Investigating The Impact Of Intragenic Dna Methylation On Gene Expression, And The Clinical Implications On Tumor Cells And Associated Stroma

Author

Michael McGuire, The University of Texas M D Anderson Cancer Center UTHealth Graduate School of Biomedical SciencesFollow

Author ORCID Identifier

0000-0003-2699-8430

Date of Graduation

5-2018

Document Type

Dissertation (PhD)

Program Affiliation

Cancer Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Anil Sood

Committee Member

Menashe Bar-Eli

Committee Member

Sharon Dent

Committee Member

Mien-Chie Hung

Committee Member

Keith Baggerly

Abstract

Investigations into the function of non-promoter DNA methylation have yielded new insights into epigenetic regulation of gene expression. Previous studies have highlighted the importance of distinguishing between DNA methylation in discrete functional regions; however, integrated non-promoter DNA methylation and gene expression analyses across a wide number of tumor types and corresponding normal tissues have not been performed. Through integrated analysis of gene expression and DNA methylation profiles, we uncovered an enrichment of DNA methylation sites within the gene body and 3’UTR in which DNA methylation is strongly positively correlated with gene expression. We examined 32 tumor types and identified 57 tumor suppressors and oncogenes out of 224 genes containing a correlation of > 0.5 between gene body methylation and gene expression in at least 1 tumor type. The lymphocyte-specific gene CARD11 exhibits robust association between gene body methylation and expression across 19 of 32 tumor types examined. It is significantly overexpressed in KIRC and LUAD, and has a z-score of 4 in KIRC, meaning that high expression of CARD11 in this tumor type was associated with lower patient overall survival. Contrary to its canonical function in lymphocyte NF-kB activation, CARD11 activates the mTOR pathway in KIRC and LUAD, resulting in suppressed autophagy, and demethylation of a CpG island within the gene body of CARD11 decreases gene expression. In addition to methylation of the open reading frame portion of a gene, other regions of site-specific DNA methylation along the gene body remain to be explored. Upon segregating the gene body into discrete functional units (5’UTR, 1^st exon, 3’UTR), it was noted that the 3’UTR contained an enrichment of probes positively correlated between DNA methylation and gene expression. In 5 of 10 tumor types examined, DNA methylation of the 3’UTR is associated with patient survival in a significant number of genes. Filtering for genes in which 3’UTR DNA methylation, relative to gene body DNA methylation, is more strongly correlated with gene expression yields a list of 156 genes, enriched for functions involving T cell activation. Activating T cells ex vivo caused the immune checkpoint gene HAVCR2, but not other genes examined, to show a substantial increase in 3’UTR DNA methylation, but not adjacent exonic/intronic, or promoter DNA methylation, upon upregulation of gene expression. Furthermore, this increase in HAVCR2 gene expression can be abrogated by treatment with demethylating agents. These findings implicate the 3’UTR as a functionally relevant DNA methylation site, particularly regarding T cell activity. Additionally, they reveal a novel mechanism by which HAVCR2 is upregulated in T cells, providing a new molecular target for immune checkpoint blockade.

Keywords

3'UTR, DNA methylation, T cells, Immune checkpoint