Unknown,Transcriptomics,Genomics,Proteomics

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DNMT3B7, an aberrant DNMT3B isoform, suppresses growth, induces differentiation, and alters DNA methylation in human neuroblastoma

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Kelly Ostler

PROVIDER: E-GEOD-36350 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Truncated DNMT3B isoform DNMT3B7 suppresses growth, induces differentiation, and alters DNA methylation in human neuroblastoma.

Ostler Kelly R KR Yang Qiwei Q Looney Timothy J TJ Zhang Li L Vasanthakumar Aparna A Tian Yufeng Y Kocherginsky Masha M Raimondi Stacey L SL DeMaio Jessica G JG Salwen Helen R HR Gu Song S Chlenski Alexandre A Naranjo Arlene A Gill Amy A Peddinti Radhika R Lahn Bruce T BT Cohn Susan L SL Godley Lucy A LA

Cancer research 20120718 18

Epigenetic changes in pediatric neuroblastoma may contribute to the aggressive pathophysiology of this disease, but little is known about the basis for such changes. In this study, we examined a role for the DNA methyltransferase DNMT3B, in particular, the truncated isoform DNMT3B7, which is generated frequently in cancer. To investigate if aberrant DNMT3B transcripts alter DNA methylation, gene expression, and phenotypic character in neuroblastoma, we measured DNMT3B expression in primary tumor ...[more]

PMID: 22815530

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Project description:Objectives: The sequencing by the PolyA selection is the most common approach for library preparation. With limited amount or degraded RNA, alternative protocols such as the NuGEN have been developed. However, it is not yet clear how the different library preparations affect the downstream analyses of the broad applications of RNA sequencing. Methods and Materials: Eight human mammary epithelial cell (HMEC) lines with high quality RNA were sequenced by Illumina’s mRNA-Seq PolyA selection and NuGEN ENCORE library preparation. The following analyses and comparisons were conducted: 1) the numbers of genes captured by each protocol; 2) the impact of protocols on differentially expressed gene detection between biological replicates; 3) expressed single nucleotide variant (SNV) detection; 4) non-coding RNAs, particularly lincRNA detection; and 5) intragenic gene expression. Results: Sequences from the NuGEN protocol had lower (75%) alignment rate than the PolyA (over 90%). The NuGEN protocol detected fewer genes (12-20% less) with a significant portion of reads mapped to non-coding regions. A large number of genes were differentially detected between the two protocols. About 17-20% of the differentially expressed genes between biological replicates were commonly detected between the two protocols. Significantly higher numbers of SNVs (5-6 times) were detected in the NuGEN samples, which were largely from intragenic and intergenic regions. The NuGEN captured fewer exons (25% less) and had higher base level coverage variance. While 6.3% of reads were mapped to intragenic regions in the PolyA samples, the percentages were much higher (20-25%) for the NuGEN samples. The NuGEN protocol did not detect more known non-coding RNAs such as lincRNAs, but targeted small and “novel” lincRNAs. Conclusion: Different library preparations can have significant impacts on downstream analysis and interpretation of RNA-seq data. The NuGEN provides an alternative for limited or degraded RNA but it has limitations for some RNA-seq applications. Eight human mammary epithelial cell (HMEC) lines with high quality RNA were sequenced by Illumina’s mRNA-Seq PolyA selection and NuGEN ENCORE library preparation. The goal was to look the impacts of protocols on results and intepretation.

Dataset Information

DNMT3B7, an aberrant DNMT3B isoform, suppresses growth, induces differentiation, and alters DNA methylation in human neuroblastoma

Publications

Truncated DNMT3B isoform DNMT3B7 suppresses growth, induces differentiation, and alters DNA methylation in human neuroblastoma.

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