Small RNAs target their complementary chromatin regions for gene silencing through nascent long non-coding RNAs (lncRNAs). In the ciliated protozoan
, the interaction between Piwi-associated small ...RNAs (scnRNAs) and the nascent lncRNA transcripts from the somatic genome has been proposed to induce target-directed small RNA degradation (TDSD), and scnRNAs not targeted for TDSD later target the germline-limited sequences for programmed DNA elimination. In this study, we show that the SUMO E3 ligase Ema2 is required for the accumulation of lncRNAs from the somatic genome and thus for TDSD and completing DNA elimination to make viable sexual progeny. Ema2 interacts with the SUMO E2 conjugating enzyme Ubc9 and enhances SUMOylation of the transcription regulator Spt6. We further show that Ema2 promotes the association of Spt6 and RNA polymerase II with chromatin. These results suggest that Ema2-directed SUMOylation actively promotes lncRNA transcription, which is a prerequisite for communication between the genome and small RNAs.
DNA combing is a powerful method developed by Bensimon and colleagues to stretch DNA molecules on silanized glass coverslips. This technique provides a unique way to monitor the activation of ...replication origins and the progression of replication forks at the level of single DNA molecules, after incorporation of thymidine analogs, such as 5-bromo-2′-deoxyuridine (BrdU), 5-iodo-2′-deoxyuridine (IdU) and 5-chloro-2′-deoxyuridine (CldU) in newly-synthesized DNA. Unlike microarray-based approaches, this assay gives access to the variability of replication profiles in individual cells. It can also be used to monitor the effect of DNA lesions on fork progression, arrest and restart. In this review, we propose standard DNA combing methods to analyze DNA replication in budding yeast and in human cells. We also show that 5-ethynyl-2′-deoxyuridine (EdU) can be used as a good alternative to BrdU for DNA combing analysis, as unlike halogenated nucleotides, it can be detected without prior denaturation of DNA.
The NuA4 histone acetyltransferase complex is required for gene regulation, cell cycle progression, and DNA repair. Dissection of the 13‐subunit complex reveals that the Eaf7 subunit bridges Eaf5 ...with Eaf3, a H3K36me3‐binding chromodomain protein, and this Eaf5/7/3 trimer is anchored to NuA4 through Eaf5. This trimeric subcomplex represents a functional module, and a large portion exists in a native form outside the NuA4 complex. Gene‐specific and genome‐wide location analyses indicate that Eaf5/7/3 correlates with transcription activity and is enriched over the coding region. In agreement with a role in transcription elongation, the Eaf5/7/3 trimer interacts with phosphorylated RNA polymerase II and helps its progression. Loss of Eaf5/7/3 partially suppresses intragenic cryptic transcription arising in set2 mutants, supporting a role in nucleosome destabilization. On the other hand, loss of the trimer leads to an increase of replication‐independent histone exchange over the coding region of transcribed genes. Taken together, these results lead to a model where Eaf5/7/3 associates with elongating polymerase to promote the disruption of nucleosomes in its path, but also their refolding in its wake.
Synopsis
The multisubunit NuA4 complex is an essential gene regulatory acetyltransferase in yeast. Dissection of its subunits reveals a trimeric submodule independently associating with gene coding regions, which may act as a histone chaperone during RNA polymerase II transcriptional elongation.
Eaf5/7/3 forms a submodule within the NuA4 acetyltransferase complex and also functions as an independent native trimer.
High‐resolution genome‐wide analysis reveals NuA4 binding to promoters and coding regions of actively transcribed genes.
The Eaf5/7/3 trimer independently associates with the coding region of transcriptionally active genes and affects RNA polymerase II processivity.
Eaf5/7/3 interacts with phosphorylated, elongating RNA polymerase II and H3K36me3‐containing nucleosomes.
Eaf5/7/3 cooperates with FACT to promote transcription‐coupled nucleosomes disruption, as well as their refolding behind the polymerase to suppress histone exchange.
Dissection of the essential multisubunit NuA4 acetyltransferase reveals a trimeric subcomplex that interacts with elongating RNA polymerase II and functions independently to control transcription‐coupled histone exchange.
The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is ...labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluorescence using specific antibodies. Unlike biochemical and NGS-based methods, DNA combing provides unique information on cell-to-cell variations in DNA replication profiles, including initiation and elongation. Finally, this assay can be used to monitor the effect of DNA lesions on fork progression, arrest and restart.
We have used EM and biochemistry to characterize the structure of NuA4, an essential yeast histone acetyltransferase (HAT) complex conserved throughout eukaryotes, and we have determined the ...interaction of NuA4 with the nucleosome core particle (NCP). The ATM-related Tra1 subunit, which is shared with the SAGA coactivator complex, forms a large domain joined to a second region that accommodates the catalytic subcomplex Piccolo and other NuA4 subunits. EM analysis of a NuA4-NCP complex shows the NCP bound at the periphery of NuA4. EM characterization of Piccolo and Piccolo-NCP provided further information about subunit organization and confirmed that histone acetylation requires minimal contact with the NCP. A small conserved region at the N terminus of Piccolo subunit enhancer of Polycomb-like 1 (Epl1) is essential for NCP interaction, whereas the subunit yeast homolog of mammalian Ing1 2 (Yng2) apparently positions Piccolo for efficient acetylation of histone H4 or histone H2A tails. Taken together, these results provide an understanding of the NuA4 subunit organization and the NuA4-NCP interactions.
DNA replication during S phase is accompanied by establishment of sister chromatid cohesion to ensure faithful chromosome segregation. The Eco1 acetyltransferase, helped by factors including Ctf4 and ...Chl1, concomitantly acetylates the chromosomal cohesin complex to stabilize its cohesive links. Here we show that Ctf4 recruits the Chl1 helicase to the replisome via a conserved interaction motif that Chl1 shares with GINS and polymerase α. We visualize recruitment by EM analysis of a reconstituted Chl1-Ctf4-GINS assembly. The Chl1 helicase facilitates replication fork progression under conditions of nucleotide depletion, partly independently of Ctf4 interaction. Conversely, Ctf4 interaction, but not helicase activity, is required for Chl1’s role in sister chromatid cohesion. A physical interaction between Chl1 and the cohesin complex during S phase suggests that Chl1 contacts cohesin to facilitate its acetylation. Our results reveal how Ctf4 forms a replisomal interaction hub that coordinates replication fork progression and sister chromatid cohesion establishment.
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•Ctf4 is a replisomal interaction hub that recruits CIP box client proteins•Chl1 helicase is the Ctf4 client that links replication and cohesion establishment•Ctf4 interaction, but not helicase activity, conveys Chl1 cohesion function•Chl1 physically engages with cohesin during cohesion establishment
Eukaryotic DNA replication is accompanied by the establishment of sister chromatid cohesion. Samora et al. show that the central replisome component Ctf4 makes this link by recruiting the Chl1 helicase to the replication fork. Chl1 in turn engages in a direct physical contact with the cohesin complex during cohesion establishment.
Structural and functional analyses of nucleosomes containing histone variant H2A.Z have drawn a lot of interest over the past few years. Important work in budding yeast has shown that H2A.Z ...(Htz1)-containing nucleosomes are specifically located on the promoter regions of genes, creating a specific chromatin structure that is poised for disassembly during transcription activation. The SWR1 complex is responsible for incorporation of Htz1 into nucleosomes through ATP-dependent exchange of canonical H2A-H2B dimers for Htz1-H2B dimers. Interestingly, the yeast SWR1 complex is functionally linked to the NuA4 acetyltransferase complex in vivo. NuA4 and SWR1 are physically associated in higher eukaryotes as they are homologous to the TIP60/p400 complex, which encompasses both histone acetyltransferase (Tip60) and histone exchange (p400/Domino) activities. Here we present work investigating the impact of NuA4-dependent acetylation on SWR1-driven incorporation of H2A.Z into chromatin. Using in vitro histone exchange assays with native chromatin, we demonstrate that prior chromatin acetylation by NuA4 greatly stimulates the exchange of H2A for H2A.Z. Interestingly, we find that acetylation of H2A or H4 N-terminal tails by NuA4 can independently stimulate SWR1 activity. Accordingly, we demonstrate that mutations of H4 or H2A N-terminal lysine residues have similar effects on H2A.Z incorporation in vivo, and cells carrying mutations in both tails are nonviable. Finally, depletion experiments indicate that the bromodomain-containing protein Bdf1 is important for NuA4-dependent stimulation of SWR1. These results provide important mechanistic insight into the functional cross-talk between chromatin acetylation and ATP-dependent exchange of histone H2A variants.
Methylation of histone H3 lysine 4 (H3K4) by Set1 complex/COMPASS is a hallmark of eukaryotic chromatin, but it remains poorly understood how this post-translational modification contributes to the ...regulation of biological processes like the cell cycle. Here, we report a H3K4 methylation-dependent pathway in Saccharomyces cerevisiae that governs toxicity toward benomyl, a microtubule destabilizing drug. Benomyl-sensitive growth of wild-type cells required mono- and dimethylation of H3K4 and Pho23, a PHD-containing subunit of the Rpd3L complex. Δset1 and Δpho23 deletions suppressed defects associated with ipl1-2 aurora kinase mutant, an integral component of the spindle assembly checkpoint during mitosis. Benomyl resistance of Δset1 strains was accompanied by deregulation of all four tubulin genes and the phenotype was suppressed by tub2-423 and Δtub3 mutations, establishing a genetic link between H3K4 methylation and microtubule function. Most interestingly, sine wave fitting and clustering of transcript abundance time series in synchronized cells revealed a requirement for Set1 for proper cell-cycle-dependent gene expression and Δset1 cells displayed delayed entry into S phase. Disruption of G1/S regulation in Δmbp1 and Δswi4 transcription factor mutants duplicated both benomyl resistance and suppression of ipl1-2 as was observed with Δset1 Taken together our results support a role for H3K4 methylation in the coordination of cell-cycle progression and proper assembly of the mitotic spindle during mitosis.
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