The main research emphasis of our laboratory is to understand how tumor suppressors exert their function as transcription factors in the repressive environment of chromatin in eukaryotic cells. Our research is focused on dissecting the molecular mechanisms regulating activity of p53.

The p53 tumor suppressor protein is a sequence-specific transcription factor that modulates the response of cells to genotoxic stress and is responsible for the development of 50% of all human tumors. As a DNA sequence-specific transcription factor, p53 exerts its functions by activating or repressing transcription of its target genes (7). p53 activates transcription of several genes, including p21/WAF1/CIP1, GADD45, Bax, PIGs, and Mdm2, whose gene products are required to regulate the cell cycle progression, apoptosis or the function of p53 itself. p53 activity is regulated through multiple post-translational modifications that largely occur in the amino and carboxyl termini of the protein, including phosphorylation, acetylation, ubiquitilation, and glycosylation.

Recently, we have identified an additional p53 modification, also found in histones, methylation. P53 is methylated by Set9 protein, which is a histone methyltransferase (HMTase) also specific for Lys 4 of histone H3. Methylation may affect the p53-dependent transcriptional response at several levels: a) at the level of chromatin by modifying histones in the p53-dependent promoters, b) at the level of p53 itself by stabilizing the protein and increasing its association with other transcriptional coactivators/chromatin remodelers. Our research design is to probe the methylation state of nucleosomes and p53 in vivo and utilize in vivo assays of p53 function to elucidate the role of this modification in respect to the transcriptional and tumor suppressor function of p53.

In addition to methylation, we and others have shown that p53 is acetylated. Functionally, acetylation regulates p53 activity and stability. Acetylation of p53 is mediated by two distinct classes of histone acetyltransferases (HATs): CBP/p300 and PCAF/hGcn5. CBP/p300 and PCAF/Gcn5 are recruited to specific promoters to modify amino terminal domains of the core histone proteins in nucleosomes resulting in de-repression of chromatin. Also, these coactivator proteins associate with numerous activators, and acetylate many of them, although the in vivo consequences of this modification are largely unknown. It has become clear in recent years that histone acetylation and chromatin remodeling work together to antagonize chromatin-mediated transcriptional repression. In mammals, there are two related homologues of the SWI2/SNF2 gene, BRG1 and hBRM, each contained within a distinct protein complex. Notably, both Swi/Snf and Gcn5-containing complexes can bind the same transcriptional activators, including c-Myc, estrogen receptor, E2A, and E2F. Recently, we have reported a characterization of a new human homologue of yeast Ada2, called hAda2b, identified as Gcn5/PCAF-interacting partner in a yeast two-hybrid system. hAda2b interacted specifically, although not stably, with the Gcn5-containing histone acetylation complex STAGA/TFTC. Biochemical fractionation suggested that Ada2b was a part of separate, multi-subunit complex. hAda2b also bound to Baf57 (a component of the hSwi/Snf complex) in the yeast two-hybrid screen, and interacted with hSwi/Snf in vitro and in vivo. Similar to yeast Ada2, overexpressed hAda2b was able to coactivate transcription of p53 in the luciferase reporter assay.

These findings suggest a new mechanism of coactivator function, where a single adaptor protein can target both histone acetylation and chromatin remodeling to the same gene promoter.

We are planning to further our observations on the role of Ada2b on the p53-dependent transcription. Ada2b may serve as a bridging factor between p53, histone acetyltransferases and chromatin remodelers. Alternatively, Ada2b may increase p53 stability through more efficient recruitment of SAGA/STAGA to p53 and hence increased acetylation. Consequently, p53 acetylation may protect the p53 protein from ubiquitilation and subsequent proteosomal degradation.