Shiming Jiang

Histone Deacetylase 7 and FoxA1 in Estrogen-Mediated Repression of RPRM

ABSTRACT Activation of estrogen receptor α (ERα) results in both induction and repression of gene transcription; while mechanistic details of estrogen induction are well described, details of repression remain largely unknown. We characterized several ERα-repressed targets and examined in detail the mechanism for estrogen repression of Reprimo (RPRM), a cell cycle inhibitor. Estrogen repression of RPRM is rapid and robust and requires a tripartite interaction between ERα, histone deacetylase 7 (HDAC7), and FoxA1. HDAC7 is the critical HDAC needed for repression of RPRM; it can bind to ERα and represses ERαs transcriptional activity—this repression does not require HDAC7s deacetylase activity. We further show that the chromatin pioneer factor FoxA1, well known for its role in estrogen induction of genes, is recruited to the RPRM promoter, is necessary for repression of RPRM, and interacts with HDAC7. Like other FoxA1 recruitment sites, the RPRM promoter is characterized by H3K4me1/me2. Estrogen treatment causes decreases in H3K4me1/me2 and release of RNA polymerase II (Pol II) from the RPRM proximal promoter. Overall, these data implicate a novel role for HDAC7 and FoxA1 in estrogen repression of RPRM, a mechanism which could potentially be generalized to many more estrogen-repressed genes and hence be important in both normal physiology and pathological processes.

Epigenetic reprogramming of HOXC10 in endocrine-resistant breast cancer.

Genome-wide screen identifies methylation of the estrogen-repressed HOXC10 gene as a determinant of resistance to aromatase inhibitors in breast cancer. Playing Tug-of-War with HOXC10 Aromatase inhibitors are drugs that prevent androgens from being converted into estrogen, and they are frequently used to treat breast cancers that express the estrogen receptor. Unfortunately, some patients’ tumors never respond to these drugs, and others gradually become resistant over time. Although the development of resistance to aromatase inhibitors has been investigated in some previous studies and some potential mechanisms have been proposed, much about this process remains unknown. Pathiraja and colleagues began by performing a genome-wide methylation screen in breast cancer cells, which identified the developmental gene HOXC10 as a target of epigenetic silencing in the context of long-term estrogen withdrawal. When HOXC10 is active, it interferes with proliferation and can stimulate apoptosis, but estrogen suppresses its activity, thereby promoting tumor growth. By decreasing estrogen production, aromatase inhibitors up-regulate HOXC10, accounting for some of their antitumor activity. However, long-term estrogen deprivation eventually has the opposite effect, leading to methylation of HOXC10 and its long-term suppression even in the absence of estrogen. These findings suggest that a rational approach for overcoming aromatase resistance in breast cancer may involve the addition of demethylating drugs to overcome the methylation of HOXC10 and take advantage of its antitumor effects, although this remains to be demonstrated directly. Resistance to aromatase inhibitors (AIs) is a major clinical problem in the treatment of estrogen receptor (ER)–positive breast cancer. In two breast cancer cell line models of AI resistance, we identified widespread DNA hyper- and hypomethylation, with enrichment for promoter hypermethylation of developmental genes. For the homeobox gene HOXC10, methylation occurred in a CpG shore, which overlapped with a functional ER binding site, causing repression of HOXC10 expression. Although short-term blockade of ER signaling caused relief of HOXC10 repression in both cell lines and breast tumors, it also resulted in concurrent recruitment of EZH2 and increased H3K27me3, ultimately transitioning to increased DNA methylation and silencing of HOXC10. Reduced HOXC10 in vitro and in xenografts resulted in decreased apoptosis and caused antiestrogen resistance. Supporting this, we used paired primary and metastatic breast cancer specimens to show that HOXC10 was reduced in tumors that recurred during AI treatment. We propose a model in which estrogen represses apoptotic and growth-inhibitory genes such as HOXC10, contributing to tumor survival, whereas AIs induce these genes to cause apoptosis and therapeutic benefit, but long-term AI treatment results in permanent repression of these genes via methylation and confers resistance. Therapies aimed at inhibiting AI-induced histone and DNA methylation may be beneficial in blocking or delaying AI resistance.

Scaffold Attachment Factor B1 Functions in Development, Growth, and Reproduction

ABSTRACT Scaffold attachment factor B1 (SAFB1) is a multifunctional protein that can bind both DNA and RNA and is involved in RNA processing and stress response. In addition, SAFB1 contains a transcriptional repression domain and can bind certain hormone receptors and repress their activity. To assess the role of SAFB1 in vivo, we generated SAFB1 mutant mice through targeted deletion in embryonic stem cells. While viable homozygous mutant (SAFB1−/−) mice were obtained, genotypic distribution indicated that homozygous deficiency resulted in both prenatal and neonatal lethality. Mice lacking SAFB1 exhibited dwarfism, as a result of in utero growth retardation, and had low serum insulin-like growth factor 1 (IGF1) levels. In agreement with the previous characterization of SAFB1 as a corepressor for hormone receptors, we found that SAFB1−/− mice displayed dramatic defects in the development and function of the reproductive system. Male SAFB1 null mice were infertile, apparently because of low circulating levels of testosterone. SAFB1−/− testes were small and showed progressive degeneration of the germinal epithelium, increased apoptosis of germ cells, and Leydig cell hyperplasia. SAFB−/− female mice were subfertile and showed progressive infertility, in part because of defects in oviductal transport and reduced numbers of follicles. Immortalized SAFB1−/− mouse embryonic fibroblasts showed cell-intrinsic defects including increased transcriptional estrogen receptor α activity and enhanced responsiveness to IGF1. Together, these in vivo findings establish a critical role for SAFB1 in development, growth regulation, and reproduction.

SAFB1 Mediates Repression of Immune Regulators and Apoptotic Genes in Breast Cancer Cells

The scaffold attachment factors SAFB1 and SAFB2 are paralogs, which are involved in cell cycle regulation, apoptosis, differentiation, and stress response. They have been shown to function as estrogen receptor corepressors, and there is evidence for a role in breast tumorigenesis. To identify their endogenous target genes in MCF-7 breast cancer cells, we utilized a combined approach of chromatin immunoprecipitation (ChIP)-on-chip and gene expression array studies. By performing ChIP-on-chip on microarrays containing 24,000 promoters, we identified 541 SAFB1/SAFB2-binding sites in promoters of known genes, with significant enrichment on chromosomes 1 and 6. Gene expression analysis revealed that the majority of target genes were induced in the absence of SAFB1 or SAFB2 and less were repressed. Interestingly, there was no significant overlap between the genes identified by ChIP-on-chip and gene expression array analysis, suggesting regulation through regions outside the proximal promoters. In contrast to SAFB2, which shared most of its target genes with SAFB1, SAFB1 had many unique target genes, most of them involved in the regulation of the immune system. A subsequent analysis of the estrogen treatment group revealed that 12% of estrogen-regulated genes were dependent on SAFB1, with the majority being estrogen-repressed genes. These were primarily genes involved in apoptosis, such as BBC3, NEDD9, and OPG. Thus, this study confirms the primary role of SAFB1/SAFB2 as corepressors and also uncovers a previously unknown role for SAFB1 in the regulation of immune genes and in estrogen-mediated repression of genes.

Disruption of Scaffold Attachment Factor B1 Leads to TBX2 Up-regulation, Lack of p19ARF Induction, Lack of Senescence, and Cell Immortalization

Scaffold attachment factor B1 (SAFB1) is a multifunctional protein, which has previously been implicated in breast cancer. Here, we show that genetic deletion of SAFB1 in mouse embryonic fibroblasts (MEF) leads to spontaneous immortalization and altered expression of two proteins involved in immortalization and escape from senescence: low levels of p19(ARF) and high levels of TBX2. Inactivation of TBX2 using a dominant-negative TBX2 resulted in up-regulation of p19(ARF) in SAFB1 knockout MEFs. SAFB1 loss also caused lack of contact inhibition, increased foci formation, and increased oncogene-induced anchorage-independent growth. These findings suggest that SAFB1 is a novel player in cellular immortalization and transformation.

Scaffold attachment factor B2 (SAFB2)-null mice reveal non-redundant functions of SAFB2 compared with its paralog, SAFB1

ABSTRACT Scaffold attachment factors SAFB1 and SAFB2 are multifunctional proteins that share >70% sequence similarity. SAFB1-knockout (SAFB1−/−) mice display a high degree of lethality, severe growth retardation, and infertility in male mice. To assess the in vivo role of SAFB2, and to identify unique functions of the two paralogs, we generated SAFB2−/− mice. In stark contrast to SAFB1−/−, SAFB2−/− offspring were born at expected Mendelian ratios and did not show any obvious defects in growth or fertility. Generation of paralog-specific antibodies allowed extensive expression analysis of SAFB1 and SAFB2 in mouse tissues, showing high expression of both SAFB1 and SAFB2 in the immune system, and in hormonally controlled tissues, with especially high expression of SAFB2 in the male reproductive tract. Further analysis showed a significantly increased testis weight in SAFB2−/− mice, which was associated with an increased number of Sertoli cells. Our data suggest that this is at least in part caused by alterations in androgen-receptor function and expression upon deletion of SAFB2. Thus, despite a high degree of sequence similarity, SAFB1−/− and SAFB2−/− mice do not totally phenocopy each other. SAFB2−/− mice are viable, and do not show any major defects, and our data suggest a role for SAFB2 in the differentiation and activity of Sertoli cells that deserves further study. Summary: Analysis of SAFB2−/− mice reveals lack of redundancy between the closely related paralogs SAFB1 and SAFB2, and a role for SAFB2 in the male reproductive system, specifically in Sertoli cells.

Analysis of SAFB1 and SAFB2 Knockout Mice Reveals Non-Redundant Functions of the Two Estrogen Receptor Co-Repressor Paralogs.

The Scaffold Attachment Factor B1 (SAFB1) and B2 (SAFB2) have been shown to be involved in chromatin organization, transcriptional regulation, and RNA processing. The paralogs SAFB1 and SAFB2 share 74% similarity at the amino acid level, with up to 98% similarity in some functional domains. These functional domains include DNA and RNA-binding domains, and a C-terminal repression domain. We have previously shown that SAFB1 and SAFB2 function as estrogen receptor (ER) corepressors – they directly bind to ER, and repress its transcriptional activity. There is also evidence that both proteins play a role in breast cancer; their shared chromosomal locus on chromosome 19p13 displays high rates of LOH, and protein loss was associated with worse survival of breast cancer patients.To understand the roles of SAFB1 and SAFB2 in development and function of hormone responsive tissues, we generated gene-specific knockout (KO) mouse models. Deletion of SAFB1 resulted in a high degree of embryonic and perinatal lethality. Surviving SAFB1 KO mice displayed severe growth retardation associated with low serum IGF-I levels, male infertility and female subfertility. In contrast, SAFB2 KO mice were born at the expected Mendelian ratio and did not show any obvious defects in growth or fertility. Initial gross pathology analysis has not yet revealed any significant defects.Since the SAFB proteins are known to play a role in estrogen response, we analyzed mammary gland development in the two mouse models. Young virgin SAFB1 KO mice showed delayed mammary gland development with a significantly reduced number of terminal end buds and decreased outgrowth compared to wild type (WT) controls. This was likely a result of delayed puberty, due to low IGF-I levels, since at the four month time point, mammary gland growth was restored in the virgin SAFB1 KO mice to that seen in WT controls. Interestingly, the KO glands exhibited increased alveolar development and side branching. To measure proliferation directly, we performed mammary gland transplantation experiments, thereby excluding secondary effects due to systemic defects. These studies showed a significant increase in proliferation in SAFB1 KO glands compared to WT control glands. In contrast to the SAFB1-null mice, we did not observe any obvious mammary gland defects in pubertal or adult SAFB2-null mice. However, mammary glands from aged virgin SAFB2 mice (1.5 years old) showed extensive side branching and precocious development of alveolar buds resembling glands of late pregnant mice. This was associated with increased proliferation of alveolar cells in the SAFB2 KO glands.In summary, genetic ablation of the ER co-repressors SAFB1 and SAFB2 results in defects in mammary gland development. In general, the two mouse models have very different phenotypes, revealing diverse and non-redundant functions of SAFB1 and SAFB2, findings that were unexpected based on their high sequence similarity. We are currently performing additional studies to finalize characterization of the in vivo phenotypes, focusing on hormone responsive tissues, and also to understand the mechanism underlying the observed phenotypes. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2162.

Estrogen Receptor-Mediated Repression of Target Genes: Mechanism of Action, and Biological Significance.

Estrogen-bound estrogen receptor (ER) can regulate the transcription of a large number of genes. Although estrogen represses as many genes as it induces, induction has been studied in detail while the repression mechanisms remain largely unknown. In this study we characterized several estrogen-repressed ERα target genes that are directly repressed by estrogen in breast cancer cells. Interestingly, many repressed genes are tumor suppressor genes, playing critical roles in cell cycle inhibition and/or apoptosis. One gene that particularly piqued our interest is Reprimo (RPRM) because of its robust repression in a number of cell lines, and its role as a cell cycle inhibitor. Additionally, it is highly methylated in a variety of cancers including breast cancer and maps to a locus that displays loss of heterozygosity, suggesting that it may be a tumor suppressor gene. As a result, E2-mediated repression of this gene may be a crucial step in the progression of breast cancer. We did show that RPRM decrease (by siRNA) enhances estrogen-mediated S-phase entry, strongly suggesting a biological role of its repression. RPRM was actively and strongly repressed by estrogen and this repression was not due to a quicker turnover of the RPRM mRNA by estrogen. E2-mediated repression of RPRM levels did not require new protein synthesis since it is also repressed in the presence of the translation inhibitor, cycloheximide. We find that estrogen repression of RPRM requires a tripartite interplay between ERα, FoxA1, and HDAC7 as knockdown of these proteins abrogates its repression. Remarkably, silencing of HDAC7 significantly relieved repression of the majority of eleven estrogen-repressed genes tested. Further examination of the interplay between HDAC7 and ERα revealed that HDAC7 can interact with ERα and repress its transcriptional activity in a deacetylase-independent manner. Chromatin immunoprecipitation assays demonstrate that ERα, FoxA1, and HDAC7 are all recruited to a cis-regulatory enhancer (-4.8 kb) in the RPRM gene in the presence of estrogen, which was associated with a release of RNA Pol II from the proximal promoter. Repression requires FoxA1 to be recruited to an epigenetic signature characterized by H3K4 mono- and di-methylation. In summary, we have uncovered a unique requirement for a deacetylase-independent function of HDAC7 in estrogen repression of genes such as RPRM. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4136.