ING1 represses transcription by direct DNA binding and through effects on p53.

The ING family of proteins is involved in the regulation of diverse processes ranging from cell cycle and cellular senescence to apoptosis. These effects are most likely through activation of acetylation-dependent pathways that ultimately alter gene expression. Despite reports linking ING to p53 activation, the molecular basis of how ING activates p53 function has not been elucidated. In this study, we found that a subset of ING family members strongly repressed human alpha-fetoprotein (AFP) promoter activity but stimulated the p21(WAF1) promoter in parallel experiments in the same cell type, similar to the effects of p53. The p47(ING1a) isoform also repressed AFP promoter activity, but in contrast to other ING isoforms, it repressed the p21(WAF1) promoter. p47(ING3) up-regulated p21(WAF1) promoter activity, but it did not have any effect on the AFP promoter. ING1b and ING2 also repressed the AFP promoter in Hep3B p53-null cell lines, and p53 coexpression enhanced this transcriptional repression. Suppression of AFP gene transcription by ING was strongly dependent on AT-motifs that bind to the hepatocyte nuclear factor 1 (HNF1) transcription factor. Indeed, electrophoretic mobility shift assays confirmed that HNF1 binds to AT-motifs, but we found, surprisingly, that the ING1 complexes binding to these AT-motifs were devoid of HNF1 protein. Both ING1 and p53 were able to suppress AFP transcription and cause p21 induction; hSIR2, a negative regulator of the p53 protein, showed the opposite effects on the AFP promoter and, like HDAC1, repressed p21 promoter activity. In addition, we found that p33(ING1b) physically interacts with hSIR2, reverses its ability to induce the AFP promoter, and induces acetylation of p53 residues at Lys(373) and/or Lys(382). These findings provide novel evidence that p33(ING1b) represses AFP transcription by at least two mechanisms, one of which includes p53. The first is by binding to the AT-motif and excluding HNF1 binding while possibly targeting HAT activity to promoter regions, and the second is by increasing the levels of active, acetylated p53 via binding and inhibiting the ability of hSIR2 to deacetylate p53 protein.

ING1a expression increases during replicative senescence and induces a senescent phenotype.

The ING family of tumor suppressor proteins affects cell growth, apoptosis and response to DNA damage by modulating chromatin structure through association with different HAT and HDAC complexes. The major splicing isoforms of the ING1 locus are ING1a and INGlb. While INGlb plays a role in inducing apoptosis, the function of ING1a is currently unknown. Here we show that alternative splicing of the ING1 message alters the INGla:INGlb ratio by approximately 30-fold in senescent compared to low passage primary fibroblasts. INGla antagonizes INGlb function in apoptosis, induces the formation of structures resembling senescence-associated heterochromatic foci containing heterochromatin protein 1 gamma, the accumulation of senescence-associated beta-galactosidase activity and promotes senescent cell morphology and cell cycle arrest. Phenotypic effects may result from differential effects on gene expression since ING1a increases levels of both retinoblastoma and the p16 cyclin-dependent kinase inhibitor and ING1a and ING1b have opposite effects on the expression of proliferating nuclear cell antigen (PCNA), which is required for cell growth. Gene expression appears to be altered by targeting of HDAC complexes to gene promoters since INGla associates with several-fold higher levels of HDAC1 in senescent, compared to replication-competent cells and ING1 is found on the PCNA promoter by chromatin immunoprecipitation analysis. These data demonstrate a novel role for the ING1 proteins in differentially regulating senescence-associated chromatin remodeling vs. apoptosis and support the idea that altered ratios of the ING1 splicing isoforms may contribute to establishing the senescent phenotype through HDAC and HAT complex-mediated effects on chromatin structure.

Human ING1 proteins differentially regulate histone acetylation.

ING1 proteins are nuclear, growth inhibitory, and regulate apoptosis in different experimental systems. Here we show that similar to their yeast homologs, human ING1 proteins interact with proteins associated with histone acetyltransferase (HAT) activity, such as TRRAP, PCAF, CBP, and p300. Human ING1 immunocomplexes contain HAT activity, and overexpression of p33(ING1b), but not of p47(ING1a), induces hyperacetylation of histones H3 and H4, in vitro and in vivo at the single cell level. p47(ING1a) inhibits histone acetylation in vitro and in vivo and binds the histone deacetylase HDAC1. Finally, we present evidence indicating that p33(ING1b) 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 damage-dependent manner, these data suggest that ING1 proteins provide a direct linkage between DNA repair, apoptosis, and chromatin remodeling via multiple HAT.ING1.PCNA protein complexes.