Subunits of mammalian SWI/SNF (mSWI/SNF, also called BAF) complexes have recently been implicated as tumor suppressors in a number of human malignancies. To understand the full extent of their involvement, we conducted a proteomic analysis of purified endogenous mSWI/SNF complexes. Our studies revealed several new dedicated, stable subunits not found in the yeast SWI/SNF complex including Bcl7a, b and c, Bcl11a and b, Brd9 and SS18. Incorporating these novel members, we determined the frequency of mSWI/SNF subunit mutations in recent exome- and whole-genome sequencing studies of primary human tumors. Surprisingly, mSWI/SNF subunits are mutated in 19.6% of all human tumors reported in 44 exome sequencing studies. Our analysis suggests that specific subunits protect against cancer in specific tissues. In addition, we find that mutations to more than one subunit, which we define as a type of 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 and that in contrast to other known tumor suppressors and oncogenes surveyed, mSWI/SNF is broadly mutated, similar to TP53. Thus, proper functioning of these polymorphic chromatin regulatory complexes may constitute a major mechanism of human tumor suppression.

Summary 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 (CiA) Assay system employing chemically-induced proximity to initiate and terminate chromatin modifications in living cells. We selectively recruited HP1α 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 rates of ~0.18 nucleosomes/hr to produce domains of up to 10kb. After removal of the HP1α 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. Applying this framework, we were able to predict the steady-state dynamics and spatial features of the majority of euchromatic H3K9me3 domains.

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.Current understanding of the ribosome and the mechanism of translation has been significantly strengthened and expanded by recent advances in crystallography 1-6 and cryo-electron microscopy 7-10 . The ribosome undergoes several dynamical structural changes as it moves relative to the mRNA and transfer RNAs during translation 8,11 . Kinetic experiments have given a quantitative description of some of these dynamics during the main steps of the elongation cycle of protein synthesis 12 . During elongation, the secondary structures present in all mRNAs are disrupted to allow movement of the mRNA through the 30S subunit, and the reading of each codon. This task is aided by the mRNA helicase activity of the ribosome that has been localized to the downstream tunnel of the 30S subunit 13 . Moreover, interactions of mRNA pseudoknots or hairpins with the helicase region of the ribosome can shift the reading frame of the mRNA to the -1 frame, and play an important role in regulating gene expression in retroviruses 14-16 . It is extremely difficult to follow the steps of ribosomes during translational elongation using ensemble methods, because the dynamics of individual ribosomes are stochastic 17,18 and it is impossible to synchronize their activity. Here, we have used optical tweezers to follow the step-by-step translation of a single hairpin-forming mRNA molecule by a single ribosome. This approach has allowed us to characterize the dynamics of ribosome translation, measuringCorrespondence and requests for materials should be addressed to I.T. (intinoco@lbl.gov).. the time the ribosome spends at each codon, the number of mRNA nucleotides that move through the ribosome in each translocation step, and the time required per step. We have also determined the effects of mRNA structure on step size and rate, and have studied the effects of internal Shine-Dalgarno sequences 19 on translation arrest. These experiments provide a dynamic picture of the movement of a messenger RNA through a ribosome. NIH Public AccessIn these experiments, we used a single mRNA hairpin with a ribosome stalled at the 5′ end by omission of a required aminoacyl-tRNA; the RNA was attached to two micrometre-...

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 pausefree velocity of Pol II. The polymerase, rather than actively separating DNA from histones, functions instead as a ratchet that rectifies nucleosomal fluctuations. We also obtain 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 regulation of eukaryotic transcription.During transcription elongation in eukaryotes, RNA polymerase II must overcome the transcriptional barriers imposed by nucleosomes in chromatin. In vitro, a single nucleosome is sufficient to halt or greatly slow transcription by Pol II (1-5), and factors that restrict transcriptional backtracking relieve nucleosome-induced pauses and arrests, suggesting that the influence of the nucleosome is mediated through polymerase backtracking (4). Pol II also affects nucleosomal dynamics: depletion and turnover of histones are seen in actively transcribed genes in vivo (6,7), and histones are often transferred behind transcribing polymerases in vitro (2). However, the mechanisms underlying the mutual influence between nucleosome and polymerase are not well understood.Here a dual-trap optical tweezers assay revealed real-time trajectories of individual Pol II complexes as they transcribed through single nucleosomes. A tether was created between two trapped beads-one attached to a stalled polymerase, the other to the upstream DNA ( Figure 1a and Supporting Material). Addition of ribonucleotide triphosphates induced the polymerase to move towards the nucleosomal positioning sequence (NPS), causing the force between the * This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the