@article {67992, title = {Bromodomains regulate dynamic targeting of the PBAF chromatin remodeling complex to chromatin hubs.}, journal = {Biophysical Journal}, volume = {121}, year = {2022}, month = {2022 May 3}, pages = {1738-1752}, abstract = {

Chromatin remodelers actively target arrays of acetylated nucleosomes at select enhancers and promoters to facilitate or shut down the repeated recruitment of RNA Pol II during transcriptional bursting. It is poorly understood how chromatin remodelers such as PBAF dynamically target different chromatin states inside a live cell. Our live-cell single molecule fluorescence microscopy study reveals chromatin hubs throughout the nucleus where PBAF rapidly cycles on and off the genome. Deletion of PBAF{\textquoteright}s bromodomains impairs targeting and stable engagement of chromatin in hubs. Dual color imaging reveals that PBAF targets both euchromatic and heterochromatic hubs with distinct genome binding kinetic profiles that mimic chromatin stability. Removal of PBAF{\textquoteright}s bromodomains stabilizes H3.3 binding within chromatin indicating that bromodomains may play a direct role in remodeling of the nucleosome. Our data suggests that PBAF{\textquoteright}s dynamic bromodomain mediated engagement of a nucleosome may reflect the chromatin remodeling potential of differentially bound chromatin states.

}, issn = {1542-0086}, doi = {10.1016/j.bpj.2022.03.027}, author = {Kenworthy, Charles A and Haque, Nayem and Liou, Shu-Hao and Chandris, Panagiotis and Wong, Vincent and Dziuba, Patrycja and Lavis, Luke D and Liu, Wei-Li and Singer, Robert H and Coleman, Robert A} } @article {67811, title = {Micro-Meta App: an interactive tool for collecting microscopy metadata based on community specifications.}, journal = {Nature Methods}, volume = {18}, year = {2021}, month = {2021 Dec 03}, pages = {1489-1495}, abstract = {

For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. Micro-Meta App is an intuitive, highly interoperable, open-source software tool that was developed in the context of the 4D Nucleome (4DN) consortium and is designed to facilitate the extraction and collection of relevant microscopy metadata as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training purposes.

}, issn = {1548-7105}, doi = {10.1038/s41592-021-01315-z}, author = {Rigano, Alessandro and Ehmsen, Shannon and {\"O}zt{\"u}rk, Serkan Utku and Ryan, Joel and Balashov, Alexander and Hammer, Mathias and Kirli, Koray and Boehm, Ulrike and Brown, Claire M and Bellve, Karl and Chambers, James J and Cosolo, Andrea and Coleman, Robert A and Faklaris, Orestis and Fogarty, Kevin E and Guilbert, Thomas and Hamacher, Anna B and Itano, Michelle S and Keeley, Daniel P and Kunis, Susanne and Lacoste, Judith and Laude, Alex and Ma, Willa Y and Marcello, Marco and Montero-Llopis, Paula and Nelson, Glyn and Nitschke, Roland and Pimentel, Jaime A and Weidtkamp-Peters, Stefanie and Park, Peter J and Alver, Burak H and Grunwald, David and Strambio-De-Castillia, Caterina} } @article {47734, title = {Imaging transcription: past, present, and future.}, journal = {Cold Spring Harbor Symposia on Quantitative Biology}, volume = {80}, year = {2015}, month = {2015}, pages = {1-8}, abstract = {

Transcription, the first step of gene expression, is exquisitely regulated in higher eukaryotes to ensure correct development and homeostasis. Traditional biochemical, genetic, and genomic approaches have proved successful at identifying factors, regulatory sequences, and potential pathways that modulate transcription. However, they typically only provide snapshots or population averages of the highly dynamic, stochastic biochemical processes involved in transcriptional regulation. Single-molecule live-cell imaging has, therefore, emerged as a complementary approach capable of circumventing these limitations. By observing sequences of molecular events in real time as they occur in their native context, imaging has the power to derive cause-and-effect relationships and quantitative kinetics to build predictive models of transcription. Ongoing progress in fluorescence imaging technology has brought new microscopes and labeling technologies that now make it possible to visualize and quantify the transcription process with single-molecule resolution in living cells and animals. Here we provide an overview of the evolution and current state of transcription imaging technologies. We discuss some of the important concepts they uncovered and present possible future developments that might solve long-standing questions in transcriptional regulation.

}, issn = {1943-4456}, doi = {10.1101/sqb.2015.80.027201}, author = {Coleman, Robert A and Liu, Zhe and Darzacq, Xavier and Tjian, Robert and Singer, Robert H and Lionnet, Timoth{\'e}e} } @article {6518, title = {Transcription initiation by human RNA polymerase II visualized at single-molecule resolution.}, journal = {Genes \& Development}, volume = {26}, year = {2012}, month = {2012 Aug 1}, pages = {1691-702}, abstract = {

Forty years of classical biochemical analysis have identified the molecular players involved in initiation of transcription by eukaryotic RNA polymerase II (Pol II) and largely assigned their functions. However, a dynamic picture of Pol II transcription initiation and an understanding of the mechanisms of its regulation have remained elusive due in part to inherent limitations of conventional ensemble biochemistry. Here we have begun to dissect promoter-specific transcription initiation directed by a reconstituted human Pol II system at single-molecule resolution using fluorescence video-microscopy. We detected several stochastic rounds of human Pol II transcription from individual DNA templates, observed attenuation of transcription by promoter mutations, observed enhancement of transcription by activator Sp1, and correlated the transcription signals with real-time interactions of holo-TFIID molecules at individual DNA templates. This integrated single-molecule methodology should be applicable to studying other complex biological processes.

}, keywords = {Fluorescence, Genetic, Humans, Microscopy, Molecular Imaging, Mutation, Promoter Regions, RNA Polymerase II, Sp1 Transcription Factor, Transcription, Transcription Factor TFIID, Video}, issn = {1549-5477}, doi = {10.1101/gad.194936.112}, author = {Revyakin, Andrey and Zhang, Zhengjian and Coleman, Robert A and Li, Yan and Inouye, Carla and Lucas, Julian K and Park, Sang-Ryul and Chu, Steven and Tjian, Robert} } @article {2389, title = {Structures of three distinct activator-TFIID complexes.}, journal = {Genes \& Development}, volume = {23}, year = {2009}, month = {2009 Jul 1}, pages = {1510-21}, abstract = {

Sequence-specific DNA-binding activators, key regulators of gene expression, stimulate transcription in part by targeting the core promoter recognition TFIID complex and aiding in its recruitment to promoter DNA. Although it has been established that activators can interact with multiple components of TFIID, it is unknown whether common or distinct surfaces within TFIID are targeted by activators and what changes if any in the structure of TFIID may occur upon binding activators. As a first step toward structurally dissecting activator/TFIID interactions, we determined the three-dimensional structures of TFIID bound to three distinct activators (i.e., the tumor suppressor p53 protein, glutamine-rich Sp1 and the oncoprotein c-Jun) and compared their structures as determined by electron microscopy and single-particle reconstruction. By a combination of EM and biochemical mapping analysis, our results uncover distinct contact regions within TFIID bound by each activator. Unlike the coactivator CRSP/Mediator complex that undergoes drastic and global structural changes upon activator binding, instead, a rather confined set of local conserved structural changes were observed when each activator binds holo-TFIID. These results suggest that activator contact may induce unique structural features of TFIID, thus providing nanoscale information on activator-dependent TFIID assembly and transcription initiation.

}, keywords = {Hela Cells, Humans, Imaging, Immunoglobulins, Models, Molecular, Protein Binding, Protein Multimerization, Protein Structure, Proto-Oncogene Proteins c-jun, Quaternary, TATA-Binding Protein Associated Factors, Three-Dimensional, Transcription Factor TFIID, Tumor Suppressor Protein p53}, issn = {1549-5477}, doi = {10.1073/pnas.1100640108}, author = {Liu, Wei-Li and Coleman, Robert A and Ma, Elizabeth and Grob, Patricia and Yang, Joyce L and Zhang, Yixi and Dailey, Gina and Nogales, Eva and Tjian, Robert} } @article {2399, title = {Structural changes in TAF4b-TFIID correlate with promoter selectivity.}, journal = {Molecular Cell}, volume = {29}, year = {2008}, month = {2008 Jan 18}, pages = {81-91}, abstract = {

Proper ovarian development requires the cell type-specific transcription factor TAF4b, a subunit of the core promoter recognition complex TFIID. We present the 35 A structure of a cell type-specific core promoter recognition complex containing TAF4b and TAF4 (4b/4-IID), which is responsible for directing transcriptional synergy between c-Jun and Sp1 at a TAF4b target promoter. As a first step toward correlating potential structure/function relationships of the prototypic TFIID versus 4b/4-IID, we have compared their 3D structures by electron microscopy and single-particle reconstruction. These studies reveal that TAF4b incorporation into TFIID induces an open conformation at the lobe involved in TFIIA and putative activator interactions. Importantly, this open conformation correlates with differential activator-dependent transcription and promoter recognition by 4b/4-IID. By combining functional and structural analysis, we find that distinct localized structural changes in a megadalton macromolecular assembly can significantly alter its activity and lead to a TAF4b-induced reprogramming of promoter specificity.

}, keywords = {B-Lymphocytes, Cell Line, Computer-Assisted, Electron, Genetic, Hela Cells, Humans, Image Processing, Imaging, Microscopy, Models, Molecular, Organ Specificity, Promoter Regions, Protein Conformation, Protein Interaction Mapping, Proto-Oncogene Proteins c-jun, Sp1 Transcription Factor, Structure-Activity Relationship, TATA-Binding Protein Associated Factors, Three-Dimensional, Transcription Factor TFIIA, Transcription Factor TFIID, Transcriptional Activation, Tumor}, issn = {1097-2765}, doi = {10.1073/pnas.1100640108}, author = {Liu, Wei-Li and Coleman, Robert A and Grob, Patricia and King, David S and Florens, Laurence and Washburn, Michael P and Geles, Kenneth G and Yang, Joyce L and Ramey, Vincent and Nogales, Eva and Tjian, Robert} }