Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing Methylation profiling by high throughput sequencing
Summary
The AID (activation induced deaminase)/APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) family of proteins are zinc-dependent cytidine deaminases active on polynucleotides and involved in RNA editing, DNA demethylation or DNA editing. The enzymatic activity, substrate or physiological target(s) of APOBEC2, a member of the AID/APOBEC, remain elusive. Here, we combined next generation sequencing (NGS) techniques with state-of-the-art molecular biology to comprehensively examine the physiological effects of APOBEC2 on the transcriptome and methylome using C2C12 myoblasts differentiating in culture. We also examined APOBEC2’s genome wide-binding specificity. Using RNA sequencing (RNA-seq) by polyA capture, we detected no evidence that APOBEC2 is an RNA editor. In the same system, enhanced reduced representation bisulfite sequencing (eRRBS) data did not support the proposed role of APOBEC2 as a 5-methyl-C (5mC) deaminase. However, chromatin immunoprecipitation sequencing (ChIP-Seq) did reveal specific locations of genomic occupancy of APOBEC2 with a specific motif preference. Combining biochemical, ChIP-Seq and RNA-Seq gene expression analyses we demonstrate that APOBEC2 acts as a negative regulator of gene expression in muscle cells. Our data support a model of APOBEC2 acting as a chromatin-binding factor that leads to inhibition of transcription of genes involved in cell cycle regulation during C2C12 differentiation.
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Overall design
Here we have combined Next Generation Sequencing (NGS) techniques with state-of-the-art molecular biology to determine the physiological targets of APOBEC2. We use a cell culture muscle differentiation system (C2C12), a system where APOBEC2 protein expression progressively increases. Control (GFP shRNA) and APOBEC2 deficient (A2shRNA, where Apobec2 is knocked down) C2C12 cells were evaluated at different time points following induction of differentiation. We profiled these cells for: gene expression changes and RNA editing changes between control and APOBEC2 deficient groups (n=3) using RNA sequencing (RNA-Seq) by polyA capture; 5-methylCytosine changes between control and APOBEC2 deficient group (n=5) in DNA using enhanced Reduced Representation Bisulfite Sequencing (ERRBS). Lastly, we identify specific APOBEC2 binding regions in the DNA using Chromatin Immunoprecipitation Sequencing (ChIP-Seq) (n=3).
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