Epigenetic Regulations and Promoter Characterization of CERIG (Cancer Endoplasmic Reticulum Gene-KIAA1199)

Abstract

Although human genome project is complete, the biological function of most genes remains obscure. In this thesis project, the regulatory mechanisms of a novel endoplasmic reticulum (ER) resident protein identified by a PCR-subtraction hybridization technique, designated as Cancer ER Invasion Gene (CERIG), is investigated. CERIG is found to be highly expressed in human invasive breast cancer specimens examined by immunohistochemistry and real-time RT-PCR. This up-regulation of CERIG is associated with poor prognosis of patients with breast cancer assessed by a DNA microarray data-mining approach from three publicly available cohorts containing a total of 696 breast cancer patients. In study of the regulation mechanism of CERIG, a 3.3 kb fragment of human genomic DNA containing the 5'-flanking sequence of the CERIG is found to possess both suppressive and activating elements. Employing a deletion mutagenesis approach, a 1.4 kb proximal region is defined as the basic CERIG promoter containing a TATA-box close to the transcription start site. A combination of 5'-primer extension approach with a bioinformatics analysis from the Cap-Analysis Gene Expression database reveals a single transcription start site in the human CERIG gene. Bioinformatics analysis suggests that the 1.4 kb CERIG promoter contains putative activating regulatory elements, including activator protein-1(AP-1), Twist-1, and NF-?ÂB sites. Sequential deletion and site-direct mutagenesis analysis demonstrate that the AP-1 and distal NF-?ÂB sites are required for CERIG gene expression. Further analyses using an electrophoretic mobility-shift assay and chromatin immunoprecipitation confirmed the requirement of these cis- and trans-acting elements in controlling CERIG gene expression. In further analysis of the regulatory mechanism of CERIG in cancer progression, we examine whether CERIG mRNA expression is regulated by the DNA methylation and/or histone H3 modification. Aberrant DNA methylation of CERIG 5'-untranslated region is identified as an important regulatory mechanism in the deregulation of CERIG. In vitro promoter methylation experiments and the demethylating reagent, 5'azacytidine, are used to determine the role of DNA methylation in CERIG expression. Pyrosequencing analysis revealed demethylation of CERIG in human breast cancer specimen that correlated with high expression of CERIG. The role of chromatin modifications in CERIG expression is also determined. Correlation between the level of tri-methylated H3K4 (activation marker) and the higher level of CERIG expression has been demonstrated in breast cancer cells. Substitution of H3K4me3 with H3K27me3 on histone tail has been found in cancer cells or primary cell lines in which CERIG expression declines. Additionally, the effects of hypoxia was analyzed on both epigenetic mechanisms for CERIG expression and determined that hypoxia increases the activation marker and decreases the repression marker on the CERIG promoter without changing the level of DNA methylation. Taken together, this study uncovers the regulatory mechanism of CERIG in breast cancer progression and suggests that CERIG may be used as a prognostic marker and potential therapeutic target in prevention of cancer metastasis.