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.

中文翻译：

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癌症相关基因LOXL2启动子的鉴定和表征。

像赖氨酰氧化酶（LOX）家族的成员一样，像2的赖氨酰氧化酶也显示出通过促进胶原蛋白和弹性蛋白的交联而与细胞外基质（ECM）中的LOX相似的功能。LOXL2还参与转录调节，细胞信号转导和细胞粘附调节。据报道，LOXL2在几种类型的肿瘤中高表达，并促进各种癌细胞中的细胞增殖和迁移。但是，LOXL2表达的调节机制仍然很大程度上未知。为了进一步研究其转录调控机制，在本研究中已经克隆并鉴定了LOXL2启动子区域。染色质状态分析显示，LOXL2基因位点在其第一个外显子附近包含一个活性启动子。然后，我们构建了五个不同的LOXL2基因启动子荧光素酶报告基因构建体，覆盖了LOXL2基因转录起始位点上游1.7 kb。系列萤光素酶报告基因测定表明，所有五个构建体均显示出显着的启动子活性，并且LOXL2核心启动子位于转录起始位点附近185 bp的区域。转录因子结合分析表明，LOXL2启动子缺乏经典的TATA框，但包含经典转录因子如Sp1和NF-κB的推定结合位点。Sp1的异位过表达显着增强了LOXL2启动子活性及其在细胞中的内源性表达。相反，光神霉素A（选择性Sp1抑制剂）处理会抑制LOXL2启动子及其内源性转录。定点诱变测定进一步证实Sp1结合位点对于LOXL2基因的近端提示活性是必不可少的。染色质免疫沉淀（ChIP）分析显示Sp1在体内结合了LOXL2启动子。值得注意的是，Sp1和LOXL2的表达呈正相关，而LOXL2的较高表达与结直肠癌的不良预后相关，强烈暗示了Sp1介导的LOXL2反式激活在结直肠癌发病中的意义。