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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.

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