Evidence for Eviction and Rapid Deposition of Histones upon Transcriptional Elongation by RNA Polymerase II

Abstract

Biochemical experiments indicate that transcriptional elongation by RNA polymerase II (Pol II) is inhibited by nucleosomes and hence requires chromatin-modifying activities. Here, we examine the fate of histones upon passage of elongating Pol II in vivo. Histone density throughout the entire Saccharomyces cerevisiae GAL10 coding region is inversely correlated with Pol II association and transcriptional activity, suggesting that the elongating Pol II machinery efficiently evicts core histones from the DNA. Furthermore, new histones appear to be deposited onto DNA less than 1 min after passage of Pol II. Transcription-dependent deposition of histones requires the FACT complex that travels with elongating Pol II. Our results suggest that Pol II transcription generates a highly dynamic equilibrium of histone eviction and histone deposition and that there is significant histone exchange throughout most of the yeast genome within a single cell cycle.The classical view of eukaryotic genomes is that essentially all DNA is stably associated with histone octamers in the form of nucleosome arrays. During S phase, histones are deposited on newly synthesized DNA, but the old histones are presumed to remain associated with DNA. However, recent results indicate that histone-DNA interactions are more dynamic than originally supposed. First, transcriptional activator proteins can cause complete unfolding, and probably dissociation, of histones from promoter regions in Saccharomyces cerevisiae cells (6,13,45). Second, the yeast Swr1 complex mediates ATP-dependent exchange of the histone H2AZ variant (24,26,36), and this activity protects euchromatin from the spread of heterochromatin (35). Third, in flies and mammals, the histone H3.3 variant is deposited into chromatin in a manner independent of DNA replication (1, 2, 54) but associated with transcription (20,34).Biochemical experiments have not revealed a clear understanding for how nucleosomes affect transcriptional elongation by RNA polymerases. Bacterial SP6 and T7 RNA polymerases and yeast RNA polymerase III (Pol III) can mobilize histones and transcribe nucleosomal templates (11,23,42,(51)(52)(53). Specifically, histone octamers step around a transcribing polymerase without leaving the template, although the enzyme pauses with a pronounced periodicity due to restricted rotation in the intranucleosomal DNA loop. In contrast, although Pol II elongation rates on naked DNA templates are comparable to physiological elongation rates, elongation on chromatin templates is markedly inhibited and produces truncated transcripts (18,19). In a purified transcription assay lacking chromatin-modifying factors, nucleosomes and histone H3-H4 tetramers nearly completely block Pol II elongation (9). However, under conditions of increased ionic strength, Pol II can elongate through nucleosomes, resulting in the dissociation of a single H2A-H2B dimer but retention of the remaining six subunits of the histone octamer (22).It is presumed that Pol II elongation in vivo requires modification of ch...

Defective histone supply causes changes in RNA polymerase II elongation rate and cotranscriptional pre-mRNA splicing

“…Partial loss of core histones at gene bodies during intense transcription has been reported (33,34), suggesting that a certain degree of histone removal is a prerequisite for or a consequence of active transcription. However, recent genome-wide studies have shown either little (4) or no (3) negative correlation between nucleosome occupancy and elongation rates in mammals.…”

Chromatin remodeling: insights and intrigue from single-molecule studies

“…Studies of the yeast PHO5 gene suggest that nucleosomes are removed from the promoter upon activation 15,16 . Interestingly, genomewide analyses show that promoters are intrinsically nucleosome deficient and become more deficient during activation 73,74 . Furthermore, promoter remodeling is considerably slower in strains bearing mutations in components of the SWI/SNF complex 75 .…”

Nucleosome fragility is associated with future transcriptional response to developmental cues and stress inC. elegans

“…We next sought to test the relationship between fragile nucleosomes and trans-factors more explicitly. At extremely highly transcribed genes, such as heat-shock-responsive genes after induction, it has been proposed that RNA polymerase II (Pol II) molecules occupy the entire gene body (Schwabish and Struhl 2004;Merz et al 2008;Cole et al 2014). We hypothesized that fragility would increase at gene bodies after inducing high levels of transcription, as a result of nucleosome competition with transcribing Pol II.…”