p21 gene regulation during enterocyte differentiation

Background. Enterocyte differentiation is associated with a withdrawal from the cell cycle and the transcriptional activation of the cell cycle inhibi tor, p21. We sought to define the molecular mechanisms involved in p21 gene activation in an in vitro system. Methods. Transient transfections were performed in HT-29 cells with plasmid s containing various 5 ' deletions of the p21 promoter upstream of the luci ferase reporter -/+ the histone deacetylase 1 (HDAC1) expression plasmid. A fter 24 h, cells were treated -/+ 5 mM sodium butyrate (NaBu) or another hi stone hyperacetylating agent, trichostatin A (TSA, 0.3 muM) for 24 h. After protein extraction, luciferase activity was measured. Acid/urea/triton gel electrophoresis was performed to examine histone acetylation in cells. Results. NaBu and TSA both caused histone H4 hyperacetylation. Both NaBu an d TSA caused a marked increase in the transactivation of plasmids containin g 291 bp of the p21 promoter upstream of the transcriptional start site, si milar to that previously seen for a 2.4-kb construct. A decrease in reporte r gene induction was seen between 173 and 153 bp. This was followed by a ma rked increase in promoter induction from 143 to 117 bp. Finally, only low b asal activity was seen in the case of the 93-bp plasmid. HDAC1 blocked NaBu -mediated induction of all plasmids. Conclusions. p21 gene activation during HT-29 cell differentiation occurs v ia at least two regions of cis-acting elements: one located between -93 and -117 bp, and the other between -173 and -291 bp. Histone hyperacetylation likely plays a role in this activation. (C) 2001 Academic Press.