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Histone deacetylation by Sir2 generates a transcriptionally repressed nucleoprotein complex.

ABSTRACT: Sir2 is an NAD-dependent histone deacetylase required for transcriptional silencing. To study the mechanism of Sir2 function, we examined the biochemical properties of purified recombinant Drosophila Sir2 (dSir2). First, we performed histone deacetylation assays and found that dSir2 deacetylates a broad range of acetylated lysine residues. We then carried out in vitro transcription experiments and observed that dSir2 does not repress transcription with either naked DNA templates or chromatin assembled from native (and mostly unacetylated) histones. It was possible, however, that repression by dSir2 requires an acetylated histone substrate. We therefore tested the transcriptional effects of dSir2 with native histones that were hyperacetylated by treatment with acetic anhydride. Assembly of the hyperacetylated histones onto DNA yields a soluble histone-DNA complex that differs from canonical nucleosomal chromatin. With this hyperacetylated histone-DNA complex, we observed potent (50- to 100-fold) NAD-dependent transcriptional repression by purified dSir2. In contrast, repression by dSir2 was not observed in parallel experiments in which histones were hyperpropionylated with propionic anhydride. We also found that dSir2 mediates the formation of a nuclease-resistant fast-sedimenting histone-DNA complex in an NAD-dependent manner. Unlike dSir2, the dHDAC1 deacetylase does not strongly repress transcription or generate a nuclease-resistant histone-DNA complex. Furthermore, with yeast Sir2, the transcriptional repression we observe correlates with deacetylation activity in vitro and silencing activity in vivo. These findings suggest that deacetylation by Sir2 causes a conformational change or rearrangement of histones into a transcriptionally repressive chromatin structure.

SUBMITTER: Parsons XH

PROVIDER: S-EPMC149880 | biostudies-literature | 2003 Feb

REPOSITORIES: biostudies-literature

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Histone deacetylation by Sir2 generates a transcriptionally repressed nucleoprotein complex.

Parsons Xuejun Huang XH Garcia Sandra N SN Pillus Lorraine L Kadonaga James T JT

Proceedings of the National Academy of Sciences of the United States of America 20030205 4

Sir2 is an NAD-dependent histone deacetylase required for transcriptional silencing. To study the mechanism of Sir2 function, we examined the biochemical properties of purified recombinant Drosophila Sir2 (dSir2). First, we performed histone deacetylation assays and found that dSir2 deacetylates a broad range of acetylated lysine residues. We then carried out in vitro transcription experiments and observed that dSir2 does not repress transcription with either naked DNA templates or chromatin ass ...[more]

PMID: 12571358

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Project description:OBJECTIVES:The present study aims to elucidate the underlying mechanism how PFKP is regulated by BRCA1 and the clinical significance of PFKP in breast cancer. METHODS:MEF-BRCA1?/? and the wild type counterpart MEF-BRCA1+/+ cell lines were used to test the sensitivity of glucose depletion in culture medium. Glucose Assay Kit was used to quantify glucose levels in cultural supernatant and cell lysate. Real time PCR was used to measure the mRNA expression levels of genes. Western blot was used to detect protein levels. Chromatin immunoprecipitation was used to verify the bindings between transcription factors and DNA elements. Luciferase reporter assay was performed to determine the transcriptional activity. Histochemistry assay was performed on tissue microarray. RESULTS:We found that MEF-BRCA1?/? cells consumed more glucose and were more vulnerable to glucose-deprived culture medium. The mRNA profiles and qPCR assay of MEF-BRCA1?/? and MEF-BRCA1+/+ cells revealed that PFKP, the rate-limiting enzyme of glycolysis, was significantly upregulated in MEF-BRCA1?/? cells. Consistently, the repressive effects of BRCA1 on PFKP were confirmed by overexpression or knockdown of BRCA1. Moreover, we also demonstrated that PFKP was suppressed by ZBRK1 as well, which was the co-repression partner of BRCA1. Mechanistically, we figured out that BRCA1 formed a transcriptional repression complex with ZBRK1 on the promoter of PFKP and consequently restrained its expression. Importantly, the expression levels of PFKP were demonstrated to associate with poor survival of patients with breast cancer. CONCLUSION:Our study provided a new insight into the dysregulation of glycolysis in breast cancer, which might be partially due to the deficiency of BRCA1/ZBRK1 axis and subsequently reversed the transcriptional repressive effect on PFKP. We also found that PFKP overexpressed in a subset of breast cancer patients and could serve as a prognostic factor, which represented a potential target for BC therapy.

Dataset Information

Histone deacetylation by Sir2 generates a transcriptionally repressed nucleoprotein complex.

Publications

Histone deacetylation by Sir2 generates a transcriptionally repressed nucleoprotein complex.

Similar Datasets

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