DNA methylation and histone modifications in tumor hypoxia

Hypoxia, an inherent condition to tumors, contributes to cancer development, metastasis, and therapy resistance. Cancer cells employ epigenetic remodelling for survival under hypoxia. DNA methylation and various histone modifications are two key mechanisms of epigenetic regulation of gene expression. Histone modifications in collaboration with DNA methylation play a vital role in controlling gene expression and modulating chromatin structure. Recently it has become evident that hypoxia plays key roles in the EMT, angiogenesis, autophagy, and ECM remodeling via (i) modulating DNA methylation at the promoter of the gene or at loci undergoing alternative splicing; and (ii) locus-specific histone modifications such as H3K27Ac, H3K9Ac, H3K4me3, H4R3me2s, and H3R8me2s. Thus, it is important to mechanistically assess how hypoxia-mediated DNA methylation changes and histone modification affect tumorigenesis using suitable methods. In this chapter, we describe the methods used in our laboratory to assess the locus-specific DNA methylation and histone modifications profiling. The goals of this chapter are twofold. First, we discuss immunoprecipitation-based approaches applied to assess DNA methylation or hydroxymethylation, histone modification, and transcription factor binding profile at specific loci. Second, we discuss methods used to modify the epigenetic landscape, such as DNA methylation, binding of transcription factors such as HIF1α and CTCF, and histone marks on a given locus using dCas9 fused with epigenetic modifiers such as DNMT3A, TET1, PRMT5, etc. Collectively, these approaches provide a template that can be applied to evaluate roles of any epigenetic mark involved in development and/or progression of cancer.