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 inhibitor, 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 plasmids containing various 5′ deletions of the p21 promoter upstream of the luciferase reporter −/+ the histone deacetylase 1 (HDAC1) expression plasmid. After 24 h, cells were treated −/+ 5 mM sodium butyrate (NaBu) or another histone hyperacetylating agent, trichostatin A (TSA, 0.3 μM) 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 and TSA caused a marked increase in the transactivation of plasmids containing 291 bp of the p21 promoter upstream of the transcriptional start site, similar to that previously seen for a 2.4-kb construct. A decrease in reporter gene induction was seen between 173 and 153 bp. This was followed by a marked increase in promoter induction from 143 to 117 bp. Finally, only low basal 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 via 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.