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

abstract Article history:Received 2 April 2013Received in revised form 28 August 2013Accepted 30 August 2013Available online 7 September 2013Keywords:LMX1ASp1EZH2Histone modificationDNA methylationCervical cancer WehavereportedpreviouslythatLIMhomeoboxtranscriptionfactor1α(LMX1A)ishypermethylatedandfunc-tionsasa metastasissuppressor incervical cancer cells. However, the regulation of LMX1A incarcinogenesis hasnotbeenreported.Weaimtoclarifywhetherspecificityprotein1(Sp1)andenhancerofzestehomolog2(EZH2)are involved in the regulation of LMX1A in cervical cancer. First we characterizedthe LMX1A promoter and usedoverexpression, knockdown, and reporter assaysto show that Sp1 increasedLMX1A promoter activity. Next, weused site-directed mutagenesis and electrophoresis mobility shift assays (EMSAs) to demonstrate that Sp1-binding siteswereimportant forSp1-mediatedactivationoftheLMX1A promoter.Chromatinimmunoprecipita-tiondatademonstratedthatSp1couldbinddirectlytotheLMX1ApromoterandactivateendogenousLMX1Aex-pression in cellspretreated with 5-aza-2′-deoxycytidine (5-aza-dC). Knockdown of EZH2 decreasedH3K27me3histone modification but was insufficient to restore LMX1A expression. To explore the effect of EZH2 on the en-dogenous LMX1A promoter, we treated EZH2-knockdown cells with 5-aza-dC and trichostatin A (TSA) andthen depleted the cells of drugs for 3 days. H3K14ac was enriched at the LMX1A promoter in EZH2-knockdown cells and LMX1A mRNA was still expressed. Taken together, these data imply that Sp1 may activateLMX1A expression upon oncogenic stress during cervical cancer development. Moreover, suppression of EZH2may delay resilencing of LMX1A after the removal of 5-aza-dC and TSA.© 2013 Elsevier B.V. All rights reserved.