Chromatin Immunoprecipitation Analysis of Aryl Hydrocarbon Receptor Binding in a Mouse B-cell Line (CH12.LX) Activated with Lipopolysaccharide and Treated with 2,3,7,8-Tetrachlorodibenzo-p-dioxin

ABSTRACT: The aryl hydrocarbon receptor (AHR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters differentiation of B cells and suppresses antibody production. The objectives of this study was to use a combination of whole genome, microarray-based chromatin immunoprecipitation (ChIP-on-chip) and time course gene expression microarray analysis on the mouse B-cell line CH12.LX following exposure to lipopolysaccharide (LPS) or LPS and TCDD to identify the primary and downstream transcriptional elements of B-cell differentiation that are altered by the AHR. CH12.LX cells (1X10^5 cells/ml, 25ml in P150s) were treated with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)(10nM) or vehicle (0.01% DMSO) for 1 hr and cells were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine. Chromatin was isolated by adding lysis buffer followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300-500 bp. Genomic DNA was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a Nanodrop ND-1000 spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin (30 ug) was precleared with protein - agarose beads. Factor-bound DNA sequences were isolated using antibodies against AhR (Biomol, Plymouth Meeting, PA, Catalog #: SA210-0100). After incubation at 4°C overnight, protein-agarose beads were used to isolate the immune complexes. Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Following purification ChIP DNA was labeled, fragmented and hybridized to Affymetrix GeneChip Mouse Tiling 2.0 Array Sets, hybridized arrays were then washed and scanned using a GeneChip Fluidics Station 450 and a GeneChip 3000 scanner. The ChIP studies were performed in triplicate (n = 3) with the cells for each replicate treated and harvested on separate days.

ORGANISM(S): Mus musculus

SUBMITTER: Russell Thomas 

PROVIDER: E-GEOD-23708 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress