Histone deacetylase inhibitors mediate post-transcriptional regulation of p21WAF1 through novel cis-acting elements in the 3' untranslated region.

Histone deacetylase inhibitors (HDACIs) are promising anti-tumor agents that selectively induce cell cycle arrest, differentiation and/or apoptosis of tumor cells. Fundamentally, HDACIs are proposed to function by activating the transcription of genes, including the potent cyclin dependent kinase inhibitor p21(WAF1). However, HDACIs primarily increase p21(WAF1) expression at the post-transcriptional level in HepG2 cells, implying that these anti-tumor agents regulate genes at multiple levels. Here, two novel cis-acting elements in the 3' untranslated region (UTR) of p21(WAF1) are identified that control the ability of HDACIs to induce p21(WAF1) mRNA stabilization. Collectively, these studies highlight the complexity of HDACIs in gene regulation.

Determination of protein lysine deacetylation.

Histone deacetylases (HDACs) are members of a diverse family of enzymes that catalyze the removal of an acetyl moiety from an acetyl-lysine-containing substrate. HDACs target a variety of substrates, including histone and nonhistone proteins, to mediate alterations in protein localization, stability, and activity. In addition, HDACs have been shown to modulate changes in gene expression, primarily through the recruitment of transcriptional cofactors to promoter regions. Mammalian HDACs are organized into distinct classes based on their homology to yeast HDACs. Classes I, II and IV HDACs are structurally and catalytically similar, whereas, class III HDACs require NAD(+) as a cofactor in the deacetylation reaction. This unit provides guidance for choosing and preparing a substrate suitable for assaying an HDAC of interest and describes key protocols necessary for assaying HDAC activity.

The modification of Sp3 isoforms by SUMOylation has differential effects on the SRC1A promoter.

Previously, we had described a housekeeping like promoter that regulates expression of the SRC gene in many cell types. This promoter was found to be regulated by Sp1 and hnRNP-K. However, at that time we could find little evidence supporting a significant role for Sp3 in SRC activation. Interestingly, despite its first description some 12 years ago, a full length Sp3 clone has only recently been described. Previous mechanistic studies, including our own, employed a version of Sp3 that was significantly N-terminally truncated. In addition, several shorter Sp3 isoforms exist that result from internally initiated translation sites. To complicate matters further, all Sp3 isoforms can be modified by SUMO-1. Due to this newly emerging information few reports exist that systematically explore these various Sp3 isoforms (SUMOylated or not) and how they affect activity of specific mammalian promoters. We therefore undertook such a study to re-evaluate regulation of SRC by these various Sp3 isoforms. Using human and insect cells we found that the newly isolated full length version of Sp3 was only a weak to moderate activator of SRC. However, to our surprise, the more commonly used N-terminally truncated version of Sp3 was up to five times more active. We also found that mutations preventing SUMOylation of the shorter Sp3 isoforms were sufficient to convert them into potent transactivators of SRC. In contrast to other studies, however, we found that SUMOylation of full length Sp3 had little effect on its transcriptional properties. These results provide new insights into the complexity of Sp3 mediated transcription which appears to be highly dependent on the isoform bound, SUMOylation status and the promoter context.