title: The role of Sp1 and EZH2 in the regulation of LMX1A in cervical cancer cells.
abstract: We have reported previously that LIM homeobox transcription factor 1α (LMX1A) is hypermethylated and functions as a metastasis suppressor in cervical cancer cells. However, the regulation of LMX1A 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 LMX1A promoter and used overexpression, knockdown, and reporter assays to show that Sp1 increased LMX1A promoter activity. Next, we used site-directed mutagenesis and electrophoresis mobility shift assays (EMSAs) to demonstrate that Sp1-binding sites were important for Sp1-mediated activation of the LMX1A promoter. Chromatin immunoprecipitation data demonstrated that Sp1 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 3days. 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 activate LMX1A expression upon oncogenic stress during cervical cancer development. Moreover, suppression of EZH2 may delay resilencing of LMX1A after the removal of 5-aza-dC and TSA.
authors.affiliation: Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, ROC.
authors.forename: Wen-Chi
authors.initials: WC
authors.lastname: Lin
authors.affiliation: Division of Gastroenterology, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan, ROC.
authors.forename: Ming-De
authors.initials: MD
authors.lastname: Yan
authors.affiliation: Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, ROC.
authors.forename: Pei-Ning
authors.initials: PN
authors.lastname: Yu
authors.affiliation: Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, ROC.
authors.forename: Hsin-Jung
authors.initials: HJ
authors.lastname: Li
authors.affiliation: Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, ROC.
authors.forename: Chih-Chi
authors.initials: CC
authors.lastname: Kuo
authors.affiliation: Department and Graduate Institute of Microbiology and Immunology, National Defense Medical Center, Taipei 114, Taiwan, ROC.
authors.forename: Chia-Lin
authors.initials: CL
authors.lastname: Hsu
authors.affiliation: Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, ROC; Department and Graduate Institute of Microbiology and Immunology, National Defense Medical Center, Taipei 114, Taiwan, ROC. Electronic address: ndmc.yawen@msa.hinet.net.
authors.forename: Ya-Wen
authors.initials: YW
authors.lastname: Lin
mesh_terms: D001374:Azacitidine / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D000071221:Enhancer of Zeste Homolog 2 Protein; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D055785:Gene Knockdown Techniques; D020868:Gene Silencing / Q000187:drug effects; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006657:Histones / Q000378:metabolism; D006801:Humans; D060850:LIM-Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D008745:Methylation / Q000187:drug effects; D008969:Molecular Sequence Data; D063151:Polycomb Repressive Complex 2 / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D002583:Uterine Cervical Neoplasms / Q000235:genetics*
chemical_list: D056572:Histone Deacetylase Inhibitors; D006657:Histones; D060850:LIM-Homeodomain Proteins; C078021:LMX1A protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2; D001374:Azacitidine
keywords: Cervical cancer; DNA methylation; EZH2; Histone modification; LMX1A; Sp1