Identification and characterization of the promoter of cancer-related gene LOXL2

Abstract

Lysyl oxidase like 2, LOXL2, as a member of the lysyl oxidase (LOX) family, has been shown to function similarly to LOX in the extracellular matrix (ECM) by promoting crosslinking of collagen and elastin. LOXL2 is also engaged to transcription regulation, cell signaling transduction and cell adhesion regulation. It has been reported that LOXL2 is highly expressed in several types of tumors and promotes cell proliferation and migration in various cancer cells. However, the regulatory mechanism of LOXL2 expression remains largely unknown. To further investigate its transcriptional regulatory mechanism, LOXL2 promoter region has been cloned and identified in the present study. Chromatin state analysis revealed that LOXL2 gene locus contained an active promoter near its first exon. We then constructed five different LOXL2 gene promoter luciferase reporter constructs covering 1.7 kb upstream of LOXL2 gene transcription initiation site. Series luciferase reporter assay demonstrated that all the five constructs showed notable promoter activity, and LOXL2 core promoter was located in a region of 185 bp near the transcription initiation site. Transcriptional factor binding analysis indicated that, LOXL2 promoter lacked classical TATA box, but contained putative binding sites for classic transcriptional factors such as Sp1 and NF-κB. Ectopic overexpression of Sp1 significantly enhanced LOXL2 promoter activity as well as its endogenous expression in cells. In contrast, mithramycin A (a selective Sp1 inhibitor) treatment repressed LOXL2 promoter as well as its endogenous transcription. Site directed mutagenesis assay further confirmed that the Sp1 binding sites were essential for proximal prompter activity of LOXL2 gene. Chromatin immunoprecipitation (ChIP) assay revealed that Sp1 bound LOXL2 promoter in vivo. Of note, the expression of Sp1 and LOXL2 are positively correlated, and the higher expression of LOXL2 is associated with poor prognosis in colorectal cancer, strongly suggesting the implication of Sp1-mediated LOXL2 transactivation in the pathogenesis of colorectal cancer.

Keywords: LOXL2; Promoter; Sp1; Transcriptional regulation.