The Rpd3 histone deacetylase (HDAC) functions in a large complex containing many proteins including Sin3 and Sap30. Previous evidence indicates that the pho23, rpd3, sin3, and sap30 mutants exhibit similar defects in PHO5 regulation. We report that pho23 mutants like rpd3, sin3, and sap30 are hypersensitive to cycloheximide and heat shock and exhibit enhanced silencing of rDNA, telomeric, and HMR loci, suggesting that these genes are functionally related. Based on these observations, we explored whether Pho23 is a component of the Rpd3 HDAC complex. Our results demonstrate that MycPho23 co-immunoprecipitates with HA-Rpd3 and HASap30. Furthermore, similar levels of HDAC activity were detected in immunoprecipitates of HA-Pho23, HARpd3, or HA-Sap30. In contrast, HDAC activity was not detected in immunoprecipitates of HA-Pho23 or HASap30 from strains lacking Rpd3, suggesting that Rpd3 is the HDAC associated with these proteins. However, HDAC activity was detected in immunoprecipitates of HA-Sap30 or HA-Rpd3 from cells lacking Pho23, although levels were significantly lower than those detected in wild-type cells, indicating that Rpd3 activity is compromised in the absence of Pho23. Together, our genetic and biochemical studies provide strong evidence that Pho23 is a component of the Rpd3 HDAC complex, and is required for the normal function of this complex.
Modifications of chromatin by histone acetyltransferases (HATs)1 and histone deacetylases (HDACs) play important roles in transcriptional regulation (1-4). Many proteins possessing intrinsic HAT activity have been identified from various organisms, and many of these proteins have been shown to be transcriptional coactivators or have other transcription-related functions. Similarly, several HDACs have been identified in different organisms as multiprotein complexes that are associated with transcriptional repressors and co-repressors (5-7). In many cases, HATs and HDACs are targeted to specific promoters through their interaction with DNA-binding transcription factors, suggesting that they regulate transcriptional activity by modifying the local chromatin structure at target promoters (8 -10). However, recent reports suggest that HATs also function in an untargeted manner to acetylate histones on a genome-wide scale (11,12).Packaging of DNA into chromatin is thought to affect transcription by impeding the access of transcription factors to DNA regulatory sequences. HATs acetylate lysine residues on core histones, thereby neutralizing the positive charge of the histone tails and decreasing their affinity for DNA and/or adjacent nucleosomes in higher order chromosomal structures (7, 13). Such a modification of chromatin is thought to increase the accessibility of DNA to transcription regulatory complexes (14,15). Thus, in general, hyperacetylation of histones correlates with activation of gene expression, whereas deacetylation represses transcription (16,17). Consistent with this model, the targeted recruitment of the Gcn5 HAT to specific promoters correlates with bo...
