Local Chromatin Motion and Transcription.

Eukaryotic chromatin is a complex of nucleic acids and proteins that is central to interpreting the information coded in the genome. Chromatin is rather irregularly folded inside the nucleus in a fluid-like state that exhibits dynamic local movement. The highly dynamic nature of chromatin has become increasingly appreciated, particularly in DNA-templated processes including transcription, because this dynamic property ensures a degree of DNA accessibility, even in compacted chromatin. Many proteins globally constrain local chromatin movements, which seem to be driven essentially by thermal fluctuation in living cells. For instance, loss of the cohesin complex, which can capture chromatin fibers, leads to an increase in chromatin motion. Another constraining factor of chromatin motion is the transcription machinery. Although the previously held view is that transcription requires open and highly dynamic chromatin, a number of studies are now pointing to a more nuanced role of transcription in constraining chromatin movement: dynamic clustering of active RNA polymerase II and other transcription factors can serve as a hub that transiently bridges active DNA regions to be transcribed, thereby loosely networking chromatin and constraining chromatin motion. In contrast, outside heterochromatin, the transcriptionally less active regions might be less constrained, more dynamic and accessible, implying a high competency state for rapid and efficient recruitment of protein factors. This new view on the interplay of local chromatin motion and transcription reflects traditional models of the transcription factories and, more recently, liquid droplets of transcription factors, providing new insight into chromatin function.

Dynamic chromatin organization without the 30-nm fiber.

Chromatin in eukaryotic cells is a negatively charged polymer composed of DNA, histones, and various associated proteins. Over the past ten years, our view of chromatin has shifted from a static regular structure to a dynamic and highly variable configuration. While the details are not fully understood yet, chromatin forms numerous compact domains that act as dynamic functional units of the genome in higher eukaryotes. By altering DNA accessibility, the dynamic nature of chromatin governs various genome functions including RNA transcription, DNA replication, and DNA repair/recombination. Based on the new evidence coming from both genomics and imaging studies, we discuss the structural and dynamic aspects of chromatin and their biological relevance in the living cell.

Single nucleosome imaging reveals loose genome chromatin networks via active RNA polymerase II.

Although chromatin organization and dynamics play a critical role in gene transcription, how they interplay remains unclear. To approach this issue, we investigated genome-wide chromatin behavior under various transcriptional conditions in living human cells using single-nucleosome imaging. While transcription by RNA polymerase II (RNAPII) is generally thought to need more open and dynamic chromatin, surprisingly, we found that active RNAPII globally constrains chromatin movements. RNAPII inhibition or its rapid depletion released the chromatin constraints and increased chromatin dynamics. Perturbation experiments of P-TEFb clusters, which are associated with active RNAPII, had similar results. Furthermore, chromatin mobility also increased in resting G0 cells and UV-irradiated cells, which are transcriptionally less active. Our results demonstrated that chromatin is globally stabilized by loose connections through active RNAPII, which is compatible with models of classical transcription factories or liquid droplet formation of transcription-related factors. Together with our computational modeling, we propose the existence of loose chromatin domain networks for various intra-/interchromosomal contacts via active RNAPII clusters/droplets.

Repeat-Specific Functions for the C-Terminal Domain of RNA Polymerase II in Budding Yeast.

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) is required to regulate transcription and to integrate it with other essential cellular processes. In the budding yeast Saccharomyces cerevisiae, the CTD of Rpb1p consists of 26 conserved heptad repeats that are post-translationally modified to orchestrate protein factor binding at different stages of the transcription cycle. A long-standing question in the study of the CTD is if there are any functional differences between the 26 repeats. In this study, we present evidence that repeats of identical sequence have different functions based on their position within the CTD. We assembled plasmids expressing Rpb1p with serine to alanine substitutions in three defined regions of the CTD and measured a range of phenotypes for yeast expressing these constructs. Mutations in the beginning and middle regions of the CTD had drastic, and region-specific effects, while mutating the distal region had no observable phenotype. Further mutational analysis determined that Ser5 within the first region of repeats was solely responsible for the observed growth differences and sequencing fast-growing suppressors allowed us to further define the functional regions of the CTD. This mutational analysis is consistent with current structural models for how the RNAPII holoenzyme and the CTD specifically would reside in complex with Mediator and establishes a foundation for studying regioselective binding along the repetitive RNAPII CTD.

DNA Instability Maintains the Repeat Length of the Yeast RNA Polymerase II C-terminal Domain.

The C-terminal domain (CTD) of RNA polymerase II in eukaryotes is comprised of tandemly repeating units of a conserved seven-amino acid sequence. The number of repeats is, however, quite variable across different organisms. Furthermore, previous studies have identified evidence of rearrangements within the CTD coding region, suggesting that DNA instability may play a role in regulating or maintaining CTD repeat number. The work described here establishes a clear connection between DNA instability and CTD repeat number in Saccharomyces cerevisiae First, analysis of 36 diverse S. cerevisiae isolates revealed evidence of numerous past rearrangements within the DNA sequence that encodes the CTD. Interestingly, the total number of CTD repeats was relatively static (24-26 repeats in all strains), suggesting a balancing act between repeat expansion and contraction. In an effort to explore the genetic plasticity within this region, we measured the rates of repeat expansion and contraction using novel reporters and a doxycycline-regulated expression system for RPB1 In efforts to determine the mechanisms leading to CTD repeat variability, we identified the presence of DNA secondary structures, specifically G-quadruplex-like DNA, within the CTD coding region. Furthermore, we demonstrated that mutating PIF1, a G-quadruplex-specific helicase, results in increased CTD repeat length polymorphisms. We also determined that RAD52 is necessary for CTD repeat expansion but not contraction, identifying a role for recombination in repeat expansion. Results from these DNA rearrangements may help explain the CTD copy number variation seen across eukaryotes, as well as support a model of CTD expansion and contraction to maintain CTD integrity and overall length.