Assistant Professor, Department of Anatomy & Structural Biology
The focus of our laboratory is to develop single molecule systems to understand the molecular mechanistic basis of actions of disease driven transcription factors on their respective target genes. Specifically, we are using a powerful combination of single molecule imaging, biochemistry, proteomics and molecular biology to understand how these key transcription factors dynamically discriminate their various targets in vitro and also within the complicated milieu of the cell. In addition we are collaborating with the Liu lab here at Einstein to utilize High-Resolution Cryo-EM to study the structural features within these transcription factors necessary for recognizing various key gene promoters and co-factors involved in tumor suppression. Ultimately we would like to discern the mechanistic basis for how cancer or disease associated mutations within these transcription factors affect gene regulatory networks with the hope of designing therapeutics to counter their negative affects in vivo.
Gene expression initiated by mammalian RNA polymerase II (Pol II) involves a highly coordinated assembly of over 100 different polypeptides residing within several multi-subunit complexes to form the pre-initiation complex (PIC). One critical step in gene activation involves direction of the core recognition complexes to specific target promoters. The multi-subunit TFIID complex is a principle component within the transcriptional machinery capable of recognizing and targeting specific promoter DNA. Current models suggest that the binding of TFIID to the core promoter is followed by a sequential recruitment of other general transcription factors including TF-IIA, -IIB, -IIE, -IIH, -IIF, Mediator and Pol II that culminates with transcription initiation and promoter escape of Pol II.
An ordered assembly pathway provides multiple points that can be regulated to ultimately affect gene expression. Indeed, activators, such as the tumor suppressor p53 protein and the onco-protein c-Jun, can stimulate transcription by targeting multiple factors, such as TFIID, TFIIB, TFIIF, Mediator, and Pol II, along with multiple steps (TFIID recruitment, Pol II promoter escape, elongation and re-initiation). Despite over 20 years of intense study, we still do not understand how transcriptional activators select their various targets within the transcription machinery to modulate levels of gene expression.
Probing the dynamic assembly of the transcription PIC in real time
Single molecule-fluorescence has become an advanced and sensitive tool to study protein dynamics of individual molecules in a population in real time. It is therefore a technique particularly suited to study complex behavior within populations of these precious megadalton sized endogenous macromolecular machines. To gain a better mechanistic understanding of how the p53 and c-Jun activators dynamically regulate transcription pre-initiation complex (PIC) formation at the single molecule level, we developed a system to specifically label multisubunit human transcription complexes with Quantum dots to image their real-time assembly on promoter DNA. Total Internal Reflection Microscopy is used to examine the recruitment of single quantum dot labeled TFIID and AlexaFluor labeled p53 molecules to various promoters of key tumor suppression genes. Time resolved studies revealed that p53 helps recruit TFIID to the promoter DNA, in addition to affecting a step in the transcription cycle after TFIID has arrived at the promoter DNA. Current efforts are focused on examining the role of p53 in targeting additional factors within the PIC. Additional projects involve proteomic screening and development of therapeutics that target key surfaces within our multisubunit complexes involved in regulation of transcription.
Revyakin A, Zhang Z,, Coleman RA, Li Y, Inouye C, Lucas, JK, Park SR, Chu S, TjianR. Transcription initiation by human RNA polymerase II visualized at single-molecule resolution. Genes Dev. 2012 Aug1;26(15):1691-702. PubMed PMID: 22810624; PubMed Central PMCID: PMC3418587.
Liu WL, Coleman RA, Ma E, Grob P, Yang JL, Zhang Y, Dailey G, Nogales E, TjianR. Structures of three distinct activator-TFIID complexes. Genes Dev. 2009 Jul1;23(13):1510-21. PubMed PMID: 19571180; PubMed Central PMCID: PMC2704470.
Liu WL, Coleman RA, Grob P, King DS, Florens L, Washburn MP, Geles KG, YangJL, Ramey V, Nogales E, Tjian R. Structural changes in TAF4b-TFIID correlate withpromoter selectivity. Mol Cell. 2008 Jan 18;29(1):81-91. PubMed PMID: 18206971;PubMed Central PMCID: PMC2486835.
Coleman RA, Taggart AK, Burma S, Chicca JJ 2nd, Pugh BF. TFIIA regulates TBPand TFIID dimers. Mol Cell. 1999 Sep;4(3):451-7. PubMed PMID: 10518227.
Jackson-Fisher AJ, Burma S, Portnoy M, Schneeweis LA, Coleman RA, Mitra M,Chitikila C, Pugh BF. Dimer dissociation and thermosensitivity kinetics of theSaccharomyces cerevisiae and human TATA binding proteins. Biochemistry. 1999 Aug 31;38(35):11340-8. PubMed PMID: 10471284.
Weideman CA, Netter RC, Benjamin LR, McAllister JJ, Schmiedekamp LA, ColemanRA, Pugh BF. Dynamic interplay of TFIIA, TBP and TATA DNA. J Mol Biol. 1997 Aug8;271(1):61-75. PubMed PMID: 9300055.
Coleman RA, Pugh BF. Slow dimer dissociation of the TATA binding proteindictates the kinetics of DNA binding. Proc Natl Acad Sci U S A. 1997 Jul8;94(14):7221-6. PubMed PMID: 9207072; PubMed Central PMCID: PMC23798.
Coleman RA, Pugh BF. Evidence for functional binding and stable sliding of theTATA binding protein on nonspecific DNA. J Biol Chem. 1995 Jun 9;270(23):13850-9.PubMed PMID: 7775443.
Coleman RA, Taggart AK, Benjamin LR, Pugh BF. Dimerization of the TATA bindingprotein. J Biol Chem. 1995 Jun 9;270(23):13842-9. PubMed PMID: 7775442.
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