Dataset Information

Control of replication initiation by the Sum1/Rfm1/Hst1 histone deacetylase.

ABSTRACT:

Background

Replication initiation at origins of replication in the yeast genome takes place on chromatin as a template, raising the question how histone modifications, for instance histone acetylation, influence origin firing. Initiation requires binding of the replication initiator, the Origin Recognition Complex (ORC), to a consensus sequence within origins. In addition, other proteins bind to recognition sites in the vicinity of ORC and support initiation. In previous work, we identified Sum1 as an origin-binding protein that contributes to efficient replication initiation. Sum1 is part of the Sum1/Rfm1/Hst1 complex that represses meiotic genes during vegetative growth via histone deacetylation by the histone deacetylase (HDAC) Hst1.

Results

In this study, we investigated how Sum1 affected replication initiation. We found that it functioned in initiation as a component of the Sum1/Rfm1/Hst1 complex, implying a role for histone deacetylation in origin activity. We identified several origins in the yeast genome whose activity depended on both Sum1 and Hst1. Importantly, sum1Delta or hst1Delta caused a significant increase in histone H4 lysine 5 (H4 K5) acetylation levels, but not other H4 acetylation sites, at those origins. Furthermore, mutation of lysines to glutamines in the H4 tail, which imitates the constantly acetylated state, resulted in a reduction of origin activity comparable to that in the absence of Hst1, showing that deacetylation of H4 was important for full initiation capacity of these origins.

Conclusion

Taken together, our results demonstrate a role for histone deacetylation in origin activity and reveal a novel aspect of origin regulation by chromatin. These results suggest recruitment of the Sum1/Rfm1/Hst1 complex to a number of yeast origins, where Hst1 deacetylated H4 K5.

SUBMITTER: Weber JM

PROVIDER: S-EPMC2585588 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Publications

Control of replication initiation by the Sum1/Rfm1/Hst1 histone deacetylase.

Weber Jan M JM Irlbacher Horst H Ehrenhofer-Murray Ann E AE

BMC molecular biology 20081106

<h4>Background</h4>Replication initiation at origins of replication in the yeast genome takes place on chromatin as a template, raising the question how histone modifications, for instance histone acetylation, influence origin firing. Initiation requires binding of the replication initiator, the Origin Recognition Complex (ORC), to a consensus sequence within origins. In addition, other proteins bind to recognition sites in the vicinity of ORC and support initiation. In previous work, we identif ...[more]

PMID: 18990212

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Project description:BACKGROUND:HIV-1 hijacks the cellular machinery for its own replication through protein-protein interactions between viral and host cell factors. One strategy against HIV-1 infection is thus to target these key protein complexes. As the integration of reverse transcribed viral cDNA into a host cell chromosome is an essential step in the HIV-1 life cycle, catalyzed by the viral integrase and other important host factors, we aimed at identifying new integrase binding partners through a novel approach. METHODS:A LTR-derived biotinylated DNA fragment complexed with the integrase on magnetic beads was incubated with extracts from integrase-expressing 293?T cells. Liquid chromatography-mass spectrometry/mass spectrometry and co-immunoprecipitation/pull-down experiments were used for the identification of binding partners. Transfections of histone deacetylase 1 (HDAC1) expression vectors and/or specific siRNA were conducted in HeLa-CD4 and 293?T cells followed by infection with fully infectious NL4-3 and luciferase-expressing pseudotyped viruses or by proviral DNA transfection. Fully infectious and pseudotyped viruses produced from HDAC1-silenced 293?T cells were tested for their infectivity toward HeLa-CD4 cells, T cell lines and primary CD4+ T cells. Late RT species and integrated viral DNA were quantified by qPCR and infectivity was measured by luciferase activity and p24 ELISA assay. Results were analyzed by the Student's t-test. RESULTS:Using our integrase-LTR bait approach, we successfully identified new potential integrase-binding partners, including HDAC1. We further confirmed that HDAC1 interacted with the HIV-1 integrase in co-immunoprecipitation and pull-down experiments. HDAC1 knockdown in infected HeLa cells was shown to interfere with an early preintegration step of the HIV-1 replication cycle, which possibly involves reverse transcription. We also observed that, while HDAC1 overexpression inhibited HIV-1 expression after integration, HDAC1 knockdown had no effect on this step. In virus producer cells, HDAC1 knockdown had a limited impact on virus infectivity in either cell lines or primary CD4+ T cells. CONCLUSIONS:Our results show that HDAC1 interacts with the HIV-1 integrase and affects virus replication before and after integration. Overall, HDAC1 appears to facilitate HIV-1 replication with a major effect on a preintegration step, which likely occurs at the reverse transcription step.

Dataset Information

Control of replication initiation by the Sum1/Rfm1/Hst1 histone deacetylase.

Background

Results

Conclusion

Publications

Control of replication initiation by the Sum1/Rfm1/Hst1 histone deacetylase.

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