Dynamics of inherently bounded histone modification domains
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (33): 13296-13301
Nucleosomal Fluctuations Govern the Transcription Dynamics of RNA Polymerase II
2009; 325 (5940): 626-628
A central goal of chromatin biology is to reveal how posttranslational histone marks modulate gene expression; however, relatively little is known about the spatial or temporal dynamics of these marks. We previously showed that a dynamic model of histone mark nucleation, propagation, and turnover fits the mean enrichment profiles from 99% of noncentromeric histone H3 lysine 9 trimethylation (H3K9me3) domains in mouse embryonic stem cells without the need for boundary or insulator elements. Here we report the full details of this "inherently bounded" model of histone modification dynamics and describe several dynamic features of the model using H3K9me3 as a paradigm. By analyzing the kinetic and structural constraints that drive formation of inherently bounded domains, we find that such domains are optimized when the rates of marking and turnover are comparable. Additionally, we find that to establish such domains, propagation of the histone marks must occur primarily through local contacts.
View details for DOI 10.1073/pnas.1211172109
View details for Web of Science ID 000307807000039
View details for PubMedID 22847427
Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy.
2013; 45 (6): 592-601
RNA polymerase II (Pol II) must overcome the barriers imposed by nucleosomes during transcription elongation. We have developed an optical tweezers assay to follow individual Pol II complexes as they transcribe nucleosomal DNA. Our results indicate that the nucleosome behaves as a fluctuating barrier that locally increases pause density, slows pause recovery, and reduces the apparent pause-free velocity of Pol II. The polymerase, rather than actively separating DNA from histones, functions instead as a ratchet that rectifies nucleosomal fluctuations. We also obtained direct evidence that transcription through a nucleosome involves transfer of the core histones behind the transcribing polymerase via a transient DNA loop. The interplay between polymerase dynamics and nucleosome fluctuations provides a physical basis for the regulation of eukaryotic transcription.
View details for DOI 10.1126/science.1172926
View details for Web of Science ID 000268493000054
View details for PubMedID 19644123
Dynamics and Memory of Heterochromatin in Living Cells
2012; 149 (7): 1447-1460
Subunits of mammalian SWI/SNF (mSWI/SNF or BAF) complexes have recently been implicated as tumor suppressors in human malignancies. To understand the full extent of their involvement, we conducted a proteomic analysis of endogenous mSWI/SNF complexes, which identified several new dedicated, stable subunits not found in yeast SWI/SNF complexes, including BCL7A, BCL7B and BCL7C, BCL11A and BCL11B, BRD9 and SS18. Incorporating these new members, we determined mSWI/SNF subunit mutation frequency in exome and whole-genome sequencing studies of primary human tumors. Notably, mSWI/SNF subunits are mutated in 19.6% of all human tumors reported in 44 studies. Our analysis suggests that specific subunits protect against cancer in specific tissues. In addition, mutations affecting more than one subunit, defined here as compound heterozygosity, are prevalent in certain cancers. Our studies demonstrate that mSWI/SNF is the most frequently mutated chromatin-regulatory complex (CRC) in human cancer, exhibiting a broad mutation pattern, similar to that of TP53. Thus, proper functioning of polymorphic BAF complexes may constitute a major mechanism of tumor suppression.
View details for DOI 10.1038/ng.2628
View details for PubMedID 23644491
The elongation rate of RNA polymerase determines the fate of transcribed nucleosomes
NATURE STRUCTURAL & MOLECULAR BIOLOGY
2011; 18 (12): 1394-1399
Posttranslational histone modifications are important for gene regulation, yet the mode of propagation and the contribution to heritable gene expression states remains controversial. To address these questions, we developed a chromatin in vivo assay (CiA) system employing chemically induced proximity to initiate and terminate chromatin modifications in living cells. We selectively recruited HP1? to induce H3K9me3-dependent gene silencing and describe the kinetics and extent of chromatin modifications at the Oct4 locus in fibroblasts and pluripotent cells. H3K9me3 propagated symmetrically and continuously at average rates of ~0.18 nucleosomes/hr to produce domains of up to 10 kb. After removal of the HP1? stimulus, heterochromatic domains were heritably transmitted, undiminished through multiple cell generations. Our data enabled quantitative modeling of reaction kinetics, which revealed that dynamic competition between histone marking and turnover, determines the boundaries and stability of H3K9me3 domains. This framework predicts the steady-state dynamics and spatial features of the majority of euchromatic H3K9me3 domains over the genome.
View details for DOI 10.1016/j.cell.2012.03.052
View details for Web of Science ID 000305753800012
View details for PubMedID 22704655
ClpX(P) Generates Mechanical Force to Unfold and Translocate Its Protein Substrates
2011; 145 (3): 459-469
Upon transcription, histones can either detach from DNA or transfer behind the polymerase through a process believed to involve template looping. The details governing nucleosomal fate during transcription are not well understood. Our atomic force microscopy images of yeast RNA polymerase II-nucleosome complexes confirm the presence of looped transcriptional intermediates and provide mechanistic insight into the histone-transfer process through the distribution of transcribed nucleosome positions. Notably, we find that a fraction of the transcribed nucleosomes are remodeled to hexasomes, and this fraction depends on the transcription elongation rate. A simple model involving the kinetic competition between transcription elongation, histone transfer and histone-histone dissociation quantitatively explains our observations and unifies them with results obtained from other polymerases. Factors affecting the relative magnitude of these processes provide the physical basis for nucleosomal fate during transcription and, therefore, for the regulation of gene expression.
View details for DOI 10.1038/nsmb.2164
View details for Web of Science ID 000298011600028
View details for PubMedID 22081017
Following translation by single ribosomes one codon at a time
2008; 452 (7187): 598-603
AAA(+) unfoldases denature and translocate polypeptides into associated peptidases. We report direct observations of mechanical, force-induced protein unfolding by the ClpX unfoldase from E. coli, alone, and in complex with the ClpP peptidase. ClpX hydrolyzes ATP to generate mechanical force and translocate polypeptides through its central pore. Threading is interrupted by pauses that are found to be off the main translocation pathway. ClpX's translocation velocity is force dependent, reaching a maximum of 80 aa/s near-zero force and vanishing at around 20 pN. ClpX takes 1, 2, or 3 nm steps, suggesting a fundamental step-size of 1 nm and a certain degree of intersubunit coordination. When ClpX encounters a folded protein, it either overcomes this mechanical barrier or slips on the polypeptide before making another unfolding attempt. Binding of ClpP decreases the slip probability and enhances the unfolding efficiency of ClpX. Under the action of ClpXP, GFP unravels cooperatively via a transient intermediate.
View details for DOI 10.1016/j.cell.2011.04.010
View details for Web of Science ID 000290022900014
View details for PubMedID 21529717
We have followed individual ribosomes as they translate single messenger RNA hairpins tethered by the ends to optical tweezers. Here we reveal that translation occurs through successive translocation--and-pause cycles. The distribution of pause lengths, with a median of 2.8 s, indicates that at least two rate-determining processes control each pause. Each translocation step measures three bases--one codon-and occurs in less than 0.1 s. Analysis of the times required for translocation reveals, surprisingly, that there are three substeps in each step. Pause lengths, and thus the overall rate of translation, depend on the secondary structure of the mRNA; the applied force destabilizes secondary structure and decreases pause durations, but does not affect translocation times. Translocation and RNA unwinding are strictly coupled ribosomal functions.
View details for DOI 10.1038/nature06716
View details for Web of Science ID 000254567200038
View details for PubMedID 18327250