@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 {65176, title = {Rational design of fluorogenic and spontaneously blinking labels for super-resolution imaging.}, journal = {ACS Central Science}, volume = {5}, year = {2019}, month = {2019 Sep 25}, pages = {1602-1613}, abstract = {

Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equilibrium toward the nonfluorescent lactone form can improve cell-permeability and allow creation of "fluorogenic" compounds-ligands that shift to the fluorescent zwitterion upon binding a biomolecular target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, we develop a quantitative framework for the development of new fluorogenic dyes, determining that the lactone-zwitterion equilibrium constant () is sufficient to predict fluorogenicity. This rubric emerged from our analysis of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging experiments. We then designed a novel fluorophore-Janelia Fluor 526 (JF)-with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resolution immunofluorescence. JF constitutes a new label for advanced microscopy experiments, and our quantitative framework will enable the rational design of other fluorogenic probes for bioimaging.

}, issn = {2374-7943}, doi = {10.1021/acscentsci.9b00676}, author = {Zheng, Qinsi and Ayala, Anthony X and Chung, Inhee and Weigel, Aubrey V and Ranjan, Anand and Falco, Natalie and Grimm, Jonathan B and Tkachuk, Ariana N and Wu, Carl and Lippincott-Schwartz, Jennifer and Singer, Robert H and Lavis, Luke D} } @article {65034, title = {Comment on "A histone acetylation switch regulates H2A.Z deposition by the SWR-C remodeling enzyme".}, journal = {Science}, volume = {353}, year = {2016}, month = {2016 Jul 22}, pages = {358}, abstract = {

Watanabe et al (Reports, 12 April 2013, p. 195) study the yeast SWR1/SWR-C complex responsible for depositing the histone variant H2A.Z by replacing nucleosomal H2A with H2A.Z. They report that reversal of H2A.Z replacement is mediated by SWR1 and related INO80 on an H2A.Z nucleosome carrying H3K56Q. Using multiple assays and reaction conditions, we find no evidence of such reversal of H2A.Z exchange.

}, keywords = {Adenosine Triphosphatases, Chromatin Assembly and Disassembly, Histones, Multienzyme Complexes, Nucleosomes, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins}, issn = {1095-9203}, doi = {10.1126/science.aad5921}, author = {Wang, Feng and Ranjan, Anand and Wei, Debbie and Wu, Carl} } @article {46973, title = {H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast.}, journal = {eLife}, volume = {4}, year = {2015}, month = {2015 Jun 27}, abstract = {

The histone variant H2A.Z is a universal mark of gene promoters, enhancers, and regulatory elements in eukaryotic chromatin. The chromatin remodeler SWR1 mediates site-specific incorporation of H2A.Z by a multi-step histone replacement reaction, evicting histone H2A-H2B from the canonical nucleosome and depositing the H2A.Z-H2B dimer. Binding of both substrates, the canonical nucleosome and the H2A.Z-H2B dimer, is essential for activation of SWR1. We found that SWR1 primarily recognizes key residues within the α2 helix in the histone-fold of nucleosomal histone H2A, a region not previously known to influence remodeler activity. Moreover, SWR1 interacts preferentially with nucleosomal DNA at superhelix location 2 on the nucleosome face distal to its linker-binding site. Our findings provide new molecular insights on recognition of the canonical nucleosome by a chromatin remodeler and have implications for ATP-driven mechanisms of histone eviction and deposition.

}, issn = {2050-084X}, doi = {10.7554/eLife.06845}, author = {Ranjan, Anand and Wang, Feng and Mizuguchi, Gaku and Wei, Debbie and Huang, Yingzi and Wu, Carl} } @article {6910, title = {The catalytic subunit of the SWR1 remodeler is a histone chaperone for the H2A.Z-H2B dimer.}, journal = {Molecular Cell}, volume = {53}, year = {2014}, month = {2014 Feb 6}, pages = {498-505}, abstract = {

Histone variant H2A.Z-containing nucleosomes exist at most eukaryotic promoters and play important roles in gene transcription and genome stability. The multisubunit nucleosome-remodeling enzyme complex SWR1, conserved from yeast to mammals, catalyzes the ATP-dependent replacement of histone H2A in canonical nucleosomes with H2A.Z. How SWR1 catalyzes the replacement reaction is largely unknown. Here, we determined the crystal structure of the N-terminal region (599-627) of the catalytic subunit Swr1, termed Swr1-Z domain, in complex with the H2A.Z-H2B dimer at 1.78 {\r A} resolution. The Swr1-Z domain forms a 310 helix and an irregular chain. A conserved LxxLF motif in the Swr1-Z 310 helix specifically recognizes the αC helix of H2A.Z. Our results show that the Swr1-Z domain can deliver the H2A.Z-H2B dimer to the DNA-(H3-H4)2 tetrasome to form the nucleosome by a histone chaperone mechanism.

}, issn = {1097-4164}, doi = {10.1016/j.molcel.2014.01.010}, author = {Hong, Jingjun and Feng, Hanqiao and Wang, Feng and Ranjan, Anand and Chen, Jianhong and Jiang, Jiansheng and Ghirlando, Rodolfo and Xiao, T Sam and Wu, Carl and Bai, Yawen} } @article {6413, title = {Molecular architecture of the ATP-dependent chromatin-remodeling complex SWR1.}, journal = {Cell}, volume = {154}, year = {2013}, month = {2013 Sep 12}, pages = {1220-31}, abstract = {

The ATP-dependent chromatin-remodeling complex SWR1 exchanges a variant histone H2A.Z/H2B dimer\ for a canonical H2A/H2B dimer at nucleosomes flanking histone-depleted regions, such as promoters. This localization of H2A.Z is conserved throughout eukaryotes. SWR1 is a 1 megadalton complex containing 14 different polypeptides, including the AAA+ ATPases Rvb1 and Rvb2. Using electron microscopy, we obtained the three-dimensional structure of SWR1 and mapped its major functional components. Our data show that SWR1 contains a single heterohexameric Rvb1/Rvb2 ring that, together with the catalytic subunit Swr1, brackets two independently assembled multisubunit modules. We also show that SWR1 undergoes a large conformational change upon engaging a limited region of the nucleosome core particle. Our work suggests an important structural role for the Rvbs and a distinct substrate-handling mode by SWR1, thereby providing a structural framework for understanding the complex dimer-exchange reaction.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2013.08.018}, author = {Nguyen, Vu Q and Ranjan, Anand and Stengel, Florian and Wei, Debbie and Aebersold, Ruedi and Wu, Carl and Leschziner, Andres E} } @article {6412, title = {Nucleosome-free region dominates histone acetylation in targeting SWR1 to promoters for H2A.Z replacement.}, journal = {Cell}, volume = {154}, year = {2013}, month = {2013 Sep 12}, pages = {1232-45}, abstract = {

The histone variant H2A.Z is a genome-wide signature of nucleosomes proximal to eukaryotic regulatory DNA. Whereas the multisubunit chromatin remodeler SWR1 is known to catalyze ATP-dependent deposition of H2A.Z, the mechanism of SWR1 recruitment to S.\ cerevisiae promoters has been unclear. A sensitive assay for competitive binding of dinucleosome substrates revealed that SWR1 preferentially binds long nucleosome-free DNA and the adjoining nucleosome core particle, allowing discrimination of gene promoters over gene bodies. Analysis of mutants indicates that the conserved Swc2/YL1 subunit and the adenosine triphosphatase domain of Swr1 are mainly responsible for binding to substrate. SWR1 binding is enhanced on nucleosomes acetylated by the NuA4 histone acetyltransferase, but recognition of nucleosome-free and nucleosomal DNA is dominant over interaction with acetylated histones. Such hierarchical cooperation between DNA and histone signals expands the dynamic range of genetic switches, unifying classical gene regulation by DNA-binding factors with ATP-dependent nucleosome remodeling and posttranslational histone modifications.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2013.08.005}, author = {Ranjan, Anand and Mizuguchi, Gaku and Fitzgerald, Peter C and Wei, Debbie and Wang, Feng and Huang, Yingzi and Luk, Ed and Woodcock, Christopher L and Wu, Carl} }