The role of Sp1 and EZH2 in the regulation of LMX1A in cervical cancer cells

We have reported previously that LIM homeobox transcription factor 1 alpha (LMX1A) is hypermethylated and functions as a metastasis suppressor in cervical cancer cells. However, the regulation of LMXIA in carcinogenesis has not been reported. We aim to clarify whether specificity protein 1 (Sp1) and enhancer of zeste homolog 2 (EZH2) are involved in the regulation of LMX1A in cervical cancer. First we characterized the L11/1X1A promoter and used overexpression, knockdown, and reporter assays to show that Spl increased LMX1A promoter activity. Next, we used site-directed mutagenesis and electrophoresis mobility shift assays (EMSAs) to demonstrate that Splbinding sites were important for Sp1-mediated activation of the LMX1A promoter. Chromatin immunoprecipitation data demonstrated that Spl could bind directly to the LMX1A promoter and activate endogenous LMX1A expression in cells pretreated with 5-aza-2'-deoxycytidine (5-aza-dC). Knockdown of EZH2 decreased H3K27me3 histone modification but was insufficient to restore LMX1A expression. To explore the effect of EZH2 on the endogenous LMX1A promoter, we treated EZH2-knockdown cells with 5-aza-dC and trichostatin A (TSA) and then depleted the cells of drugs for 3 days. H3K14ac was enriched at the LMX1A promoter in EZH2knockdown cells and LIVIX1A mRNA was still expressed. Taken together, these data imply that Spl may activate LMX1A expression upon oncogenic stress during cervical cancer development Moreover, suppression of EZH2 may delay resilencing of LMXIA after the removal of 5-aza-dC and TSA. (C) 2013 Elsevier B.V. All rights reserved.