Single Molecule Imaging of Transcription Factors Involved in Cancer, Hematopoiesis and Ageing

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 cancer and additional diseases related to hematopoietic dysfunction, eye development and ageing 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, our live cell imaging studies find that transcription factors and chromatin remodelers, including p53, RNA Polymerase II and PBAF, dynamically cycle on and off the genome in spatial hubs of activity on the timescale of seconds to minutes. The chromatin structure of underlying target sequences and enzymatic activities associated with the transcription factors/chromatin remodelers dictate their dynamic cycling and binding kinetics. Oncogenic mutations in transcription factors can also affect the binding kinetics of associated proteins that are being co-loaded onto the genome. In addition, we find that single molecule kinetic binding profiles of transcription factors are associated with different cellular states during development.  Furthermore, we are also adapting our imaging systems to directly assess the enzymatic activity of chromatin remodelers and enzymes that translocate along the genome.

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. Using these imaging systems, we find that patterns of transcription factor binding and transcriptional bursting display memory effects to fine-tune expression of genes. Future work will be to develop this high resolution imaging in live mice and live-cell image based drug screening platforms that rapidly determine combinatorial effects of epigenetic inhibitors in different cell types and diseased cells.

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 how these complexes engage nucleosomes surrounding the core promoter.

1.     Kenworthy CA, Haque N, Liou S-H, Chandris P, Wong V, Dziuba P, Lavis LD, Liu WL, Singer RH, and Coleman RA. Bromodomains regulate dynamic targeting of the PBAF chromatin remodeling complex to chromatin hubs. Biophys J. 2022 Mar 30;S0006-3495(22)00237-5. doi: 10.1016/j.bpj.2022.03.027. PMID: 35364106.

2.     Senecal A, *Singer RH and *Coleman RA. Transcriptional burst kinetics are linked to short term transcriptional memory. *corresponding authors. bioRxiv. 2021 Nov. 2. doi: https://doi.org/10.1101/2021.10.31.466715 .

3.     Desai RV, Chen X, Martin B, Chaturvedi S, Hwang DW, Li W, Yu C, Ding S, Thomson M, Singer RH, Coleman RA, Hansen M, and Weinberger LS. Discovery of a Cellular Mechanism Regulating Transcriptional Noise. Science. 2021 July 22:eabc6505. doi: 10.1126/science.abc6506. Online ahead of print. PMID: 34301855.

4.     Liou S-H, Singh SK, Singer RH, *Coleman RA, and *Liu WL. Structure of the p53/RNA Polymerase II Assembly. Commun Biol. 2021 Mar 25;4(1):397. doi: 10.1038/s42003-021-01934-4.PMID: 33767390 *corresponding authors.

5.     Drosopoulos W, Vierra DA, Kenworthy CA, *Coleman RA, and *Schildkraut CL. Dynamic assembly and disassembly of the human DNA Polymerase δ on the genome in vivo. Cell Rep. 2020 Feb 4;30(5):1329-1341.e5. doi: 10.1016/j.celrep.2019.12.101. Pubmed PMID: 32023453. *corresponding authors

6.     Carvajal LA, Ben Neriah D, Senecal A, Benard L, Thiruthuvanathan V, Yatsenko T, Narayanagari SR, Wheat JC, Todorova TI, Mitchell K, Kenworthy C, Guerlavais V, Annis DA, Bartholdy B, Will B, Anampa JD, Mantzaris I, Aivado M, Singer RH, Coleman RA, Verma A, and Steidl U.  Dual inhibition of MDMX and MDM2 as a therapeutic strategy in leukemia. Science Translational Medicine 11 Apr 2018 Vol. 10, Issue 436, eaao3003. DOI: 10.1126/scitranslmed.aao3003. Pubmed PMID: 29643228.

7.     *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.*corresponding authors

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