Abstract:SUMMARY
H2A.Z is a highly conserved histone variant involved in several key nuclear processes. It is incorporated into promoters by SWR-C-related chromatin remodeling complexes, but whether it is also actively excluded from non-promoter regions is not clear. Here, we provide genomic and biochemical evidence that RNA polymerase II (RNAPII) elongation-associated histone chaperones FACT and Spt6 both contribute to restricting H2A.Z from intragenic regions. In the absence of FACT or Spt6, the lack of efficient nuc… Show more
“…Importantly, the direction of shift at these nucleosomal positions are consistent with the published results of ino80 ∆ and are reproducible in both biological replicates (Yen et al, 2012) (Figure 4—figure supplement 1E). Overall, our data is in agreement with the INO80 remodeler functioning as a histone octamer slider but not an evictor of H2A.Z nucleosomes in vivo (Jeronimo et al, 2015; Shen et al, 2003; Yao et al, 2016; Yen et al, 2012). 10.7554/eLife.14243.032Figure 4.The effects of Ino80 depletion on H2A.Z occupancy and nucleosomal positions.( A ) Sequencing tag coverage of H2A.Z ( green ), H2A ( red ), and input ( gray ) nucleosomes, as well as the corresponding ∆(Z/A) values were plotted as described in Figure 1A.…”
The assembly of the preinitiation complex (PIC) occurs upstream of the +1 nucleosome which, in yeast, obstructs the transcription start site and is frequently assembled with the histone variant H2A.Z. To understand the contribution of the transcription machinery in the disassembly of the +1 H2A.Z nucleosome, conditional mutants were used to block PIC assembly. A quantitative ChIP-seq approach, which allows detection of global occupancy change, was employed to measure H2A.Z occupancy. Blocking PIC assembly resulted in promoter-specific H2A.Z accumulation, indicating that the PIC is required to evict H2A.Z. By contrast, H2A.Z eviction was unaffected upon depletion of INO80, a remodeler previously reported to displace nucleosomal H2A.Z. Robust PIC-dependent H2A.Z eviction was observed at active and infrequently transcribed genes, indicating that constitutive H2A.Z turnover is a general phenomenon. Finally, sites with strong H2A.Z turnover precisely mark transcript starts, providing a new metric for identifying cryptic and alternative sites of initiation.DOI:
http://dx.doi.org/10.7554/eLife.14243.001
“…Importantly, the direction of shift at these nucleosomal positions are consistent with the published results of ino80 ∆ and are reproducible in both biological replicates (Yen et al, 2012) (Figure 4—figure supplement 1E). Overall, our data is in agreement with the INO80 remodeler functioning as a histone octamer slider but not an evictor of H2A.Z nucleosomes in vivo (Jeronimo et al, 2015; Shen et al, 2003; Yao et al, 2016; Yen et al, 2012). 10.7554/eLife.14243.032Figure 4.The effects of Ino80 depletion on H2A.Z occupancy and nucleosomal positions.( A ) Sequencing tag coverage of H2A.Z ( green ), H2A ( red ), and input ( gray ) nucleosomes, as well as the corresponding ∆(Z/A) values were plotted as described in Figure 1A.…”
The assembly of the preinitiation complex (PIC) occurs upstream of the +1 nucleosome which, in yeast, obstructs the transcription start site and is frequently assembled with the histone variant H2A.Z. To understand the contribution of the transcription machinery in the disassembly of the +1 H2A.Z nucleosome, conditional mutants were used to block PIC assembly. A quantitative ChIP-seq approach, which allows detection of global occupancy change, was employed to measure H2A.Z occupancy. Blocking PIC assembly resulted in promoter-specific H2A.Z accumulation, indicating that the PIC is required to evict H2A.Z. By contrast, H2A.Z eviction was unaffected upon depletion of INO80, a remodeler previously reported to displace nucleosomal H2A.Z. Robust PIC-dependent H2A.Z eviction was observed at active and infrequently transcribed genes, indicating that constitutive H2A.Z turnover is a general phenomenon. Finally, sites with strong H2A.Z turnover precisely mark transcript starts, providing a new metric for identifying cryptic and alternative sites of initiation.DOI:
http://dx.doi.org/10.7554/eLife.14243.001
“…It is important to emphasize that we did not find evidence for a redistribution of H2A.Z, which could occur via a hypothetical arp6-independent H2A.Z deposition pathway (Jeronimo et al, 2015) ( Figure 6C; Supplemental Figure 10) or passive H2A.Z incorporation during nucleosome reassembly (no correlation with expression rate was observed) (Hardy and Robert, 2010). This indicates that the SWR1 complex is the major factor controlling spatial distribution of H2A.Z in chromatin in plants.…”
The influence of the histone variant H2A.Z on transcription remains a long-standing conundrum. Here, by analyzing the actinrelated protein6 mutant, which is impaired in H2A.Z deposition, and by H2A.Z profiling in stress conditions, we investigated the impact of this histone variant on gene expression in Arabidopsis thaliana. We demonstrate that the arp6 mutant exhibits anomalies in response to osmotic stress. Indeed, stress-responsive genes are overrepresented among those hyperactive in arp6. In wild-type plants, these genes exhibit high levels of H2A.Z in the gene body. Furthermore, we observed that in droughtresponsive genes, levels of H2A.Z in the gene body correlate with transcript levels. H2A.Z occupancy, but not distribution, changes in parallel with transcriptional changes. In particular, we observed H2A.Z loss upon transcriptional activation and H2A.Z gain upon repression. These data suggest that H2A.Z has a repressive role in transcription and counteracts unwanted expression in noninductive conditions. However, reduced activity of some genes in arp6 is associated with distinct behavior of H2A.Z at their +1 nucleosome, which exemplifies the requirement of this histone for transcription. Our data support a model where H2A.Z in gene bodies has a strong repressive effect on transcription, whereas in +1 nucleosomes, it is important for maintaining the activity of some genes.
“…This effect, however, was relatively modest compared with that of depleting the Head subunit Med18 from the nucleus using the anchor-away technique (Haruki et al, 2008) (Figure 4A, solid trace). Note that this result represents an underestimation of the contribution of Med18 on TFIIB occupancy, because nuclear depletion is often incomplete using this technique (Jeronimo and Robert, 2014; Jeronimo et al, 2015) and because TFIIB may not have had enough time to completely turn over from the promoter during the short time used for nuclear depletion (90 min). Taken together, these data suggest that the Tail module positively contributes to, but is not absolutely required for, the positive role of Mediator in PIC assembly/stability.…”
Summary
Mediator is a highly conserved transcriptional coactivator organized into four modules, namely Tail, Middle, Head, and Kinase (CKM). Previous work suggests regulatory roles for Tail and CKM, but an integrated model for these activities is lacking. Here, we analyzed the genome-wide distribution of Mediator subunits in wild-type and mutant yeast cells in which RNA polymerase II promoter escape is blocked, allowing detection of transient Mediator forms. We found that although all modules are recruited to upstream activated regions (UAS), assembly of Mediator within the pre-initiation complex is accompanied by the release of CKM. Interestingly, our data show that CKM regulates Mediator-UAS interaction rather than Mediator-promoter association. In addition, although Tail is required for Mediator recruitment to UAS, Tailless Mediator nevertheless interacts with core promoters. Collectively, our data suggest that the essential function of Mediator is mediated by Head and Middle at core promoters, while Tail and CKM play regulatory roles.
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