of adipogenesis. However, the mechanism of PPARγ gene regulation by glucocorticoids, the glucocorticoid receptor (GR), and its coregulators is poorly understood. Here we show that two GR binding regions (GBRs) in the mouse PPARγ gene were responsive to glucocorticoid, and treatment of 3T3-L1 preadipocytes with glucocorticoid alone induced GR occupancy and chromatin remodeling at PPARγ GBRs, which also contain binding sites for C/EBP and PPARγ proteins. GR recruited cell cycle and apoptosis regulator 1 (CCAR1), a transcription coregulator, to the PPARγ gene GBRs. Notably, CCAR1 was required for GR occupancy and chromatin remodeling at one of the PPARγ gene GBRs. Moreover, depletion of CCAR1 markedly suppressed differentiation of mouse mesenchymal stem cells and 3T3-L1 preadipocytes to mature adipocytes and decreased induction of PPARγ, C/EBPα, and C/EBPδ. Although CCAR1 was required for stimulation of several GR-regulated adipogenic genes in 3T3-L1 preadipocytes by glucocorticoid, it was not required for GR-activated transcription of certain anti-inflammatory genes in human A549 lung epithelial cells. Overall, our results highlighted the novel and specific roles of GR and CCAR1 in adipogenesis.

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.

Establishment of active chromatin structure at enhancer elements by mixed-lineage leukemia 1 to initiate estrogen-dependent gene expression.

A number of genome-wide analyses have revealed that estrogen receptor α binding to and regulation of its target genes correlate with binding of FOXA1, a pioneer factor, to nearby DNA sites in MCF-7 breast cancer cells. The enhancer element-specific histone H3K4me1/2 mark is enriched at the specific FOXA1/ERα recruitment sites in chromatin, but the mechanism by which these enhancer marks are established in chromatin before hormone treatment is unclear. Here, we show that mixed-lineage leukemia 1 (MLL1) protein is a key determinant that maintains permissive chromatin structure of the TFF1 enhancer region. MLL1 occupies the TFF1 enhancer region and methylates H3K4 before hormone stimulation. In vitro, MLL1 binds directly to the CpG-rich region of the TFF1 enhancer, and its binding is dependent on hypomethylation of DNA. Furthermore, the depletion of MLL1 in MCF-7 cells results in a dramatic decrease of chromatin accessibility and recruitment of FOXA1 and ERα to the enhancer element. Our study defines the mechanism by which MLL1 nucleates histone H3K4 methylation marks in CpG-enriched regions to maintain permissive chromatin architecture and allow FOXA1 and estrogen receptor α binding to transcriptional regulatory sites in breast cancer cells.

Aberrant BAF57 signaling facilitates prometastatic phenotypes.

PURPOSE: BAF57, a component of the switching-defective and sucrose nonfermenting (SWI/SNF) chromatin-remodeling complex conglomerate, modulates androgen receptor activity to promote prostate cancer. However, the molecular consequences of tumor-associated BAF57 expression have remained undefined in advanced disease such as castration-resistant prostate cancer and/or metastasis. EXPERIMENTAL DESIGN: Clinical human specimens of primary and metastatic prostate cancer were immunohistochemically examined for tumor-grade association of BAF57 expression. Global gene expression analyses were conducted in models mimicking tumor-associated BAF57 expression. Aberrant BAF57-dependent gene expression changes, bypass of androgen-mediated signaling, and chromatin-specific SWI/SNF complex alterations with respect to cytoskeletal remodelers such as integrins were validated. Cell migration assays were used to profile the biologic phenotypes conferred under conditions simulating tumor-derived BAF57 expression. RESULTS: Immunohistochemical quantitation of primary human specimens revealed that BAF57 was significantly and aberrantly elevated as a function of tumor grade. Critically, gene expression analyses showed that BAF57 deregulation circumvented androgen-mediated signaling, elicited α2 integrin upregulation, and altered other SWI/SNF complex components at the α2 integrin locus. BAF57-dependent α2 integrin induction conferred a prometastatic migratory advantage, which was attenuated by anti-α2 integrin antibody blockade. Furthermore, BAF57 was found to be markedly upregulated in human prostate cancer metastases of the lung, lymph node, and dura. CONCLUSION: The findings herein, identifying tumor-associated BAF57 perturbation as a means to bypass androgen-signaling events that facilitate novel prometastatic phenotypes, link BAF57 upregulation to tumor dissemination. These data thereby establish BAF57 as a putative marker of metastatic potential that could be leveraged for therapeutic intervention.

cAMP response element-binding protein interacts with and stimulates the proteasomal degradation of the nuclear receptor coactivator GRIP1.

The glucocorticoid receptor interacting protein (GRIP1) belongs to the p160 steroid receptor coactivator family that plays essential roles in nuclear receptor-dependent transcriptional regulation. Previously, we reported that the cAMP-dependent protein kinase (PKA) induces ubiquitination leading to degradation of GRIP1. Here we show that the cAMP response element-binding protein (CREB) downregulates GRIP1 and is necessary for the PKA-stimulated degradation of GRIP1, which leads to changes in the expression of a subset of genes regulated by estrogen receptor-α in MCF-7 breast cancer cells. Our data of domain-mapping and ubiquitination analyses suggest that CREB promotes the proteasomal breakdown of ubiquitinated GRIP1 through 2 functionally independent protein domains containing amino acids 347 to 758 and 1121 to 1462. We provide evidence that CREB interacts directly with GRIP1 and that CREB Ser-133 phosphorylation or transcriptional activity is not required for GRIP1 interaction and degradation. The basic leucine zipper domain (bZIP) of CREB is important for the interaction with GRIP1, and deletion of this domain led to an inability to downregulate GRIP1. We propose that CREB mediates the PKA-stimulated degradation of GRIP1 through protein-protein interaction and stimulation of proteasomal degradation of ubiquitinated GRIP1.

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.