Challenges for optimizing chromatin immunoprecipitations studies

Inflammatory bowel disease, including ulcerative colitis, affects millions of Americans with recurring visceral pain and inflammation. Also, with this number increasing drastically each year, it makes it a necessary area of research. The most widely used animal model to mimic colitis is TNBS-induced colitis model where the chemical 2,4,6-trinitrobenzenesulfonic acid (TNBS) in ethanol is intrarectally infused in rat colon. Ethanol degrades the mucosal epithelial barrier allowing entry for TNBS to mount a localized immune response in the colon. The TNBS-induced colitis peaks on day three and appears to subside within a week. Twenty-four hours post-treatment, TNBS-induced colitis results in DNA hypermethylation of CpG dinucleotides in the promoter region of glutaminase (GLS1) gene. GLS1 catalyzes the production of glutamate, an excitatory neurotransmitter that plays a crucial role in neuro-immune pathway. This hypermethylation results in recruitment of methyl CpG binding protein 2 (MeCP2) to hypermethylated CpG dinucleotides. MeCP2 protein has been shown to interact with corepressors to suppress or turn off gene transcription, however, in TNBS-induced colitis recruitment of MeCP2 to GLS1 promoter results in increased gene expression indicating interaction with coactivators rather than corepressors. For this study, we are discussing new challenges in using chromatin immunoprecipitation (ChIP) assay to confirm protein - DNA interactions and identifying interacting proteins by mass spectrometry. Aim: In this current study, we are identifying the challenges in optimizing chromatin immunoprecipitation and proteomics/mass spectrometry.

Method: The key component of the chromatin immunoprecipitation assay is the size of the DNA, which needs to be fragmented in a reliable consistent and repeatable manner. The collected tissue from treated rats were first crosslinked followed by lysing the cells to release the cellular component. We then used benchtop sonicator to fragment the genomic DNA. We also used commercially available enzymes for DNA fragmentation, like MNase and Fragmentase (both from New England Biolabs). These enzymes were used according to manufacturer’s methods. After fragmentation, genomic DNA was extracted and analyzed on 2% agarose gel electrophoresis. Fuurther, we performed the ChIP assay followed by bisulfite conversion and methylation specific PCR to confirm protein-DNA interaction. For MS, we extracted proteins from ChIP assay using either MeCP2 or IgG antibodies. For some samples, we performed PAGE and confirmed the protein bands after pulldown. We are planning to send these samples for MS analysis. 43 12th Annual OSU CHS Research Day Abstract Boo