TBP facilitates RNA Polymerase I transcription following mitosis.

The TATA-box binding protein (TBP) is the sole transcription factor common in the initiation complexes of the three major eukaryotic RNA Polymerases (Pol I, II and III). Although TBP is central to transcription by the three RNA Pols in various species, the emergence of TBP paralogs throughout evolution has expanded the complexity in transcription initiation. Furthermore, recent studies have emerged that questioned the centrality of TBP in mammalian cells, particularly in Pol II transcription, but the role of TBP and its paralogs in Pol I transcription remains to be re-evaluated.

A dynamic role for transcription factors in restoring transcription through mitosis.

Mitosis involves intricate steps, such as DNA condensation, nuclear membrane disassembly, and phosphorylation cascades that temporarily halt gene transcription. Despite this disruption, daughter cells remarkably retain the parent cell's gene expression pattern, allowing for efficient transcriptional memory after division. Early studies in mammalian cells suggested that transcription factors (TFs) mark genes for swift reactivation, a phenomenon termed 'mitotic bookmarking', but conflicting data emerged regarding TF presence on mitotic chromosomes.

Heat shock transcription factors demonstrate a distinct mode of interaction with mitotic chromosomes.

A large number of transcription factors have been shown to bind and interact with mitotic chromosomes, which may promote the efficient reactivation of transcriptional programs following cell division. Although the DNA-binding domain (DBD) contributes strongly to TF behavior, the mitotic behaviors of TFs from the same DBD family may vary. To define the mechanisms governing TF behavior during mitosis in mouse embryonic stem cells, we examined two related TFs: Heat Shock Factor 1 and 2 (HSF1 and HSF2).

Using human induced pluripotent stem cell-derived cardiomyocytes to understand the mechanisms driving cardiomyocyte maturation.

Cardiovascular diseases are the leading cause of mortality and reduced quality of life globally. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide a personalized platform to study inherited heart diseases, drug-induced cardiac toxicity, and cardiac regenerative therapy. However, the immaturity of CMs obtained by current strategies is a major hurdle in utilizing hiPSC-CMs at their fullest potential.

43rd International Asilomar Chromatin, Chromosomes, and Epigenetics Conference.

The 43rd Asilomar Chromatin, Chromosomes, and Epigenetics Conference was held in an entirely online format from 9 to 11 December 2021. The conference enabled presenters at various career stages to share promising new findings, and presentations covered modern chromatin research across an array of model systems. Topics ranged from the fundamental principles of nuclear organization and transcription regulation to key mechanisms underlying human disease.

System reset: topoisomerase 1 clears mitotic DNA for transcriptional memory.

Topoisomerase 1 (Top1) relieves torsional stress on DNA, including from RNA Polymerase II (Pol II) transcription. A new study by Wiegard et al. uncovers an unexpected role of Top1 in the appropriate clearance of Pol II from mitotic DNA, allowing for a reset of transcriptional memory in the daughter cells.

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.

Function through absence: Active RNA exclusion from chromosomes leads to proper cell division.

The dynamics and functional roles of chromatin-bound RNA during cell division are largely unexplored. In this issue, Sharp et al. (2020.

Visualizing Transcription Factor Binding on Mitotic Chromosomes Using Single-Molecule Live-Cell Imaging.

For over two decades, scientists have observed that most transcription factors (TFs) become excluded from mitotic chromosomes of mammalian cells undergoing cell division. The few TFs that were observed to remain bound to chromosomes have been termed mitotic bookmarkers and were predicted to play important roles in reestablishing transcription after mitosis. Using live-cell imaging of endogenous TFs in mouse embryonic stem cells, we discovered that the observed exclusion from mitotic chromosomes is largely a result of formaldehyde cross-linking and that in fact, most TFs bind to mitotic chromosomes throughout mitosis.

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.

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.

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.

Transcribing through the nucleosome.

The packaging of DNA into chromatin limits sequence accessibility, which affects all DNA-based processes including transcription. Indeed, the fundamental unit of chromatin, the nucleosome, presents a strong barrier to transcription in vitro. Since the discovery of the nucleosome barrier, the question of how the RNA polymerase II (Pol II) machinery overcomes nucleosomes at high speeds in vivo has remained a central question in chromatin biology.

DNA torsion as a feedback mediator of transcription and chromatin dynamics.

The double helical structure of DNA lends itself to topological constraints. Many DNA-based processes alter the topological state of DNA, generating torsional stress, which is efficiently relieved by topoisomerases. Maintaining this topological balance is crucial to cell survival, as excessive torsional strain risks DNA damage.

Doxorubicin, DNA torsion, and chromatin dynamics.

Doxorubicin is one of the most important anti-cancer chemotherapeutic drugs, being widely used for the treatment of solid tumors and acute leukemias. The action of doxorubicin and other anthracycline drugs has been intensively investigated during the last several decades, but the mechanisms that have been proposed for cell killing remain disparate and controversial. In this review, we examine the proposed models for doxorubicin action from the perspective of the chromatin landscape, which is altered in many types of cancer due to recurrent mutations in chromatin modifiers.

Transcription-generated torsional stress destabilizes nucleosomes.

As RNA polymerase II (Pol II) transcribes a gene, it encounters an array of well-ordered nucleosomes. How it traverses through this array in vivo remains unresolved. One model proposes that torsional stress generated during transcription destabilizes nucleosomes ahead of Pol II.

The heat shock response: A case study of chromatin dynamics in gene regulation.

Recent studies in transcriptional regulation using the Drosophila heat shock response system have elucidated many of the dynamic regulatory processes that govern transcriptional activation and repression. The classic view that the control of gene expression occurs at the point of RNA polymerase II (Pol II) recruitment is now giving way to a more complex outlook of gene regulation. Promoter chromatin dynamics coordinate with transcription factor binding to maintain the promoters of active genes accessible.

Measuring genome-wide nucleosome turnover using CATCH-IT.

The dynamic interplay between DNA-binding proteins and nucleosomes underlies essential nuclear processes such as transcription, replication, and DNA repair. Manifestations of this interplay include the assembly, eviction, and replacement of nucleosomes. Hence, measurements of nucleosome turnover kinetics can lead to insights into the regulation of dynamic chromatin processes.

Salt fractionation of nucleosomes for genome-wide profiling.

Salt fractionation of nucleosomes, a classical method for defining "active" chromatin based on nucleosome solubility, has recently been adapted for genome-scale profiling. This method has several advantages for profiling chromatin dynamics, including general applicability to cell lines and tissues, quantitative recovery of chromatin, base-pair resolution of nucleosomes, and overall simplicity both in concept and execution. This chapter provides detailed protocols for nuclear isolation, chromatin fragmentation by micrococcal nuclease digestion, successive solubilization of chromatin fractions by addition of increasing concentrations of salt, and genome-wide analyses through microarray hybridization and next-generation sequencing.

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

Heat shock rapidly induces expression of a subset of genes while globally repressing transcription, making it an attractive system to study alterations in the chromatin landscape that accompany changes in gene regulation. We characterized these changes in Drosophila cells by profiling classical low-salt-soluble chromatin, RNA polymerase II (Pol II), and nucleosome turnover dynamics at single-base-pair resolution. With heat shock, low-salt-soluble chromatin and stalled Pol II levels were found to decrease within gene bodies, but no overall changes were detected at transcriptional start sites.

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.