Faculty Profile

Dr. Robert A. Coleman, Ph.D.

Robert A. Coleman, Ph.D.

Assistant Professor, Department of Anatomy & Structural Biology

Professional Interests

Human tumorigenesis is a complicated process marked by a loss of the cell’s ability to regulate critical cellular processes, such as transcription, RNA processing and translation, leading to uncontrollable cell growth. As our understanding of tumorigenesis becomes more sophisticated, a combinatorial approach is necessary to better understand the dynamic coordinated action of very large multi-subunit enzymes and protein complexes controlling these key cellular processes. Recent advances in single-molecule imaging provide an unprecedented spatiotemporal window into probing dynamic functional interactions between these heterogeneous large multi-subunit transcription assemblies and chromatin in real-time. In addition, high-resolution cryo-electron microscopy and genome-wide binding assays allow us to rapidly survey detailed functional interactions on physiologically relevant substrates with limited amounts of samples. Armed with these advanced in vitro and in vivo approaches, our lab seeks to gain a mechanistic understanding of how transcription complexes dynamically regulate expression of tumor suppression pathways and how these processes are altered in cancer.

Specifically we apply these tools to mechanistically dissect how the p53 tumor suppressor protein communicates with multiple transcription assemblies, such as chromatin remodeling factors (PBAF), core promoter recognition factors (TFIID), and RNA Polymerase II, to circumnavigate the repressive effects of chromatin on transcription.  We are also applying these same strategies to study how oncogenic mutants of p53 and chromatin-remodeling complexes mechanistically function to promote tumor formation. Our long-term goal is to determine the molecular origin of additional diseases such as Rett Syndrome, Huntington’s, and Diabetes using our multi-disciplinary approach centered around advanced single molecule imaging, genome-wide studies, and structural biology. 

Development of single molecule imaging systems to study how transcription factors alter chromatin structure and regulate transcriptional bursting

To understand how proteins engage chromatin at high temporal and spatial resolution, our group has established numerous in vitro and in vivo systems utilizing high-resolution co-localization, single molecule FRET, and dynamic live cell imaging. Strikingly, we find that p53 serves as pioneer factor that prefers to bind native target DNA sequences within nucleosomes versus on naked DNA. Furthermore, our studies reveal that p53 transiently alters the structure of the nucleosome, potentially paving the way for other factors to further modify chromatin. We have also adapted our in vitro single molecule assays to begin mechanistically deciphering how the PBAF chromatin-remodeling complex remodels nucleosomes. In collaboration with Rob Singer's lab at Einstein, we have also developed a live-cell multicolor single molecule imaging system to examine how our transcription factors dynamically regulate transcriptional bursting of tumor suppression genes.

Single molecule dynamics and structural studies of TFIID mediated transcription

As TFIID is a central player in regulating transcription initiation by RNA Polymerase II, we want to understand how many different regulatory factors access this key core promoter recognition factor during transcription pre-initiation complex (PIC) formation.  To this end, our in vitro single molecule studies revealed that p53 dynamically loads TFIID onto native promoters.  Interestingly, once bound to DNA, TFIID induced dissociation of p53 from the complex to allow additional p53 molecules to escort general transcription factors, such as RNA Polymerase II to the TFIID bound promoter scaffold. Future studies will use single molecule co-localization and FRET better understand the role of chromatin in regulating p53-mediated PIC formation.

My group has also collaborated with Dr. Wei-Li Liu’s lab at Einstein to establish a system to “build” up p53 and TFIID mediated assemblies involved in PIC formation for both single molecule and cryo-EM structural studies. Using this system we determined the 3D cryo-EM structure of a p53/TFIID/TFIIA ternary complex bound to two different native p53-regulated promoter DNAs.  The 3D cryo-EM analysis indicated that p53 induces a common global architecture of TFIID bound to these native promoters. We have also determined the 3D cryo-EM structure of p53 bound to RNA Polymerase II. In this structure p53 occupies a position on RNA Polymerase that is typically bound by elongation factors. In vitro biochemical assays revealed that p53 stimulates transcription elongation of RNA Polyermase II suggesting a common structural mechanism associated with regulating elongation. Our newly established systems have paved the way towards the building of large multimeric assemblies with the eventual goal of structurally examining a complete PIC on native promoters involved in tumor suppression. 

High-throughput Peptidomic studies for use as probes of transcription factor function in our single molecule systems

We have recently isolated diverse sets of peptides that interact with our transcription factor complexes by combining bacterial flagella peptide-display systems with next generation DNA sequencing. In our screens of TFIID, we have found novel interacting peptides that resemble proteins involved in DNA repair and RNA Polymerase III driven transcription, two functions that were not previously ascribed to TFIID. We further confirmed these novel interactions with TFIID through conventional immunoprecipitation assays and MudPIT mass spectrometry, suggesting additional roles of TFIID outside of Pol II transcription. Future studies in collaboration with Dr. Wei-Li Liu will focus on determining the 3D structure of TFIID bound to these novel interacting proteins using cryo-EM to determine their influence on TFIID’s function. We will also investigate the use of these peptides as small molecule therapeutics that modulate the in vivo function of both wild type and disease driven mutants of p53 and TFIID.

 

Selected Publications

a. Coleman RA, Zhen Q, Singh SK, Peng CS, Cianfrocco M, Zhang Z, Song L, Piasecka A, Aldeborgh H, Basishvili G, Rice W, and Liu WL. p53 dynamically directs TFIID assembly on target DNA. Mol Cell Biol., 2017 Jun 15;37(13). pii:e00085-17. Doi: 10.1128/MCB.00085-17. PubMed PMID: 28416636.Liu WL, Coleman RA, Ma E, Grob P, Yang JL, Zhang Y, Dailey G, Nogales E, Tjian
R. Structures of three distinct activator-TFIID complexes. Genes Dev. 2009 Jul
1;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, Yang
JL, Ramey V, Nogales E, Tjian R. Structural changes in TAF4b-TFIID correlate with
promoter 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 TBP
and 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 the
Saccharomyces 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, Coleman
RA, Pugh BF. Dynamic interplay of TFIIA, TBP and TATA DNA. J Mol Biol. 1997 Aug
8;271(1):61-75. PubMed PMID: 9300055.
Coleman RA, Pugh BF. Slow dimer dissociation of the TATA binding protein
dictates the kinetics of DNA binding. Proc Natl Acad Sci U S A. 1997 Jul
8;94(14):7221-6. PubMed PMID: 9207072; PubMed Central PMCID: PMC23798.
Coleman RA, Pugh BF. Evidence for functional binding and stable sliding of the
TATA 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 binding
protein. J Biol Chem. 1995 Jun 9;270(23):13842-9. PubMed PMID: 7775442.

Liu WL, Coleman RA, and Singh, SK. A new era of studying p53-mediated transcription activation. Transcription. 2017 Aug 10:0 doi: 10.1080/21541264.2017.1345354. PMID: 28795863

Coleman RA, Zhen Q, Singh SK, Peng CS, Cianfrocco M, Zhang Z, Song L, Piasecka A, Aldeborgh H, Basishvili G, Rice W, and Liu WL. p53 dynamically directs TFIID assembly on target DNA. Mol Cell Biol., 2017 Jun 15;37(13). pii:e00085-17. Doi: 10.1128/MCB.00085-17. PubMed PMID: 28416636.

Singh SK, Qiao Z, Song L, Jani V, Rice W, Eng E, Coleman RA, Liu WL. Structural visualization of the p53/RNA Polymerase II assembly. Genes Dev. 2016 Nov 15;30(22) 2527-2537. PubMed PMID: 27920087.

Coleman RA, Liu Z, Darzacq X, Tjian R, Singer RH, Lionnet T. Imaging Transcription: Past, Present, and Future. Cold Spring Harb Symp Quant Biol. 2015;80:1-8. PubMed PMID: 26763984; PubMed Central PMCID:PMC4915995.

Revyakin A, Zhang Z, Coleman RA, Li Y, Inouye C, Lucas JK, Park SR, Chu S, Tjian R. Transcription initiation by human RNA polymerase II visualized at single-molecule resolution. Genes Dev. 2012 Aug 1; 26(15), 1691-702. PubMed PMID: 22810624; PubMed Central PMCID: PMC3418587.

Liu WL, Coleman RA, Ma E, Grob P, Yang JL, Dailey G, Nogales E, and Tjian R. Structures of three distinct activator-TFIID complexes.  Genes Dev. 2009 Jul 1;23(13), 1510-21. PubMed PMID: 19571180PubMed Central PMCIDPMC2704470.

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.

 

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Research Information