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DNA methylation profiling of cancer cells with genetic disruption of DNA methyltransferases

ABSTRACT: Maximizing DNA methyltransferase (DNMT’s) inhibition for cancer therapy requires defining the roles and interactions between these enzymes for maintaining the widespread DNA methylation abnormalities in cancer. Combining genetic and shRNA depletion in colon and other cancer cells reveals that DNMT1 is extremely dominant in this maintenance, with DNMT’s 3A and 3B playing minor roles, at all genomic loci including promoters and enhancers. Reducing DNMT1 below a deep threshold level, especially at promoters, is required for reversing abnormal DNA methylation and re-expressing key tumor suppressor genes. Current DNMT inhibitors (DNMTis) are challenged, at tolerable patient doses, for achieving such reductions. We introduce a new approach in which reducing levels of the DNMT targeting protein, UHRF1, complements low DNMTi doses to DNA demethylate and reactivate expression of such genes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE77247 | GEO | 2017/01/13

SECONDARY ACCESSION(S): PRJNA309847

REPOSITORIES: GEO

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Project description:Atherosclerosis preferentially occurs in arterial regions of disturbed blood flow (d-flow), which alters gene expression, endothelial function, and atherosclerosis. Here, we show that d-flow regulates genome-wide DNA methylation patterns in a DNA methyltransferase (DNMT)-dependent manner. D-flow induced expression of DNMT1 in mouse arterial endothelium in vivo and in cultured endothelial cells by oscillatory shear (OS) in vitro. The DNMT inhibitor 5-Aza-2’deoxycytidine (5Aza) or DNMT1 siRNA significantly reduced OS-induced endothelial inflammation. Moreover, 5Aza reduced lesion formation in two ApoE-/- mouse atherosclerosis models. To identify the 5Aza mechanisms, we conducted two genome-wide studies: reduced representation bisulfite sequencing (RRBS) and microarray using endothelial-enriched gDNA and RNA, respectively, from the partially-ligated left carotid artery (LCA exposed to d-flow) and the right contralateral control (RCA) of mice treated with 5Aza or vehicle. Systems biological analyses using RRBS and transcriptome data revealed 11 mechanosensitive genes whose promoters were hypermethylated under d-flow conditions, but rescued by 5Aza treatment. Of those, the two transcription factors HoxA5 and Klf3 contain cAMP- response-elements, and their methylation status could serve as a mechanosensitive master switch in gene expression. Our results demonstrate that d-flow controls epigenomic DNA methylation patterns in a DNMT-dependent manner, which in turn alters endothelial gene expression and induces atherosclerosis. We used 6- to 8-week-old male C57Bl/6 mice (The Jackson Laboratory) according to the approved Institutional Animal Care and Use Committee protocol by Emory University. Mice were subjected to partial carotid ligation surgery under anesthesia. Brieﬂy, 3 of 4 caudal branches of LCA (left external carotid, internal carotid, and occipital artery) were ligated with 6-0 silk suture, although the superior thyroid artery was left intact. Development of low and oscillatory blood flow in the Left Carotid Artery of each mouse was determined by ultrasound measurements. Each sample contained total RNA from 3 pooled RCAs or LCAs. We ran 3 samples of LCA, RCA, AzaLCA, and AzaRCA on 2 microarrays.

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DNA methylation profiling of cancer cells with genetic disruption of DNA methyltransferases

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