Archer S Y, Johnson J J, Kim H J, Hodin R A
Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
J Surg Res. 2001 Jun 1;98(1):4-8. doi: 10.1006/jsre.2001.6147.
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.
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 microM) for 24 h. After protein extraction, luciferase activity was measured. Acid/urea/triton gel electrophoresis was performed to examine histone acetylation in cells.
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.
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.
肠上皮细胞分化与退出细胞周期以及细胞周期抑制剂p21的转录激活有关。我们试图在体外系统中确定参与p21基因激活的分子机制。
用含有荧光素酶报告基因上游p21启动子不同5'缺失的质粒，在HT - 29细胞中进行瞬时转染，并加入/不加入组蛋白去乙酰化酶1（HDAC1）表达质粒。24小时后，细胞用5 mM丁酸钠（NaBu）或另一种组蛋白超乙酰化剂曲古抑菌素A（TSA，0.3 microM）处理24小时。提取蛋白质后，测量荧光素酶活性。进行酸/尿素/ Triton凝胶电泳以检测细胞中的组蛋白乙酰化。
NaBu和TSA均导致组蛋白H4超乙酰化。NaBu和TSA均使含有转录起始位点上游291 bp的p21启动子的质粒的反式激活显著增加，类似于先前在2.4 kb构建体中观察到的情况。在173至153 bp之间观察到报告基因诱导的降低。随后在143至117 bp之间启动子诱导显著增加。最后，对于93 bp的质粒，仅观察到低基础活性。HDAC1阻断了NaBu介导的所有质粒的诱导。
HT - 29细胞分化过程中p21基因激活通过至少两个顺式作用元件区域发生：一个位于 - 93至 - 117 bp之间，另一个位于 - 173至 - 291 bp之间。组蛋白超乙酰化可能在这种激活中起作用。
