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Chromatin affinity-precipitation using a small metabolic molecule: its application to analysis of O-acetyl-ADP-ribose.

ABSTRACT: In the cell, many small endogenous metabolic molecules are involved in distinct cellular functions such as modulation of chromatin structure and regulation of gene expression. O-acetyl-ADP-ribose (AAR) is a small metabolic molecule that is generated during NAD-dependent deacetylation by Sir2. Sir2 regulates gene expression, DNA repair, and genome stability. Here, we developed a novel chromatin affinity-precipitation (ChAP) method to detect the chromatin fragments at which small molecules interact with binding partners. We used this method to demonstrate that AAR associated with heterochromatin. Moreover, we applied the ChAP method to whole genome tiling array chips to compare the association of AAR and Sir2. We found that AAR and Sir2 displayed similar genomic binding patterns. Furthermore, we identified 312 potential association cluster regions of AAR. The ChAP assay may therefore be a generally useful strategy to study the small molecule association with chromosomal regions. Our results further suggest that the small metabolic molecule AAR associates with silent chromatin regions in a Sir2-dependent manner and provide additional support for the role of AAR in assembly of silent chromatin.

SUBMITTER: Tung SY

PROVIDER: S-EPMC3266462 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Chromatin affinity-precipitation using a small metabolic molecule: its application to analysis of O-acetyl-ADP-ribose.

Tung Shu-Yun SY Hong Jia-Yang JY Walz Thomas T Moazed Danesh D Liou Gunn-Guang GG

Cellular and molecular life sciences : CMLS 20110728 4

In the cell, many small endogenous metabolic molecules are involved in distinct cellular functions such as modulation of chromatin structure and regulation of gene expression. O-acetyl-ADP-ribose (AAR) is a small metabolic molecule that is generated during NAD-dependent deacetylation by Sir2. Sir2 regulates gene expression, DNA repair, and genome stability. Here, we developed a novel chromatin affinity-precipitation (ChAP) method to detect the chromatin fragments at which small molecules interac ...[more]

PMID: 21796450

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Project description:Sirtuins catalyze the NAD(+)-dependent deacetylation of target proteins, which are regulated by this reversible lysine modification. During deacetylation, the glycosidic bond of the nicotinamide ribose is cleaved to yield nicotinamide and the ribose accepts the acetyl group from substrate to produce O-acetyl-ADP-ribose (OAADPr), which exists as an approximately 50:50 mixture of 2' and 3' isomers at neutral pH. Discovery of this metabolite has fueled the idea that OAADPr may play an important role in the biology associated with sirtuins, acting as a signaling molecule and/or an important substrate for downstream enzymatic processes. Evidence for OAADPr-metabolizing enzymes indicates that at least three distinct activities exist that could modulate the cellular levels of this NAD(+)-derived metabolite. In Saccharomyces cerevisiae, NUDIX hydrolase Ysa1 cleaves OAADPr to AMP and 2- and 3-O-acetylribose-5-phosphate, lowering the cellular levels of OAADPr. A buildup of OAADPr and ADPr has been linked to a metabolic shift that lowers endogenous reactive oxygen species and diverts glucose towards preventing oxidative damage. In vitro, the mammalian enzyme ARH3 hydrolyzes OAADPr to acetate and ADPr. A third nuclear-localized activity appears to utilize OAADPr to transfer the acetyl-group to another small molecule, whose identity remains unknown. Recent studies suggest that OAADPr may regulate gene silencing by facilitating the assembly and loading of the Sir2-4 silencing complex onto nucleosomes. In mammalian cells, the Trpm2 cation channel is gated by both OAADPr and ADP-ribose. Binding is mediated by the NUDIX homology (NudT9H) domain found within the intracellular portion of the channel. OAADPr is capable of binding the Macro domain of splice variants from histone protein MacroH2A, which is highly enriched at heterochromatic regions. With recently developed tools, the pace of new discoveries of OAADPr-dependent processes should facilitate new molecular insight into the diverse biological processes modulated by sirtuins.

Dataset Information

Chromatin affinity-precipitation using a small metabolic molecule: its application to analysis of O-acetyl-ADP-ribose.

Publications

Chromatin affinity-precipitation using a small metabolic molecule: its application to analysis of O-acetyl-ADP-ribose.

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