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Genome-wide methylation analysis of DNMT3B gene isoforms revealed specific methylation profiles in breast cell lines

ABSTRACT: Both DNA methylation and alternative splicing represent mechanisms closely involved in regulation of gene expression and are well known to be involved in cancer susceptibility and development. Human DNA methylation is introduced into DNA by enzymes of the DNA cytosine methyltransferases family, which includes DNMT3B. The DNMT3B protein allows a de novo DNA methylation activity, which is a key mechanism involved in the transformation of normal cell into cancer cell. The DNMT3B3 isoform is overexpressed in cancerous tissues and tumor cell lines, and could act as a dominant negative factor. This isoform was found to be highly expressed in our cohort of non-BRCA1/2 families. Using Infinium Human Methylation 450 BeadChips, we undertook the characterization of the specific methylation profile associated with this DNMT3B3 isoform and its DNMT3B2 wild type counterpart in ER/PR-positive and –negative breast cancer cell lines. A large spectrum of DNMT3B3/DNMT3B2 expression ratio values was observed in cancer and non-cancerous cell lines. Based on their methylation profiles, hierarchical clustering of parental cell lines revealed clustering of cells based on their ER/PR status. Overexpression of DNMT3B3 triggered methylation changes of thousands of CpG sites in MCF7, T-47D, MDA-MB-231 and MCF10A cells. These methylated loci were distributed in similar proportion over the 22 autosomal chromosomes and genomic locations such as promoter, gene body or intergenic regions. Pathways associated with genes containing these differentially methylated CpG sites were also determined regarding their enrichment. Moreover, based on the trend of methylation changes, the results suggest an anti-proliferative effect of the DNMT3B3 isoform through negative effect on its wild-type isoform counterpart DNMT3B2. To our knowledge, this study represents the first exhaustive analysis describing specific methylation profile triggered by modulated expression of DNMT3B isoforms and revealed specific genes and pathways, which could significantly regulate cell growth and proliferation as well as other biological and molecular mechanisms.

ORGANISM(S): Homo sapiens

PROVIDER: GSE77348 | GEO | 2016/10/31

SECONDARY ACCESSION(S): PRJNA310106

REPOSITORIES: GEO

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Project description:In adult cancers, epigenetic changes and aberrant splicing of the DNMT3B is commonly observed, and the pattern of gene methylation and expression has been shown to be modified by DNMT3B7, a truncated protein of DNMT3B. Much less is known about the mechanism of epigenetic changes in the pediatric cancer neuroblastoma. To investigate if aberrant DNMT3B transcripts alter DNA methylation, gene expression and tumor phenotype in neuroblastoma, we measured DNMT3B isoform expression in primary tumors and cell lines. Higher levels of DNMT3B7 were detected in differentiated ganglioneuroblastomas compared to undifferentiated neuroblastomas, suggesting that expression of DNMT3B7 may induce a less clinically aggressive tumor phenotype. To test this hypothesis, we investigated the effects of forced DNMT3B7 in neuroblastoma cells. We found that DNMT3B7 expression significantly inhibited neuroblastoma cell proliferation in vitro, and in neuroblastoma xenografts, DNMT3B7 decreased angiogenesis and tumor growth. DNMT3B7-positive cells had higher levels of total genomic methylation, and RNA-sequencing revealed a dramatic decrease in expression of FOS and JUN family members, AP1 complex components. Consistent with the established antagonistic relationship between AP1 expression and retinoic acid receptor activity, decreased proliferation and increased differentiation was seen in the DNMT3B7-expressing neuroblastoma cells following treatment with all trans retinoic acid (ATRA) compared to controls. Our results demonstrate that high levels of DNMT3B7 modify the epigenome in neuroblastoma cells, induce changes in gene expression, inhibit tumor growth, and increase sensitivity to ATRA. Further knowledge regarding mechanisms by which DNMT3B7 regulates gene methylation may ultimately lead to the development of therapeutic strategies that reverse the epigenetic aberrations that drive neuroblastoma pathogenesis. DNMT3B7, a truncated DNMT3B isoform, was stably transfected into an N-type neuroblastoma cell line (LA1-55n) using a Tet-off inducible system. DNMT3B7 expressing cells were compared to vector control cells after 21 days of induction.

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Genome-wide methylation analysis of DNMT3B gene isoforms revealed specific methylation profiles in breast cell lines

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