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AID-ChIP-seq Analysis of diffuse large B-cell lymphoma

ABSTRACT: Purpose: The goals of this study are to identify AID targets in DLBCL and investigate the overlapping of AID peaks and predicted G4 forming sequences (PQS) and the B-cell super enhancer (SE) U2932 DLBCL cells obtained from the American Type Culture Collection were grown in RPMI 1640 medium (Corning) supplemented with 10% FBS (Atlanta Biiologicals) and 1% Pen/Strep (Gibco). For each immunoprecipitation reaction, 4x106 chromatin cell equivalent were incubated overnight with either an AID antibody (Abcam, ab59361) or a rabbit IgG negative control antibody, subjected to protein A/G magnetic bead binding, IP elution and DNA recovery. The ChIP DNA was used to generate ChIPseq libraries with the ChIP-Seq sample preparation kit (Illumina) for sequencing at the Arkansas Children’s Research Institute Genomics Core. The sequence reads that passed quality filters were aligned to the human genome (hg19) using BWA-MEM and peakcalled using MACS2. Results: Using an optimized data analysis workflow, we mapped about 10-20 million sequence reads per sample to the human genome (build hg19) and identified 4,573 AID peaks which were filtered out if they overlap with the mock pull-down peaks and the blacklist (ENCODE blacklisted regions). We observed the intersection of AID peaks with PQS and SEs are significantly higher than expected by chance in all cases of comparisons supporting that the number of AID peaks sharing PQS and/or SEs is not a random event. Conclusions: Sequencing analyses reveal AID targets in both activated B-cells and lymphoma are highly enriched for G4 elements and validation studies confirm a representative set of these sequences form characteristic, stable G4 structures.

ORGANISM(S): Homo sapiens

PROVIDER: GSE146695 | GEO | 2020/04/08

REPOSITORIES: GEO

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Project description:The human Werner and Bloom syndromes (WS and BS) are caused by deficiencies in the WRN and BLM RecQ helicases, respectively. WRN, BLM and their S. cerevisiae homologue Sgs1, are particularly active in vitro in unwinding G-quadruplex DNA (G4-DNA), a family of non-canonical nucleic acid structures formed by certain G-rich sequences. Recently, mRNA levels from loci containing potential G-quadruplex-forming sequences (PQS) were found to be preferentially altered in sgs1 mutants, suggesting that G4-DNA targeting by Sgs1 directly affects gene expression. Here, we extend these findings to human cells. Using microarrays to measure mRNAs obtained from human fibroblasts deficient for various RecQ family helicases, we observe significant associations between loci that are upregulated in WS or BS cells and loci that have PQS. No such PQS associations were observed for control expression datasets, however. Furthermore, upregulated genes in WS and BS showed no or dramatically reduced associations with sequences similar to PQS but that have considerably reduced potential to form intramolecular G4-DNA. These findings indicate that, like Sgs1, WRN and BLM can regulate transcription globally by targeting G4-DNA. Cell culture conditions and media Human fibroblast cell strains (WS: AG05229, AG12795, AG12797; BS: GM02932, GM03402, GM16891; RTS: AG18371, AG18375, AG05013; Normal/Wild-type: AG04054, AG06310, AG09975) were obtained from the Coriell Repository (Camden, NJ), from donors matched for gender and of similar ages, and were at similar passage levels. Cells were cultured in MEM supplemented with Earle’s salts, 20% fetal bovine serum, 1x penicillin/streptomycin, and 1x fungizone in 3% O2 at 37oC and harvested for RNA extraction during active growth and at ~ 80% confluence. GeneChip microarray expression Total RNA from the 12 fibroblast cell strains was isolated by extraction with TRIzol (Invitrogen) and purified using the RNeasy system (Qiagen). Total RNA was amplified by in vitro transcription using the Ovation RNA Amplification System V2 (NuGen). The resultant cDNA was fragmented and labeled using the FL-Ovation cDNA Biotin Module V2 (NuGen), and then purified using QIAquick columns (Qiagen), as specified by the Ovation System manual. Labeled probe was hybridized to Affymetrix U133A 2.0 GeneChips, and ultimately scanned using an Axon GenePix array scanner. Statistical analysis of microarray expression experiment The output files were normalized by Robust Multiarray Average (RMA), using the R package GCRMA and gene expression levels were log2-transformed.

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AID-ChIP-seq Analysis of diffuse large B-cell lymphoma

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