Krüppel-like Factor 4 Is Acetylated by p300 and Regulates Gene Transcription via Modulation of Histone Acetylation

Paul M. Evans,Jun Yang,Wen Zhang,Chunming Liu,Xi Chen,Kishor K. Bhakat

doi:10.1074/jbc.m701847200

Abstract

Colon cancer is the second leading cause of cancer death in the United States. Krüppel-like factor 4 (KLF4) is a transcription factor involved in both proliferation and differentiation in the colon. It is down-regulated in both mouse and human colonic adenomas and has been implicated as a tumor suppressor in the gut, whereas in breast cancer, KLF4 is an oncogene. KLF4 is also involved in reprogramming differentiated cells into pluripotent stem cells. KLF4 can act as a transcriptional activator or repressor, but the underlying mechanisms are poorly understood. We found that p300, a CREB-binding protein-related protein, interacts with KLF4 both in vitro and in vivo and activates transcription. We further made the novel observation that endogenous KLF4 is acetylated by p300/CBP in vivo and that mutations of the acetylated lysines resulted in a decreased ability of KLF4 to activate target genes, suggesting that acetylation is important for KLF4-mediated transactivation. Furthermore, we found that KLF4 differentially modulates histone H4 acetylation at the promoters of target genes. Co-transfection of KLF4 and HDAC3 resulted in a synergistic repression of a cyclin B(1) reporter construct. Our results suggest that KLF4 might function as an activator or repressor of transcription depending on whether it interacts with co-activators such as p300 and CREB-binding protein or co-repressors such as HDAC3.

Highlights

Fewer goblet cells, and the remaining goblet cells are histologically and ultrastructurally abnormal [7]
Expression of p21Cip1/WAF1 increased in a similar manner, whereas expression of cyclin B1 was strongly down-regulated at 48 h, suggesting that Kruppel-like factor 4 (KLF4) represses the cell cycle by differentially regulating expression of these genes
We made the novel observation KLF4 interacts with, and is acetylated by, the histone acetyltransferase (HAT) proteins p300 and CREB-binding protein (CBP). This observation is physiologically relevant, as we found that KLF4 is acetylated in vivo as well

Summary

EXPERIMENTAL PROCEDURES

Plasmid DNA Constructs—pCS2-KLF4 (N-terminal FLAG tag), FLAG-p300, and pRC-CMV-mCBP-HA have been described previously [14, 42,43,44]. GST beads containing purified GST or GST-p300 (CH3 domain) proteins were incubated with cell lysate containing FLAG-tagged KLF4 or KLF4 mutants at 4 °C overnight, and beads were washed three times with lysis buffer and boiled in 1ϫ SDS sample buffer, followed by analysis via Western blot. HEK293T cells were transiently transfected in a 12-well plate with 0.2 ␮g of the appropriate reporter and 0.5 ␮g of empty vector (pCS2ϩ) or the appropriate KLF4 construct with or without 0.5 ␮g of p300 plasmid DNA. HT29 cells were harvested after treatment with 5 mM sodium butyrate, and 40 ␮l of cell lysate was incubated with 200 ␮l of p-nitrophenyl phosphate (Sigma, catalog number A3469) in a 96-well plate for 1–3 h. For ChIP assays, antibodies used include p300 (Santa Cruz Biotechnology, catalog number sc-584), HDAC3 (BD Biosciences, catalog number 611124), and KLF4 [14]

RESULTS