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Our long-term goal is to elucidate basic mechanisms of eukaryotic transcription control and clarify how these controls are subverted during oncogenesis. Ultimately, we hope to use such information to identify points of oncogenic susceptibility within the cell and thus identify potential targets for pharmacologic intervention. We employ biochemical, molecular, and cell biological approaches to study the regulation of eukaryotic RNA polymerase II transcription.
1. Function and regulation of the human transcriptional Mediator
Transcription is central to most cellular processes, both normal and pathologic. For protein-coding genes, transcription by RNA polymerase II is a principal step targeted for regulation within the cell. Gene-specific transcription factors regulate transcription initiation through interactions with chromatin-modifying activities or the general transcription machinery in order to enhance or repress nucleosome-mediated promoter repression and transcription pre-initiation complex formation, respectively. Work over the last decade has established that a central component of the general transcription machinery is Mediator, an evolutionarily conserved multiprotein interface between gene-specific transcription factors and RNA polymerase II. In this capacity, the Mediator occupies a centrally important role in the expression of most eukaryotic protein-coding genes. Originally identified biochemically in the yeast Saccharomyces cerevisiae, we were among the first to identify from human cells a metazoan homolog of the yeast Mediator termed human Mediator (hMediator). We were the first to link the metazoan Mediator to the control of cell-fate specific gene transcription during development and among the first to implicate the Mediator as an end-point in signal transduction pathways. Our work along with that of others has led to the current concept that metazoan Mediators specify unique transcriptional control mechanisms that underlie, at least in part, the advanced complexity of multicellular organisms. We are actively engaged in biochemical studies pertaining to the following issues with respect to hMediator: (1) the precise mechanism by which eukaryotic activators and repressors interface with hMediator to effect activation or repression of transcription; (2) the precise physical and functional interactions among individual Mediator subunits; and (3) how developmental and oncogenic signaling pathways converge on hMediator and how these signals are, in turn, relayed to RNA polymerase II. These studies should reveal novel insight into underlying mechanisms by which cellular phenotype is specified.
2. BRCA1 and transcription control in the DNA damage response.
A major focus of our ongoing research aims to understand how inactivation of the human breast cancer susceptibility gene product BRCA1 leads to breast tumorigenesis. BRCA1 ensures global genome stability through its dual participation in DNA double-strand break repair and transcriptional regulation of DNA damage-inducible genes. This work aims to understand how the caretaker function of BRCA1 is subserved by its role in transcription control. To this end, we have been studying the functional interaction between BRCA1 and ZBRK1, a sequence-specific DNA-binding transcriptional repressor of the DNA damage-inducible GADD45 gene that functions in G2/M cell cycle checkpoint control. Our studies show that BRCA1 functions as a co-repressor of ZBRK1 by targeting the two hallmark features of ZBRK1 as a sequence-specific transcriptional repressor – its ability to bind sequence-specifically to DNA and its ability to repress transcription once bound to DNA. In addition to GADD45, potential ZBRK1 binding sites have been identified in other DNA damage-inducible genes, indicating a prospective global role for ZBRK1 and BRCA1 in the coordinate regulation of DNA damage-response genes. We are actively engaged in the following biochemical and cell biological studies to further define the mechanism and regulation of BRCA1-dependent ZBRK1 transcriptional repression: (1) the influence of BRCA1 on the DNA-binding affinity and specificity of ZBRK1; (2) the identification and characterization of the complete co-repressor network through which BRCA1 mediates repression of promoter-bound ZBRK1; and (3) how BRCA1-dependent ZBRK1 repression is regulated by DNA damage-induced cell signaling. These studies should reveal novel insight into the underlying basis for the caretaker properties of BRCA1 in preserving genomic stability and identify potential therapeutic targets for future intervention in breast cancer.
3. BRCA1 and estrogen signaling in breast cancer.
Mutational inactivation of BRCA1 confers a cumulative lifetime risk of breast and ovarian cancers. However, the underlying basis for the tissue- and gender-specific tumor suppressor properties of BRCA1 remains poorly defined. We previously discovered a novel function for BRCA1 in suppressing the ligand-independent transcriptional activity of the estrogen receptor ? (ER?), a principal determinant of the growth and differentiation of breast and ovarian tissues. Importantly, we showed that clinically validated BRCA1 missense mutations abrogate this repression activity, suggesting that its ER?-specific repression function is important for the biological activity of BRCA1 in breast tumor suppression. We hypothesize that BRCA1 represents a ligand-reversible barrier to transcriptional activation by unliganded promoter-bound ER? and, further, that mutational inactivation of BRCA1 promotes mammary epithelial cell proliferation through aberrant expression of estrogen-responsive genes. To confirm and extend this hypothesis, we are currently engaged in the following biochemical and cell biological studies pertaining to the modulation of ER? function by BRCA1: (1) the precise mechanism by which BRCA1 mediates ligand-independent ER? repression; (2) the regulation of BRCA1-mediated ER? repression by both estrogen-dependent and estrogen-independent cell signals; and (3) the biological role of BRCA1 in the control of cellular proliferation through modulation of ER? activity. These studies should reveal novel insight into the tissue-specific tumor suppressor function of BRCA1 and identify defined molecular targets for future intervention in breast cancer.
- Vulto-van Silfhout AT, de Vries BB, van Bon BW, Hoischen A, Ruiterkamp-Versteeg M, Gilissen C, Gao F, van Zwam M, Harteveld CL, van Essen AJ, Hamel BC, Kleefstra T, Willemsen MA, Yntema HG, van Bokhoven H, Brunner HG, Boyer TG, de Brouwer AP. Mutations in MED12 cause X-linked Ohdo syndrome. Am J Hum Genet. 2013 Mar 7;92(3):401-6.
- Gu S, Boyer TG, and Naski MC: (2012) Basic helix-loop-helix transcription factor Twist1 inhibits transacivator function of master chondrogenic regulator Sox9. J Biol Chem. 287(25): 21082-92.
- Spaeth JM, Kim NH, and Boyer TG: (2011) Mediator and human disease. Semin Cell Dev Biol. 22(7): 776-87.
- Krebs P, Fan W, Chen YH, Tobita K, Downes MR, Wood MR, Sun L, Li X, Xia Y, Ding N, Spaeth JM, Moresco EM, Boyer TG, Lo CW, Yen J, Evans RM, and Beutler B: (2011) Lethal mitochondrial cardiomyopathy in a hypomorphic Med30 mouse mutant is ameliorated by ketogenic diet. Proc Natl Acad Sci U S A. 108(49): 19678-82.
- Xu X, Zhou H, and Boyer TG: (2011) Mediator is a transducer of amyloid-precursor-protein-dependent nuclear signaling. EMBO Rep. 12(3): 216-22.
- Trauernicht AM, Kim SJ, Kim NH, Clarke R, and Boyer TG: (2010) DBC-1 mediates endocrine resistant breast cancer cell survival. Cell Cycle. 9(6): 1218-19.
- Ding N, Tomomori-Sato C, Sato S, Conaway RC, Conaway JW, and Boyer TG: (2009) MED19 and MED26 are synergistic functional targets of the RE1 silencing transcription factor in epigenetic silencing of neuronal gene expression. J Biol Chem. 284(5): 2648-56.
- Ding N, Zhou H, Esteve P-E, Chin HG, Kim S, Xu X, Joseph SM, Friez MJ, Schwartz CE, Pradhan S, and Boyer TG: (2008) Mediator links epigenetic silencing of neuronal gene expression with X-linked mental retardation. Mol Cell. 31(3): 347-59.
- Trauernicht A, Kim SJ, Kim NH, and Boyer TG: (2007) Modulation of estrogen receptor alpha protein level and survival function by DBC-1. Mol Endocrinol. 21(7): 1526-36.
- Zhou H, Kim S, Ishii S, and Boyer TG: (2006) Mediator modulates Gli3-dependent Sonic hedgehog signaling. Mol Cell Biol. 26(23): 8667-82.
- Lu M, Chen D, Lin Z, Reierstad S, Trauernicht AM, Boyer TG, and Bulun SE: (2006) BRCA1 negatively regulates the cancer-associated aromatase promoters I.3 and II in breast adipose fibroblasts and malignant epithelial cells. J Clin Endocrinol Metab. 91(11): 4514-9.
- Kim S, Xu X, Hecht A, and Boyer TG: (2006) Mediator is a transducer of Wnt/beta-catenin signaling. J Biol Chem. 281(20): 14066-75.
- Tan W, Kim S, and Boyer TG: (2004) Tetrameric oligomerization mediates transcriptional repression by the BRCA1-dependent Kruppel-associated box-zinc finger protein ZBRK1. J Biol Chem. 279(53): 55153-60.
- Tan W, Zheng L, Lee W-H, and Boyer TG: (2004) Functional dissection of transcription factor ZBRK1 reveals zinc fingers with dual roles in DNA-binding and BRCA1-dependent transcriptional repression. J Biol Chem. 279(8): 6576-87.
- Trauernicht AM and Boyer TG: (2003) BRCA1 and estrogen signaling in breast cancer. Breast Dis. 18: 11-20.
- Zhong Q, Boyer TG, Hasty P, Chen P-L, and Lee W-H: (2002) Deficient nonhomologous end-joining activity in cell-free extracts from Brca1-null fibroblasts. Cancer Res. 62(14): 3966-70.
- Boyer TG and Lee W-H: (2002) Breast Cancer Susceptibility Genes. Science & Medicine 8: 138-149.
- Lee W-H and Boyer TG: (2001) BRCA1 and BRCA2 in breast cancer. The Lancet (Supplement), 358: S5.
- Zheng L, Annab LA, Afshari CA, Lee W-H, and Boyer TG: (2001) BRCA1 Mediates Ligand-Independent Transcriptional Repression of the Estrogen Receptor. Proc Natl Acad Sci U S A. 98(17): 9587-92.
- Zheng L, Li S, Boyer TG, and Lee W-H: (2000) Lessons learned from BRCA1 and BRCA2. Oncogene. 19(53): 6159-75.