Enhancer role of a native metabolite, O-acetyl-ADP-ribose, on the Saccharomyces cerevisiae chromatin epigenetic gene silencing.

To study the epigenetic gene silencing, yeast is an excellent model organism. Sir proteins are required for the formation of silent heterochromatin. Sir2 couples histone deacetylation and NAD hydrolysis to generate an endogenous epigenetic metabolic small molecule, O-acetyl-ADP-ribose (AAR). AAR is involved in the conformational change of SIR complexes, modulates the formation of SIR-nucleosome preheterochromatin and contributes to the spreading of SIR complexes along the chromatin fiber to form extended silent heterochromatin regions. Here, we show that AAR is capable of enhancing the chromatin silencing effect under either an extra exogenous AAR or a defect AAR metabolic enzyme situation, but decreasing the chromatin silencing effect under a defect AAR synthetic enzyme state. Our results provide an evidence of biological function importance of AAR. It is indicated that AAR does not only function in vitro but also play a role in vivo to increase the effect of heterochromatin epigenetic gene silencing. However, further investigations of AAR are warranted to expand our knowledge of epigenetics and associated small molecules.

Stabilization of Sir3 interactions by an epigenetic metabolic small molecule, O-acetyl-ADP-ribose, on yeast SIR-nucleosome silent heterochromatin.

In Saccharomyces cerevisiae, Sir proteins mediate heterochromatin epigenetic gene silencing. The assembly of silent heterochromatin requires histone deacetylation by Sir2, conformational change of SIR complexes, and followed by spreading of SIR complexes along the chromatin fiber to form extended silent heterochromatin domains. Sir2 couples histone deacetylation and NAD hydrolysis to generate an epigenetic metabolic small molecule, O-acetyl-ADP-ribose (AAR). Here, we demonstrate that AAR physically associates with Sir3 and that polySir3-AAR formation has a specific and essential role in the assembly of silent SIR-nucleosome pre-heterochromatin filaments. Furthermore, we show that AAR is capable of stabilizing binding of the Sir3 BAH domain to the Sir3 carboxyl-terminal region. Our data suggests that for the assembly of SIR-nucleosome pre-heterochromatin filament, the structural rearrangement of SIR-nucleosome is important and result in creating more stable interactions of Sir3, such as the inter-molecule Sir3-Sir3 interaction, and the Sir3-nucleosome interaction within the filaments. In conclusion, our results reveal the importance of AAR, indicating that it not only affects the conformational rearrangement of SIR complexes but also might function as a critical fine-tuning modulatory component of yeast silent SIR-nucleosome pre-heterochromatin by stabilizing the intermolecular interaction between Sir3 N- and C-terminal regions.

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

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.

The Capability of O-Acetyl-ADP-Ribose, an Epigenetic Metabolic Small Molecule, on Promoting the Further Spreading of Sir3 along the Telomeric Chromatin.

O-acetyl-ADP-ribose (AAR) is a metabolic small molecule relevant in epigenetics that is generated by NAD-dependent histone deacetylases, such as Sir2. The formation of silent heterochromatin in yeast requires histone deacetylation by Sir2, structural rearrangement of SIR complexes, spreading of SIR complexes along the chromatin, and additional maturation processing. AAR affects the interactions of the SIR-nucleosome in vitro and enhances the chromatin epigenetic silencing effect in vivo. In this study, using isothermal titration calorimetry (ITC) and dot blotting methods, we showed the direct interaction of AAR with Sir3. Furthermore, through chromatin immunoprecipitation (ChIP)-on-chip and chromatin affinity purification (ChAP)-on chip assays, we discovered that AAR is capable of increasing the extended spreading of Sir3 along telomeres, but not Sir2. In addition, the findings of a quantitative real-time polymerase chain reaction (qRT-PCR) and examinations of an in vitro assembly system of SIR-nucleosome heterochromatin filament were consistent with these results. This study provides evidence indicating another important effect of AAR in vivo. AAR may play a specific modulating role in the formation of silent SIR-nucleosome heterochromatin in yeast.

Changes in the genome-wide localization pattern of Sir3 in Saccharomyces cerevisiae during different growth stages.

In budding yeast, the Sir2, Sir3 and Sir4 proteins form SIR complexes, required for the assembly of silent heterochromatin domains, and which mediate transcription silencing at the telomeres as well as at silent mating type loci. In this study, under fluorescence microscopy, we found most Sir3-GFP expressions in the logarithmic phase cells appeared as multiple punctations as expected. However, some differences in the distribution of fluorescent signals were detected in the diauxic~early stationary phase cells. To clarify these, we then used ChIP on chip assays to investigate the genome-wide localization of Sir3. In general, Sir3 binds to all 32 telomere proximal regions, the silent mating type loci and also binds to the rDNA region. However, the genome-wide localization patterns of Sir3 are different between these two distinct growth phases. We also confirmed that Sir3 binds to a recently identified secondary binding site, PAU genes, and further identified 349 Sir3-associated cluster regions. These results provide additional support in roles for Sir3 in the modulation of gene expression during physical conditions such as diauxic~early stationary phase growing. Moreover, they imply that Sir3 may be not only involved in the formation of conventional silent heterochromatin, but also able to associate with some other chromatin regions involved in epigenetic regulation.