Three Saccharomyces cerevisiae proteins (Yng1/YOR064c, Yng2/YHR090c, and Pho23) and two Schizosaccharomyces pombe proteins (Png1/CAA15917 and Png2/CAA21250) share significant sequence identity with the human candidate tumor suppressor p33 ING1 in their C-terminal regions. The homologous regions contain PHD finger domains which have been implicated in chromatin-mediated transcriptional regulation. We show that GFP-Yng2, like human Ing1, is localized in the nucleus. Deletion of YNG2 results in several phenotypes, including an abnormal multibudded morphology, an inability to utilize nonfermentable carbon sources, heat shock sensitivity, slow growth, temperature sensitivity, and sensitivity to caffeine. These phenotypes are suppressed by expression of either human Ing1 or S. pombe Png1, suggesting that the yeast and human proteins are functionally conserved. Yng1-and Pho23-deficient cells also share some of these phenotypes. We demonstrated by yeast two-hybrid and coimmunoprecipitation tests that Yng2 interacts with Tra1, a component of histone acetyltransferase (HAT) complexes. We further demonstrated by coimmunoprecipitation that HAYng1, HA-Yng2, HA-Pho23, and HA-Ing1 are associated with HAT activities in yeast. Genetic and biochemical evidence indicate that the Yng2-associated HAT is Esa1, suggesting that Yng2 is a component of the NuA4 HAT complex. These studies suggest that the yeast Ing1-related proteins are involved in chromatin remodeling. They further suggest that these functions may be conserved in mammals and provide a possible mechanism for the human Ing1 candidate tumor suppressor. Several observations suggest that mammalian p33ING1 is involved in the regulation of cell proliferation and apoptosis (18,21,28). NIH 3T3 cells transformed by infection with a retrovirus containing a region of the Ing1 cDNA in the antisense orientation exhibit anchorage-independent growth in soft agar, and they form tumors in nude mice. Furthermore, microinjection of constructs that express Ing1 in the sense orientation results in inhibition of DNA synthesis and cell cycle progression in human diploid fibroblasts. Ing1 levels are also increased upon the induction of apoptosis in P19 cells by serum deprivation, and overexpression of Ing1 in P19 and rodent fibroblasts enhances Myc-dependent apoptosis (28). Evidence indicates that expression of Ing1 is repressed in a majority of breast and lymphoid cancer cell lines and glioblastomas and is mutated in some neuroblastoma cell lines, breast cancers, and brain tumors (21,52,74). Together, these observations suggest that Ing1 acts as a tumor suppressor and that it is involved in regulating apoptosis. This is further supported by reports that Ing1 and the p53 tumor suppressor form a complex and functionally cooperate to control cell growth (20, 83).The carboxyl-terminal 70 amino acid residues of Ing1 contain the Cys 4 -His-Cys 3 sequence of a PHD finger domain. This evolutionarily conserved domain is predicted to chelate two Zn 2ϩ ions and is similar to, but distinct from, other zinc...

affects the degree of physical association between proliferating cell nuclear antigen (PCNA) and p300, an association that has been proposed to link DNA repair to chromatin remodeling. Together with the finding that human ING1 proteins bind PCNA in a DNA damagedependent manner, these data suggest that ING1 proteins provide a direct linkage between DNA repair, apoptosis, and chromatin remodeling via multiple HAT⅐ING1⅐PCNA protein complexes.

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...