My lab has been interested in understanding the mechanisms of transcription and gene regulation in eukaryotes using primarily cell-free systems reconstituted with purified gene-specific transcription factors, general cofactors, and components of the general transcription machinery to recapitulate transcriptional events in the test tubes. The mechanistic studies based on cell-free transcription systems with DNA and chromatin templates are further verified in vivo by chromatin immunoprecipitation (ChIP), RT-PCR, reporter gene assay, co-immunoprecipitation (co-IP), and nucleosome mapping using different types of cultured cells, including knock-out and knock-down cells.
Our goals are to elucidate the general principles underlying gene activation and repression in mammalian cells and their associated viruses, in particular, DNA tumor viruses such as human papillomaviruses (HPVs). Recently, we have applied reconstituted chromatin systems to define histone modifications and chromatin remodeling involved in p53 target gene transcription implicated in cell cycle control, DNA repair, and apoptosis. In addition, we have identified bromodomain 4 protein (Brd4) as the cellular corepressor implicated in transcriptional repression of HPV E6 and E7 oncoprotein expression by HPV-encoded E2 proteins. In general, our research has been focused on the following four areas:
Role of Human General Transcription Factors and Cofactors in Eukaryotic Transcription
Several general transcription factors, including TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, are essential for transcription on most eukaryotic promoters by RNA polymerase II (pol II). The core promoter-binding factor TFIID, which is comprised of the TATA-binding protein (TBP) and a dozen or so evolutionarily conserved pol II-specific TBP-associated factors (TAFs), has intrinsic activity to recognize the TATA box, initiator and downstream promoter elements, and initiates preinitiation complex assembly on both TATA-containing and TATA-less promoters. TFIID also functions as a general cofactor in transducing regulatory signals to the general transcription machinery and plays a crucial role for transcription from chromatin templates due to multiple enzymatic activities inherent to its TAF components.
These diverse features have implicated TFIID as a central player in eukaryotic transcription. To dissect the mechanisms of gene activation and repression in mammalian cells, we have employed human cell-free transcription systems reconstituted either with individually purified general transcription components (TFIIA, TFIIB, TBP, TFIIE, TFIIF, and PC4 coactivator) and highly purified epitope-tagged protein complexes (TFIID, TFIIH, and pol II), or with TFIID and a preassembled pol II holoenzyme complex that contains components of the general transcription machinery as well as SWI/SNF chromatin remodeling factor and GCN5 histone acetyltransferase. We would like to investigate how pol II, working in conjunction with accessory proteins, such as TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and general cofactors (Mediator, USA-derived components, TBP-associated factors, and TFIIA) determines the time and location in transcribing specific gene products.
Our objectives are to define the biochemical activities of general transcription components in modulating gene activity and to uncover the combinatorial nature of eukaryotic gene regulation using well-defined cell-free transcription systems reconstituted with all purified human proteins.
Gene Regulation in Human Papillomaviruses (HPVs)
HPVs induce many human diseases, including skin warts, genital warts, and cervical cancer. We are interested in understanding the mechanisms by which virus-encoded E2 proteins activate or repress HPV transcription in the context of both DNA and chromatin templates. Using in vitro-reconstituted HPV minichromosome that faithfully recapitulates in vivo phasing of HPV chromatin, we recently identified bromodomain 4 (Brd4) protein as the cellular adaptor mediating the repressing activity of HPV E2 that in turn controls the expression of HPV E6 and E7 oncoproteins.
We are currently elucidating the repression mechanisms employed by the E2-Brd4 silencing complex and defining the role of SMC5 and SMC6 proteins in regulating E2 function in transcription, cell cycle checkpoint control, viral DNA replication, and HPV genome maintenance and segregation. In addition, we have been working on the activation mechanisms by which p53 and AP-1 modulate cellular and viral gene expression and how posttranslational modifications, such as methylation, acetylation, sumoylation, neddylation, and ubiquitination, fine-tune the transcriptional activity of these transcription factors via covalent linkage of critical lysine residues.
We are also interested in dissecting the molecular mechanism underlying p300's involvement in gene activation and repression by switching its role from activation to repression. Reconstituted histone acetyltransferase (HAT) assays and in vitro sumoylation, neddylation, and ubiquitination assays have already been established in my laboratory with purified factors for these attempts.
Mechanisms of Hormone Receptor Function
Hormone receptors are ligand-dependent transcription factors that play a central role in cellular function and physiology. Our goal is to dissect the mechanisms of transcriptional regulation mediated by hormone receptors acting through the recruitment of hormone-specific cofactors and the general transcription machinery and general cofactors.
Our establishment of a ligand-independent estrogen receptor a-dependent activation system reconstituted with recombinant TFIIB, TFIIE, TFIIF, PC4, and epitope tagged TFIID, TFIIH, and pol II has made it possible to define the functional role of Mediator and nuclear receptor-specific coactivators and corepressors in chromatin transcription.
Gene Regulation on TATA-Less Promoters
The majority of our human genes is TATA-less and contains promoters constituted from a combination of at least 7 different types of core promoter elements. Our goals are to define transcription complex assembly pathways on human TATA-less gene promoters and to investigate how transcriptional regulators modulate gene activation and repression through distinct core promoter elements. Understanding these intricate networks will uncover the regulatory mechanisms underlying differential and combinatorial expression of our human genes in a cell cycle-, cell type-, and differentiation-specific manner.
These projects are conducted in hope to uncover the mechanisms of eukaryotic transcription and to understand the general principles of combinatorial regulation in mammalian cells.
- Chiang, C.-M. 2016. Phospho-BRD4: transcription plasticity and drug targeting. Drug Discovery Today: Technologies 19: 17-22.
- Wu, S.-Y., D.S. Nin, A-Y. Lee, S. Simanski, T. Kodadek, and C.-M. Chiang. 2016. BRD4 phosphorylation regulates HPV E2-mediated viral transcription, origin replication, and cellular MMP-9 expression. Cell Reports 16: 1733-1748.
- Delcuratolo, M., J. Fertey, M. Schneider, J. Schütz, N. Leiprecht, B. Hudjez, S. Brodbeck, S. Corall, M. Dreer, R.M. Schwab, M. Grimm, S.-Y. Wu, F. Stubenrauch, C.-M. Chiang, and T. Iftner. 2016. Papillomavirus-associated tumor formation critically depends on c-fos expression induced by viral protein E2 and bromodomain protein Brd4. PLoS Pathog. 12: e1005366.
- Chiang, C.-M. 2014. Nonequivalent response to bromodomain-targeting BET inhibitors in oligodendrocyte cell fate decision. Chemistry & Biology 21: 804-806.
- Yang, X.-J. and C.-M. Chiang. 2013. Sumoylation in gene regulation, human diseases and therapeutic actions. F1000Prime Reports 5: 45.
- Wu, S.-Y., A-Y. Lee, H.-T. Lai, H. Zhang, and C.-M. Chiang. 2013. Phospho switch triggers Brd4 chromatin binding and activator recruitment for gene-specific targeting. Mol. Cell 49: 843-857. (Journal Highlight with Article Preview by Laptenko and Prives. 2013. Anything but simple: a phosphorylation-driven toggle within Brd4 triggers gene-specific transcriptional activation. Mol. Cell 49: 838-839.)
- Chiang, C.-M. 2009. Brd4 engagement from chromatin targeting to transcriptional regulation: selective contact with acetylated histone H3 and H4. F1000 Biology Reports 1:98.
- Wu, S.-Y. and C.-M. Chiang. 2009. Crosstalk between acetylation and sumoylation in regulating p53-dependent chromatin transcription and DNA binding. EMBO J. 28: 1246-1259.
- Wu, S.-Y. and C.-M. Chiang. 2007. The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regulation. J. Biol. Chem. 282: 13141-13145.
- Wu, S.-Y., A.Y. Lee, S.Y. Hou, J.K. Kemper, H. Erdjument-Bromage, P. Tempst, and C.-M. Chiang. 2006. Brd4 links chromatin targeting to HPV transcriptional silencing. Genes Dev. 20: 2383-2396.
- Thomas, M.C. and C.-M. Chiang. 2006. The general transcription machinery and general cofactors. Critical Reviews in Biochemistry and Molecular Biology 41: 105-178.
- Thomas, M.C. and C.-M. Chiang. 2005. E6 oncoprotein represses p53-dependent gene activation via inhibition of protein acetylation independently of inducing p53 degradation. Mol. Cell 17: 251-264.