Unknown,Transcriptomics,Genomics,Proteomics

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RNA-sequencing of Nedd4+/+ and Nedd4-/- mouse splenic B cells

ABSTRACT: Programmed mutagenesis of the immunoglobulin locus of B-lymphocytes during class switch recombination and somatic hypermutation requires RNA polymerase II (RNA polII) transcription complex dependent targeting of the DNA mutator, Activation Induced cytidine Deaminase (AID). AID deaminates cytidine residues on substrate sequences in the immunoglobulin (Ig) locus via a transcription-dependent mechanism and this activity is stimulated by the RNA polII stalling co-factor Spt5 and the eleven-subunit cellular non-coding RNA 3’-5’ exonucleolytic processing complex, RNA exosome. The mechanism by which the RNA exosome recognizes immunoglobulin locus RNA substrates to stimulate AID DNA deamination activity on its in vivo substrate sequences is an important question. Here we report that E3-ubiquitin ligase Nedd4 destabilizes AID-associated RNA polII by a ubiquitination event leading to generation of 3’-end free RNA exosome RNA substrates at the Ig locus and other AID target sequences genome-wide. Using highthrough-out RNA sequencing technology, we find that lack of Nedd4 activity in B cells leads to accumulation of RNA exosome substrates at AID target genes. Moreover, we find that Nedd4-deficient B cells are inefficient in undergoing class switch recombination. Taken together, our study links non-coding RNA processing following RNA polymerase II pausing with regulation of the mutator AID protein. Our study also identifies Nedd4 as a regulator of non-coding RNA that are generated by stalled RNA polII genome-wide. Splenic B cells from Nedd4+/+ and Nedd4-/- B cells fetal liver chimeric mice were were stimulated in culture for IgG1 CSR. Total RNA was isolated and evaluated with whole genome RNA-seq

ORGANISM(S): Mus musculus

SUBMITTER: Jiguang Wang

PROVIDER: E-GEOD-49027 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The activation-induced cytidine deaminase enzyme (AID) is required in germinal center (GC) B cells for somatic hyper-mutation and class switch recombination at the immunoglobulin locus. In GC-B cells, AID is highly expressed, with inherent mutator activity that helps generate antibody diversity. However, AID may also regulate gene expression epigenetically, irrespective of mutator activity, by directly deaminating 5-methylcytosine (5mC) in concert with base excision repair glycosylases to exchange unmethylated cytosine. This pathway promotes gene demethylation, thereby removing epigenetic memory. For example, AID promotes active demethylation of the genome in primordial germ cells. However, the range and mechanism by which AID promotes pluripotency is not known. Different studies have suggested either a requirement or a lack of function for promoting pluripotency in somatic nuclei following fusion with embryonic stem cells (ESC). Here we tested directly whether AID regulates epigenetic memory, by comparing the relative ability of cells lacking AID to reprogram from a differentiated cell type to an induced pluripotent stem cell (iPSC). We show that loss of AID impacts two distinct steps of reprogramming: First, AID-null cells are transiently hyper-responsive to the reprogramming process. Second, although they initiate expression of pluripotency genes, they fail to stabilize the pluripotent state. The genome of AID-null cells remains hypermethylated in reprogramming cells, and hypermethylated genes associated with pluripotency fail to be stably up-regulated. MYC target genes are highly enriched in the set of genes hypermethylated and under-expressed in reprogramming cells lacking AID. Recent studies identified a distinctive late step of reprogramming associated with methylation status. AID appears to regulate this step to stabilize the pluripotent state, removing epigenetic memory to promote expression of secondary pluripotency network genes. Transcriptome sequencing of AID-null tail fibroblasts, wildtype tail fibroblasts, AID-null and wildtype tail fibroblasts reprogrammed for three weeks by ectopic expression of transcription factors Oct4, Sox2, KLf4 and cMyc. Methylation profiling by reduced representation bisulphite seuencing of AID-null tail fibroblasts, wildtype tail fibroblasts, AID-null and wildtype tail fibroblasts reprogrammed for three weeks and AID-null and wildtype clones after three weeks of reprogramming (Picked at two weeks)

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RNA-sequencing of Nedd4+/+ and Nedd4-/- mouse splenic B cells

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