Yongfeng Shang

Cofactor Dynamics and Sufficiency in Estrogen Receptor–Regulated Transcription

Many cofactors bind the hormone-activated estrogen receptor (ER), yet the specific regulators of endogenous ER-mediated gene transcription are unknown. Using chromatin immunoprecipitation (ChIP), we find that ER and a number of coactivators rapidly associate with estrogen responsive promoters following estrogen treatment in a cyclic fashion that is not predicted by current models of hormone activation. Cycles of ER complex assembly are followed by transcription. In contrast, the anti-estrogen tamoxifen (TAM) recruits corepressors but not coactivators. Using a genetic approach, we show that recruitment of the p160 class of coactivators is sufficient for gene activation and for the growth stimulatory actions of estrogen in breast cancer supporting a model in which ER cofactors play unique roles in estrogen signaling.

Formation of the Androgen Receptor Transcription Complex

Androgen receptor (AR) is required for sexual differentiation and is implicated in the development of prostate cancer. Here we describe distinct functions for cofactor proteins and gene regulatory elements in the assembly of AR-mediated transcription complexes. The formation of an activation complex involves AR, coactivators, and RNA polymerase II recruitment to both the enhancer and promoter, whereas the formation of a repression complex involves factors bound only at the promoter and not the enhancer. These results suggest a model for the functional coordination between the promoter and enhancer in which communication between these elements is established through shared coactivators in the AR transcription complex.

LSD1 is a subunit of the NuRD complex and targets the metastasis programs in breast cancer.

Lysine-specific demethylase 1 (LSD1) exerts pathway-specific activity in animal development and has been linked to several high-risk cancers. Here, we report that LSD1 is an integral component of the Mi-2/nucleosome remodeling and deacetylase (NuRD) complex. Transcriptional target analysis revealed that the LSD1/NuRD complexes regulate several cellular signaling pathways including TGFbeta1 signaling pathway that are critically involved in cell proliferation, survival, and epithelial-to-mesenchymal transition. We demonstrated that LSD1 inhibits the invasion of breast cancer cells in vitro and suppresses breast cancer metastatic potential in vivo. We found that LSD1 is downregulated in breast carcinomas and that its level of expression is negatively correlated with that of TGFbeta1. Our data provide a molecular basis for the interplay of histone demethylation and deacetylation in chromatin remodeling. By enlisting LSD1, the NuRD complex expands its chromatin remodeling capacity to include ATPase, histone deacetylase, and histone demethylase.

The Molecular Mechanism Governing the Oncogenic Potential of SOX2 in Breast Cancer

SOX genes encode a family of high-mobility group transcription factors that play critical roles in organogenesis. The functional specificity of different SOX proteins and the tissue specificity of a particular SOX factor are largely determined by the differential partnership of SOX transcription factors with other transcription regulators, many of which have not yet been discovered. Virtually all members of the SOX family have been found to be deregulated in a wide variety of tumors. However, little is known about the cellular and molecular behaviors involved in the oncogenic potential of SOX proteins. Using cell culture experiments, tissue analysis, molecular profiling, and animal studies, we report here that SOX2 promotes cell proliferation and tumorigenesis by facilitating the G1/S transition and through its transcription regulation of the CCND1 gene in breast cancer cells. In addition, we identified β-catenin as the transcription partner for SOX2 and demonstrated that SOX2 andβ-catenin act in synergy in the transcription regulation of CCND1 in breast cancer cells. Our experiments not only determined a role for SOX2 in mammary tumorigenesis but also revealed another activity of the multifunctional protein, β-catenin.

Hypomethylation-linked activation of PAX2 mediates tamoxifen-stimulated endometrial carcinogenesis.

Tamoxifen, a selective oestrogen receptor modulator, has been used in the treatment of all stages of hormone-responsive breast cancer. However, tamoxifen shows partial oestrogenic activity in the uterus and its use has been associated with an increased incidence of endometrial cancer. The molecular explanation for these observations is not known. Here we show that tamoxifen and oestrogen have distinct but overlapping target gene profiles. Among the overlapping target genes, we identify a paired-box gene, PAX2, that is crucially involved in cell proliferation and carcinogenesis in the endometrium. Our experiments show that PAX2 is activated by oestrogen and tamoxifen in endometrial carcinomas but not in normal endometrium, and that this activation is associated with cancer-linked hypomethylation of the PAX2 promoter.

Integration of Estrogen and Wnt Signaling Circuits by the Polycomb Group Protein EZH2 in Breast Cancer Cells

ABSTRACT Essential for embryonic development, the polycomb group protein enhancer of zeste homolog 2 (EZH2) is overexpressed in breast and prostate cancers and is implicated in the growth and aggression of the tumors. The tumorigenic mechanism underlying EZH2 overexpression is largely unknown. It is believed that EZH2 exerts its biological activity as a transcription repressor. However, we report here that EZH2 functions in gene transcriptional activation in breast cancer cells. We show that EZH2 transactivates genes that are commonly targeted by estrogen and Wnt signaling pathways. We demonstrated that EZH2 physically interacts directly with estrogen receptor α and β-catenin, thus connecting the estrogen and Wnt signaling circuitries, functionally enhances gene transactivation by estrogen and Wnt pathways, and phenotypically promotes cell cycle progression. In addition, we identified the transactivation activity of EZH2 in its two N-terminal domains and demonstrated that these structures serve as platforms to connect transcription factors and the Mediator complex. Our experiments indicated that EZH2 is a dual function transcription regulator with a dynamic activity, and we provide a mechanism for EZH2 in tumorigenesis.

BRG-1 Is Recruited to Estrogen-Responsive Promoters and Cooperates with Factors Involved in Histone Acetylation

ABSTRACT Several factors that mediate activation by nuclear receptors also modify the chemical and structural composition of chromatin. Prominent in this diverse group is the steroid receptor coactivator 1 (SRC-1) family, which interact with agonist-bound nuclear receptors, thereby coupling them to multifunctional transcriptional coregulators such as CREB-binding protein (CBP), p300, and PCAF, all of which have potent histone acetyltransferase activity. Additionally factors including the Brahma-related gene 1 (BRG-1) that are involved in the structural remodeling of chromatin also mediate hormone-dependent transcriptional activation by nuclear receptors. Here, we provide evidence that these two distinct mechanisms of coactivation may operate in a collaborative manner. We demonstrate that transcriptional activation by the estrogen receptor (ER) requires functional BRG-1 and that the coactivation of estrogen signaling by either SRC-1 or CBP is BRG-1 dependent. We find that in response to estrogen, ER recruits BRG-1, thereby targeting BRG-1 to the promoters of estrogen-responsive genes in a manner that occurs simultaneous to histone acetylation. Finally, we demonstrate that BRG-1-mediated coactivation of ER signaling is regulated by the state of histone acetylation within a cell. Inhibition of histone deacetylation by trichostatin A dramatically increases BRG-1-mediated coactivation of ER signaling, and this increase is reversed by overexpression of histone deacetylase 1. These studies support a critical role for BRG-1 in ER action in which estrogen stimulates an ER–BRG-1 association coupling BRG-1 to regions of chromatin at the sites of estrogen-responsive promoters and promotes the activity of other recruited factors that alter the acetylation state of chromatin.

Molecular mechanisms of oestrogen and SERMs in endometrial carcinogenesis

Endometrial cancer is the most common gynaecological cancer, and is associated with endometrial hyperplasia, unopposed oestrogen exposure and adjuvant therapy for breast cancer using selective oestrogen-receptor modulators (SERMs), particularly tamoxifen. Oestrogen and SERMs are thought to be involved in endometrial carcinogenesis through their effects on transcriptional regulation. Ultimately, oestrogen and SERMs affect the transduction of cellular signalling pathways that govern cell growth and proliferation, through downstream effectors such as PAX2 (paired box 2).

Histone demethylase JMJD2B coordinates H3K4/H3K9 methylation and promotes hormonally responsive breast carcinogenesis

It is well-documented that the methylation of histone H3 lysine 4 (H3K4) and of H3K9 are mutually exclusive, an epigenetic phenomenon conserved from yeast to humans. How this opposed methylation modification is accomplished and coordinated in mammalian cells is poorly understood. Here we report that the H3K9 trimethyl demethylase JMJD2B is an integral component of the H3K4-specific methyltransferase, the mixed-lineage leukemia (MLL) 2 complex. We show that the JMJD2B/MLL2 complex is copurified with estrogen receptor α (ERα) and is required for ERα-regulated transcription. We demonstrate that H3K9 demethylation and H3K4 methylation are coordinated in ERα-activated transcription such that H3K9 demethylation is a prerequisite for H3K4 methylation. Significantly, depletion of JMJD2B impairs the estrogen-induced G1/S transition of the cell cycle in vitro and inhibits breast tumorigenesis in vivo. Interestingly, JMJD2B itself is an ERα target gene, and forms a feed-forward regulatory loop in regulation of the hormone response. Our results provide a molecular basis for the coordinated H3K4 methylation/H3K9 demethylation in transcription activation, link the trimethyl demethylase JMJD2B to euchromatin functions, and provide a mechanism for JMJD2B in breast carcinogenesis.

Molecular mechanisms involved in the growth stimulation of breast cancer cells by leptin

Obesity is a risk factor for breast cancer in postmenopausal women. Leptin, an adipocyte-derived cytokine, elicits proliferative effects in some cell types and potentially stimulates the growth of mammary epithelium. Here we show that leptin induced time- and dose-dependent signal transducer and activator of transcription 3 (STAT3) phosphorylation and extracellular signal-regulated kinase (ERK) 1/2 kinase activation in breast carcinoma cells. Blocking STAT3 phosphorylation with a specific inhibitor, AG490, abolished leptin-induced proliferation of MCF-7 cells, whereas blocking ERK1/2 activation by a specific ERK1/2 kinase inhibitor, U0126, did not result in any significant changes in leptin-induced cell proliferation. Our experiments also showed that one member of the p160 family of steroid receptor coactivators, steroid receptor coactivator (SRC)-1, but not glucocorticoid receptor interacting protein 1 (GRIP1) or amplified in breast cancer 1 (AIB1), also functioned in gene transactivation in response to leptin treatment. Glutathione S-transferase pull-down experiments showed that SRC-1 physically interacted with the activation domain of STAT3 and that chromatin immunoprecipitation experiments detected the occupancy of SRC-1, but not GRIP1 or AIB1, on the promoter of STAT3 target genes. Our experiments collectively showed that SRC-1 is involved in STAT3 signaling pathway that is implicated in leptin-stimulated cell growth.