Regulation of KLF4 Turnover Reveals an Unexpected Tissue-Specific Role of pVHL in Tumorigenesis

The transcription factor Krüppel-like factor 4 (KLF4) is an important regulator of cell fate decision, including cell cycle regulation, apoptosis, and stem cell renewal, and plays an ambivalent role in tumorigenesis as a tissue specific tumor suppressor or oncogene. Here we report that the Von Hippel-Lindau gene product, pVHL, physically interacts with KLF4 and regulates its rapid turnover observed in both differentiated and stem cells. We provide mechanistic insights into KLF4 degradation and show that pVHL depletion in colorectal cancer cells leads to cell cycle arrest concomitant with increased transcription of the KLF4-dependent p21 gene. Finally, immunohistochemical staining revealed elevated pVHL and reduced KLF4 levels in colon cancer tissues. We therefore propose that unexpectedly pVHL, via the degradation of KLF4, is a facilitating factor in colorectal tumorigenesis

Multivalent Binding of p53 to the STAGA Complex Mediates Coactivator Recruitment after UV Damage▿

The recruitment of transcriptional coactivators, including histone modifying enzymes, is an important step in transcription regulation. A typical activator is thought to interact with several cofactors, presumably in a sequential manner. The common use of several cofactors raises the question of how activators achieve both cofactor selectivity and diversity. Human STAGA is a multiprotein complex with the acetyltransferase GCN5L as the catalytic subunit. Here, we first show, through RNA interference-mediated knock-down and chromatin immunoprecipitation assays, that GCN5 plays a role in p53-dependent gene activation. We then employ p53 mutagenesis, in vitro binding, protein-protein cross-linking, and chromatin immunoprecipitation assays to establish a novel role for the second p53 activation subdomain (AD2) in STAGA recruitment and, further, to demonstrate that optimal binding of STAGA to p53 involves interactions of STAGA subunits TAF9, GCN5, and ADA2b, respectively, with AD1, AD2, and carboxy-terminal domains of p53. These results provide concrete evidence for mediation of transcription factor binding to coactivator complexes through multiple interactions. Based on our data, we propose a cooperative and modular binding mode for the recruitment of coactivator complexes to promoters

The STAGA Subunit ADA2b Is an Important Regulator of Human GCN5 Catalysis▿ †

Human STAGA is a multisubunit transcriptional coactivator containing the histone acetyltransferase GCN5L. Previous studies of the related yeast SAGA complex have shown that the yeast Gcn5, Ada2, and Ada3 components form a heterotrimer that is important for the enzymatic function of SAGA. Here, we report that ADA2a and ADA2b, two human homologues of yeast Ada2, each have the ability to form a heterotrimer with ADA3 and GCN5L but that only the ADA2b homologue is found in STAGA. By comparing the patterns of acetylation of several substrates, we found context-dependent requirements for ADA2b and ADA3 for the efficient acetylation of histone tails by GCN5. With human proteins, unlike yeast proteins, the acetylation of free core histones by GCN5 is unaffected by ADA2b or ADA3. In contrast, the acetylation of mononucleosomal substrates by GCN5 is enhanced by ADA2b, with no significant additional effect of ADA3, and the efficient acetylation of nucleosomal arrays (chromatin) by GCN5 requires both ADA2b and ADA3. Thus, ADA2b and ADA3 appear to act at two different levels of histone organization within chromatin to facilitate GCN5 function. Interestingly, although ADA2a forms a complex(es) with GCN5 and ADA3 both in vitro and in vivo, ADA2a-containing complexes are unable to acetylate nucleosomal H3. We have also shown the preferential recruitment of ADA2b, relative to ADA2a, to p53-dependent genes. This finding indicates that the previously demonstrated presence and function of GCN5 on these promoters reflect the action of STAGA and that the ADA2a and ADA2b paralogues have nonredundant functional roles

ATR kinase activation in G1 phase facilitates the repair of ionizing radiation-induced DNA damage. Nucleic Acids Res

ABSTRACT The kinase ATR is activated by RPA-coated singlestranded DNA generated at aberrant replicative structures and resected double strand breaks. While many hundred candidate ATR substrates have been identified, the essential role of ATR in the replicative stress response has impeded the study of ATR kinase-dependent signalling. Using recently developed selective drugs, we show that ATR inhibition has a significantly more potent effect than ATM inhibition on ionizing radiation (IR)-mediated cell killing. Transient ATR inhibition for a short interval after IR has long-term consequences that include an accumulation of RPA foci and a total abrogation of Chk1 S345 phosphorylation. We show that ATR kinase activity in G1 phase cells is important for survival after IR and that ATR colocalizes with RPA in the absence of detectable RPA S4/8 phosphorylation. Our data reveal that, unexpectedly, ATR kinase inhibitors may be more potent cellular radiosensitizers than ATM kinase inhibitors, and that this is associated with a novel role for ATR in G1 phase cells

Regulation of KLF4 Turnover Reveals an Unexpected Tissue-Specific Role of pVHL in Tumorigenesis

The transcription factor Krüppel-like factor 4 (KLF4) is an important regulator of cell fate decision, including cell cycle regulation, apoptosis, and stem cell renewal, and plays an ambivalent role in tumorigenesis as a tissue specific tumor suppressor or oncogene. Here we report that the Von Hippel-Lindau gene product, pVHL, physically interacts with KLF4 and regulates its rapid turnover observed in both differentiated and stem cells. We provide mechanistic insights into KLF4 degradation and show that pVHL depletion in colorectal cancer cells leads to cell cycle arrest concomitant with increased transcription of the KLF4-dependent p21 gene. Finally, immunohistochemical staining revealed elevated pVHL and reduced KLF4 levels in colon cancer tissues. We therefore propose that unexpectedly pVHL, via the degradation of KLF4, is a facilitating factor in colorectal tumorigenesis