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Transcriptional elongation requires the concerted action of several factors that allow RNA polymerase II to advance through chromatin in a highly processive manner. In order to identify novel elongation factors, we performed systematic yeast genetic screening based on the GLAM (Gene Length-dependent Accumulation of mRNA) assay, which is used to detect defects in the expression of long transcription units. Apart from well-known transcription elongation factors, we identified mutants in the prefoldin complex subunits, which were among those that caused the most dramatic phenotype. We found that prefoldin, so far involved in the cytoplasmic co-translational assembly of protein complexes, is also present in the nucleus and that a subset of its subunits are recruited to chromatin in a transcription-dependent manner. Prefoldin influences RNA polymerase II the elongation rate in vivo and plays an especially important role in the transcription elongation of long genes and those whose promoter regions contain a canonical TATA box. Finally, we found a specific functional link between prefoldin and histone dynamics after nucleosome remodeling, which is consistent with the extensive network of genetic interactions between this factor and the machinery regulating chromatin function. This study establishes the involvement of prefoldin in transcription elongation, and supports a role for this complex in cotranscriptional histone eviction. [ABSTRACT FROM AUTHOR]

The SWR1 complex replaces the canonical histone H2A with the variant H2A.Z (Htz1 in yeast) at specific chromatin regions. This dynamic alteration in nucleosome structure provides a molecular mechanism to regulate transcription, gene silencing, chromosome segregation and DNA repair. Here we show that genetic instability, sensitivity to drugs impairing different cellular processes and genome-wide transcriptional misregulation in htz1Δ can be partially or totally suppressed if SWR1 is not formed (swr1Δ), if it forms but cannot bind to chromatin (swc2Δ) or if it binds to chromatin but lacks histone replacement activity (swc5Δ and the ATPase-dead swr1-K727G). These results suggest that in htz1Δ the nucleosome remodelling activity of SWR1 affects chromatin integrity because of an attempt to replace H2A with Htz1 in the absence of the latter. This would impair transcription and, either directly or indirectly, other cellular processes. Specifically, we show that in htz1Δ, the SWR1 complex causes an accumulation of recombinogenic DNA damage by a mechanism dependent on phosphorylation of H2A at Ser129, a modification that occurs in response to DNA damage, suggesting that the SWR1 complex impairs the repair of spontaneous DNA damage in htz1Δ. In addition, SWR1 causes DSBs sensitivity in htz1Δ; consistently, in the absence of Htz1 the SWR1 complex bound near an endonuclease HO-induced DSB at the mating-type (MAT) locus impairs DSB-induced checkpoint activation. Our results support a stepwise mechanism for the replacement of H2A with Htz1 and demonstrate that a tight control of this mechanism is essential to regulate chromatin dynamics but also to prevent the deleterious consequences of an incomplete nucleosome remodelling. [ABSTRACT FROM AUTHOR]

Background: A complex interplay between chromatin and topological machineries is critical for genome architecture and function. However, little is known about these reciprocal interactions, even for cohesin, despite its multiple roles in DNA metabolism. Results: We have used genome-wide analyses to address how cohesins and chromatin structure impact each other in yeast. Cohesin inactivation in scc1-73 mutants during the S and G2 phases causes specific changes in chromatin structure that preferentially take place at promoters; these changes include a significant increase in the occupancy of the − 1 and + 1 nucleosomes. In addition, cohesins play a major role in transcription regulation that is associated with specific promoter chromatin architecture. In scc1-73 cells, downregulated genes are enriched in promoters with short or no nucleosome-free region (NFR) and a fragile "nucleosome − 1/RSC complex" particle. These results, together with a preferential increase in the occupancy of nucleosome − 1 of these genes, suggest that cohesins promote transcription activation by helping RSC to form the NFR. In sharp contrast, the scc1-73 upregulated genes are enriched in promoters with an "open" chromatin structure and are mostly at cohesin-enriched regions, suggesting that a local accumulation of cohesins might help to inhibit transcription. On the other hand, a dramatic loss of chromatin integrity by histone depletion during DNA replication has a moderate effect on the accumulation and distribution of cohesin peaks along the genome. Conclusions: Our analyses of the interplay between chromatin integrity and cohesin activity suggest that cohesins play a major role in transcription regulation, which is associated with specific chromatin architecture and cohesin-mediated nucleosome alterations of the regulated promoters. In contrast, chromatin integrity plays only a minor role in the binding and distribution of cohesins. [ABSTRACT FROM AUTHOR]