@article {68226, title = {Kinetic principles underlying pioneer function of GAGA transcription factor in live cells.}, journal = {Nature Structural and Molecular Biology}, volume = {29}, year = {2022}, month = {2022 Jul 01}, pages = {665-676}, abstract = {

How pioneer factors interface with chromatin to promote accessibility for transcription control is poorly understood in vivo. Here, we directly visualize chromatin association by the prototypical GAGA pioneer factor (GAF) in live Drosophila hemocytes. Single-particle tracking reveals that most GAF is chromatin bound, with a stable-binding fraction showing nucleosome-like confinement residing on chromatin for more than 2 min, far longer than the dynamic range of most transcription factors. These kinetic properties require the full complement of GAF{\textquoteright}s DNA-binding, multimerization and intrinsically disordered domains, and are autonomous from recruited chromatin remodelers NURF and PBAP, whose activities primarily benefit GAF{\textquoteright}s neighbors such as Heat Shock Factor. Evaluation of GAF kinetics together with its endogenous abundance indicates that, despite on-off dynamics, GAF constitutively and fully occupies major chromatin targets, thereby providing a temporal mechanism that sustains open chromatin for transcriptional responses to homeostatic, environmental and developmental signals.

}, keywords = {Animals, Chromatin, DNA-Binding Proteins, Drosophila, Drosophila Proteins, Kinetics, Transcription Factors}, issn = {1545-9985}, doi = {10.1038/s41594-022-00800-z}, author = {Tang, Xiaona and Li, Taibo and Liu, Sheng and Wisniewski, Jan and Zheng, Qinsi and Rong, Yikang and Lavis, Luke D and Wu, Carl} } @article {67671, title = {Spatiotemporal coordination of transcription preinitiation complex assembly in live cells.}, journal = {Molecular Cell}, volume = {81}, year = {2021}, month = {2021 Sep 02}, pages = {3560-3575}, abstract = {

Transcription initiation by RNA polymerase II (RNA Pol II) requires preinitiation complex (PIC) assembly at gene promoters. In the dynamic nucleus, where thousands of promoters are broadly distributed in chromatin, it is unclear how multiple individual components converge on any target to establish the PIC. Here we use live-cell, single-molecule tracking in S.\ cerevisiae to visualize constrained exploration of the nucleoplasm by PIC components and Mediator{\textquoteright}s key role in guiding this process. On chromatin, TFIID/TATA-binding protein (TBP), Mediator, and RNA Pol II instruct assembly of a short-lived PIC, which occurs infrequently but efficiently within a few seconds on average. Moreover, PIC exclusion by nucleosome encroachment underscores regulated promoter accessibility by chromatin remodeling. Thus, coordinated nuclear exploration and recruitment to accessible targets underlies dynamic PIC establishment in yeast. Our study provides a global spatiotemporal model for transcription initiation in live cells.

}, issn = {1097-4164}, doi = {10.1016/j.molcel.2021.07.022}, author = {Nguyen, Vu Q and Ranjan, Anand and Liu, Sheng and Tang, Xiaona and Ling, Yick Hin and Wisniewski, Jan and Mizuguchi, Gaku and Li, Kai Yu and Jou, Vivian and Zheng, Qinsi and Lavis, Luke D and Lionnet, Timoth{\'e}e and Wu, Carl} } @article {65581, title = {Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction.}, journal = {eLife}, volume = {9}, year = {2020}, month = {2020 Apr 27}, abstract = {

The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription at yeast promoters is responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.

}, issn = {2050-084X}, doi = {10.7554/eLife.55667}, author = {Ranjan, Anand and Nguyen, Vu Q and Liu, Sheng and Wisniewski, Jan and Kim, Jee Min and Tang, Xiaona and Mizuguchi, Gaku and Elalaoui, Ejlal and Nickels, Timothy J and Jou, Vivian and English, Brian P and Zheng, Qinsi and Luk, Ed and Lavis, Luke D and Lionnet, Timoth{\'e}e and Wu, Carl} } @article {48908, title = {Molecular basis of CENP-C association with the CENP-A nucleosome at yeast centromeres.}, journal = {Genes \& Development}, volume = {31}, year = {2017}, month = {2017 Oct 01}, pages = {1958-1972}, abstract = {

Histone CENP-A-containing nucleosomes play an important role in nucleating kinetochores at centromeres for chromosome segregation. However, the molecular mechanisms by which CENP-A nucleosomes engage with kinetochore proteins are not well understood. Here, we report the finding of a new function for the budding yeast Cse4/CENP-A histone-fold domain interacting with inner kinetochore protein Mif2/CENP-C. Strikingly, we also discovered that AT-rich centromere DNA has an important role for Mif2 recruitment. Mif2 contacts one side of the nucleosome dyad, engaging with both Cse4 residues and AT-rich nucleosomal DNA. Both interactions are directed by a contiguous DNA- and histone-binding domain (DHBD) harboring the conserved CENP-C motif, an AT hook, and RK clusters (clusters enriched for arginine-lysine residues). Human CENP-C has two related DHBDs that bind preferentially to DNA sequences of higher AT content. Our findings suggest that a DNA composition-based mechanism together with residues characteristic for the CENP-A histone variant contribute to the specification of centromere identity.

}, issn = {1549-5477}, doi = {10.1101/gad.304782.117}, author = {Xiao, Hua and Wang, Feng and Wisniewski, Jan and Shaytan, Alexey K and Ghirlando, Rodolfo and Fitzgerald, Peter C and Huang, Yingzi and Wei, Debbie and Li, Shipeng and Landsman, David and Panchenko, Anna R and Wu, Carl} } @article {47418, title = {Multifocus microscopy with precise color multi-phase diffractive optics applied in functional neuronal imaging.}, journal = {Biomedical Optics Express}, volume = {7}, year = {2016}, month = {2016 Feb 16}, pages = {855-69}, abstract = {

Multifocus microscopy (MFM) allows high-resolution instantaneous three-dimensional (3D) imaging and has been applied to study biological specimens ranging from single molecules inside cells nuclei to entire embryos. We here describe pattern designs and nanofabrication methods for diffractive optics that optimize the light-efficiency of the central optical component of MFM: the diffractive multifocus grating (MFG). We also implement a {\textquotedblleft}precise color{\textquotedblright} MFM layout with MFGs tailored to individual fluorophores in separate optical arms. The reported advancements enable faster and brighter volumetric time-lapse imaging of biological samples. In live microscopy applications, photon budget is a critical parameter and light-efficiency must be optimized to obtain the fastest possible frame rate while minimizing photodamage. We provide comprehensive descriptions and code for designing diffractive optical devices, and a detailed methods description for nanofabrication of devices. Theoretical efficiencies of reported designs is ≈90\% and we have obtained efficiencies of \> 80\% in MFGs of our own manufacture. We demonstrate the performance of a multi-phase MFG in 3D functional neuronal imaging in living\ C. elegans.

Additional authors include:

Xin Jin, Joan Pulupa, Christine Cho, Mustafa Mir, Mohamed El Beheiry, Xavier Darzacq, Marcelo Nollmann, Maxime Dahan, Carl Wu, Timoth{\'e}e Lionnet, J. Alexander Liddle, and Cornelia I. Bargmann

}, doi = {10.1364/BOE.7.000855}, author = {Abrahamsson, Sara and Ilic, Rob and Wisniewski, Jan and Mehl, Brian and Yu, Liya and Chen, Lei and Davanco, Marcelo and Oujedi, Laura and Fiche, Jean-Bernard and Hajj, Bassam} } @article {45166, title = {Imaging the fate of histone Cse4 reveals de novo replacement in S phase and subsequent stable residence at centromeres.}, journal = {eLife}, volume = {3}, year = {2014}, month = {2014 May 20}, pages = {e02203}, abstract = {

The budding yeast centromere contains Cse4, a specialized histone H3 variant. Fluorescence pulse-chase analysis of an internally tagged Cse4 reveals that it is replaced with newly synthesized molecules in S phase, remaining stably associated with centromeres thereafter. In contrast, C-terminally-tagged Cse4 is functionally impaired, showing slow cell growth, cell lethality at elevated temperatures, and extra-centromeric nuclear accumulation. Recent studies using such strains gave conflicting findings regarding the centromeric abundance and cell cycle dynamics of Cse4. Our findings indicate that internally tagged Cse4 is a better reporter of the biology of this histone variant. Furthermore, the size of centromeric Cse4 clusters was precisely mapped with a new 3D-PALM method, revealing substantial compaction during anaphase. Cse4-specific chaperone Scm3 displays steady-state, stoichiometric co-localization with Cse4 at centromeres throughout the cell cycle, while undergoing exchange with a nuclear pool. These findings suggest that a stable Cse4 nucleosome is maintained by dynamic chaperone-in-residence Scm3.DOI: http://dx.doi.org/10.7554/eLife.02203.001.

}, issn = {2050-084X}, doi = {10.7554/eLife.02203}, author = {Wisniewski, Jan and Hajj, Bassam and Chen, Jiji and Mizuguchi, Gaku and Xiao, Hua and Wei, Debbie and Dahan, Maxime and Wu, Carl} } @article {45778, title = {Whole-cell, multicolor superresolution imaging using volumetric multifocus microscopy.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {111}, year = {2014}, month = {2014 Dec 9}, pages = {17480-5}, abstract = {

Single molecule-based superresolution imaging has become an essential tool in modern cell biology. Because of the limited depth of field of optical imaging systems, one of the major challenges in superresolution imaging resides in capturing the 3D nanoscale morphology of the whole cell. Despite many previous attempts to extend the application of photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) techniques into three dimensions, effective localization depths do not typically exceed 1.2 {\textmu}m. Thus, 3D imaging of whole cells (or even large organelles) still demands sequential acquisition at different axial positions and, therefore, suffers from the combined effects of out-of-focus molecule activation (increased background) and bleaching (loss of detections). Here, we present the use of multifocus microscopy for volumetric multicolor superresolution imaging. By simultaneously imaging nine different focal planes, the multifocus microscope instantaneously captures the distribution of single molecules (either fluorescent proteins or synthetic dyes) throughout an \~{}4-{\textmu}m-deep volume, with lateral and axial localization precisions of \~{}20 and 50 nm, respectively. The capabilities of multifocus microscopy to rapidly image the 3D organization of intracellular structures are illustrated by superresolution imaging of the mammalian mitochondrial network and yeast microtubules during cell division.

}, issn = {1091-6490}, doi = {10.1073/pnas.1412396111}, author = {Hajj, Bassam and Wisniewski, Jan and El Beheiry, Mohamed and Chen, Jiji and Revyakin, Andrey and Wu, Carl and Dahan, Maxime} } @article {5522, title = {Fast multicolor 3D imaging using aberration-corrected multifocus microscopy.}, journal = {Nature Methods}, volume = {10}, year = {2013}, month = {2013}, pages = {60-3}, abstract = {

Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.

}, issn = {1548-7105}, doi = {10.1038/nmeth.2277}, author = {Abrahamsson, Sara and Chen, Jiji and Hajj, Bassam and Stallinga, Sjoerd and Katsov, Alexander Y and Wisniewski, Jan and Mizuguchi, Gaku and Soule, Pierre and Mueller, Florian and Darzacq, Claire Dugast and Darzacq, Xavier and Wu, Carl and Bargmann, Cornelia I and Agard, David A and Dahan, Maxime and Gustafsson, Mats G L} }