Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Abstract

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of a wide range of transcription factors (TFs) and cofactors mediating transcriptional activation or repression via changes in chromatin structure. Chromatin immunoprecipitation (ChIP) is a useful molecular biology approach for mapping histone modifications and profiling transcription factors/cofactors binding to DNA, thus providing a snapshot of the dynamic nuclear changes occurring during different biological processes. To study transcriptional regulation in adipose tissue, samples derived from in vitro cell cultures of immortalized or primary cell lines are often favored in ChIP assays because of the abundance of starting material and reduced biological variability. However, these models represent a limited snapshot of the actual chromatin state in living organisms. Thus, there is a critical need for optimized protocols to perform ChIP on adipose tissue samples derived from animal models. Here we describe a protocol for efficient ChIP-seq of both histone modifications and non-histone proteins in brown adipose tissue (BAT) isolated from a mouse. The protocol is optimized for investigating genome-wide localization of proteins of interest and epigenetic markers in the BAT, which is a morphologically and physiologically distinct tissue amongst fat depots.

Egr1 mediates the effect of insulin on leptin transcription in adipocytes

“…Purified DNA eluates were analyzed by qPCR according to Cardamone et al (40), using the primers 5Ј-TCCCGCCCCA-CCGCTAGCGAGCTC-3Ј (forward) and 5Ј-GAGCTCGCTA-GCGGTGGGGCGGGA-3Ј (reverse) to detect the Egr1 binding site. Dilutions of total lysates (1:10, 1:100, and 1:1,000) were amplified by PCR to form a standard reference curve.…”

FoxK1 associated gene regulatory network in hepatic insulin action and its relationship to FoxO1 and insulin receptor mediated transcriptional regulation

“…Cell culture media was aspirated, and the cells were washed with ice-cold PBS and cells were scrapped, and performed ChIP using FoxK1 antibody (ab18196) as described by Cardamone et al. [ 20 ]. Three biological replicates were used for the ChIP.…”

“…60 h after transfection, cells were serum starved in EMEM containing 0.1% BSA overnight, and transfected cells were stimulated with 100 nM insulin for 20 min. ChIP assays for FoxK1 were performed as described previously [ 20 ].…”