A dynamic mode of mitotic bookmarking by transcription factors.

During mitosis, transcription is shut off, chromatin condenses, and most transcription factors (TFs) are reported to be excluded from chromosomes. How do daughter cells re-establish the original transcription program? Recent discoveries that a select set of TFs remain bound on mitotic chromosomes suggest a potential mechanism for maintaining transcriptional programs through the cell cycle termed mitotic bookmarking. Here we report instead that many TFs remain associated with chromosomes in mouse embryonic stem cells, and that the exclusion previously described is largely a fixation artifact. In particular, most TFs we tested are significantly enriched on mitotic chromosomes. Studies with Sox2 reveal that this mitotic interaction is more dynamic than in interphase and is facilitated by both DNA binding and nuclear import. Furthermore, this dynamic mode results from lack of transcriptional activation rather than decreased accessibility of underlying DNA sequences in mitosis. The nature of the cross-linking artifact prompts careful re-examination of the role of TFs in mitotic bookmarking

A stable mode of bookmarking by TBP recruits RNA polymerase II to mitotic chromosomes.

Maintenance of transcription programs is challenged during mitosis when chromatin becomes condensed and transcription is silenced. How do the daughter cells re-establish the original transcription program? Here, we report that the TATA-binding protein (TBP), a key component of the core transcriptional machinery, remains bound globally to active promoters in mouse embryonic stem cells during mitosis. Using live-cell single-molecule imaging, we observed that TBP mitotic binding is highly stable, with an average residence time of minutes, in stark contrast to typical TFs with residence times of seconds. To test the functional effect of mitotic TBP binding, we used a drug-inducible degron system and found that TBP promotes the association of RNA Polymerase II with mitotic chromosomes, and facilitates transcriptional reactivation following mitosis. These results suggest that the core transcriptional machinery promotes efficient transcription maintenance globally

Evidence for DNA-mediated nuclear compartmentalization distinct from phase separation.

RNA Polymerase II (Pol II) and transcription factors form concentrated hubs in cells via multivalent protein-protein interactions, often mediated by proteins with intrinsically disordered regions. During Herpes Simplex Virus infection, viral replication compartments (RCs) efficiently enrich host Pol II into membraneless domains, reminiscent of liquid-liquid phase separation. Despite sharing several properties with phase-separated condensates, we show that RCs operate via a distinct mechanism wherein unrestricted nonspecific protein-DNA interactions efficiently outcompete host chromatin, profoundly influencing the way DNA-binding proteins explore RCs. We find that the viral genome remains largely nucleosome-free, and this increase in accessibility allows Pol II and other DNA-binding proteins to repeatedly visit nearby DNA binding sites. This anisotropic behavior creates local accumulations of protein factors despite their unrestricted diffusion across RC boundaries. Our results reveal underappreciated consequences of nonspecific DNA binding in shaping gene activity, and suggest additional roles for chromatin in modulating nuclear function and organization

Heat shock reduces stalled RNA polymerase II and nucleosome turnover genome-wide

The cellular heat-shock response involves multiple changes in gene regulation. Using single-base-pair mapping of nucleosomal and subnucleosomal particles in chromatin extracts, Teves and Henikoff explore alterations in the chromatin landscape upon heat shock. They find loss of RNA Pol II and reduced nucleosome turnover in the bodies of genes, providing insight into the interplay between nucleosome dynamics and transcriptional changes

Advances in visualizing transcription factor-DNA interactions

At the heart of the transcription process is the specific interaction between transcription factors (TFs) and their target DNA sequences. Decades of molecular biology research have led to unprecedented insights into how TFs access the genome to regulate transcription. In the last 20 years, advances in microscopy have enabled scientists to add imaging as a powerful tool in probing two specific aspects of TF-DNA interactions: structure and dynamics. In this review, we examine how applications of diverse imaging technologies can provide structural and dynamic information that complements insights gained from molecular biology assays. As a case study, we discuss how applications of advanced imaging techniques have reshaped our understanding of TF behavior across the cell cycle, leading to a rethinking in the field of mitotic bookmarking.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

A dynamic mode of mitotic bookmarking by transcription factors.

During mitosis, transcription is shut off, chromatin condenses, and most transcription factors (TFs) are reported to be excluded from chromosomes. How do daughter cells re-establish the original transcription program? Recent discoveries that a select set of TFs remain bound on mitotic chromosomes suggest a potential mechanism for maintaining transcriptional programs through the cell cycle termed mitotic bookmarking. Here we report instead that many TFs remain associated with chromosomes in mouse embryonic stem cells, and that the exclusion previously described is largely a fixation artifact. In particular, most TFs we tested are significantly enriched on mitotic chromosomes. Studies with Sox2 reveal that this mitotic interaction is more dynamic than in interphase and is facilitated by both DNA binding and nuclear import. Furthermore, this dynamic mode results from lack of transcriptional activation rather than decreased accessibility of underlying DNA sequences in mitosis. The nature of the cross-linking artifact prompts careful re-examination of the role of TFs in mitotic bookmarking

A stable mode of bookmarking by TBP recruits RNA polymerase II to mitotic chromosomes.

Maintenance of transcription programs is challenged during mitosis when chromatin becomes condensed and transcription is silenced. How do the daughter cells re-establish the original transcription program? Here, we report that the TATA-binding protein (TBP), a key component of the core transcriptional machinery, remains bound globally to active promoters in mouse embryonic stem cells during mitosis. Using live-cell single-molecule imaging, we observed that TBP mitotic binding is highly stable, with an average residence time of minutes, in stark contrast to typical TFs with residence times of seconds. To test the functional effect of mitotic TBP binding, we used a drug-inducible degron system and found that TBP promotes the association of RNA Polymerase II with mitotic chromosomes, and facilitates transcriptional reactivation following mitosis. These results suggest that the core transcriptional machinery promotes efficient transcription maintenance globally

Changes in H2A.Z occupancy and DNA methylation during B-cell lymphomagenesis

The histone variant H2A.Z has been implicated in the regulation of gene expression, and in plants antagonizes DNA methylation. Here, we ask whether a similar relationship exists in mammals, using a mouse B-cell lymphoma model, where chromatin states can be monitored during tumorigenesis. Using native chromatin immunoprecipitation with microarray hybridization (ChIP-chip), we found a progressive depletion of H2A.Z around transcriptional start sites (TSSs) during MYC-induced transformation of pre-B cells and, subsequently, during lymphomagenesis. In addition, we found that H2A.Z and DNA methylation are generally anticorrelated around TSSs in both wild-type and MYC-transformed cells, as expected for the opposite effects of these chromatin features on promoter competence. Depletion of H2A.Z over TSSs both in cells that are induced to proliferate and in cells that are developing into a tumor suggests that progressive loss of H2A.Z during tumorigenesis results from the advancing disease state. These changes were accompanied by increases in chromatin salt solubility. Surprisingly, ∼30% of all genes showed a redistribution of H2A.Z from around TSSs to bodies of active genes during the transition from MYC-transformed to tumor cells, with DNA methylation lost from gene bodies where H2A.Z levels increased. No such redistributions were observed during MYC-induced transformation of wild-type pre-B cells. The documented role of H2A.Z in regulating transcription suggests that 30% of genes have the potential to be aberrantly expressed during tumorigenesis. Our results imply that antagonism between H2A.Z deposition and DNA methylation is a conserved feature of eukaryotic genes, and that transcription-coupled H2A.Z changes may play a role in cancer initiation and progression