Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Summary

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in vivo.

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.

Introduction

While white adipose tissue (WAT) is specialized for energy storage, brown adipose tissue (BAT) dissipates energy in the form of heat due to its ability to convert carbohydrates and lipids into thermal energy via mitochondrial uncoupling¹. Because of this specialized function, the BAT depot is required for maintenance of body temperature in physiological conditions and in response to cold exposure. While gene expression changes during BAT differentiation and upon thermogenic stress have been extensively studied in vivo and in vitro, the molecular mechanisms underlying these changes have been mostly dissected in immortalized cell lines a....

Protocol

The animal handling steps of the protocol have been approved by Boston University’s Institutional Animal Care and Use Committee (IACUC).

1. Day 1: Dissection and Preparation of BAT for Chromatin Immunoprecipitation (ChIP)

1. Euthanize mice using a carbon dioxide (CO₂) chamber, and perform the dissection immediately afterwards. Spray mouse fur with 70% ethanol before incision. Place the mouse with its back facing up, then cut open the skin along the neck. Locate the BAT directly under the skin between the shoulders (interscapular; it appears as two lobes, butterfly-shaped, with a thin layer of white fat that needs t....

Results

Figure 1: ChIP validation by qPCR. ChIP-qPCR analysis of representative GPS2 target genes NDUFV1 (left) and TOMM20 (right) in the BAT of WT and GPS2-AKO mice, showing relative changes in the level of H3K9 methylation and GPS2 and Pol2 binding. The bar graphs represent the sample mean of 3 replicates with *p < 0.05 and **p < 0.01 as calculated with Student'.......

Discussion

The protocol described here represents a valuable tool for performing ChIP from murine tissues, specifically optimized for brown adipose tissue. One of the greater challenges in performing ChIP from tissue is recovering a sufficient number of cells during sample preparation. Shearing the BAT using a tissue homogenizer blender coupled with stainless steel beads instead of a canonical glass pestle significantly reduces the number of cells lost due to unbroken tissue. Moreover, homogenizing the tissue directly in a hypotoni.......

Materials

Name	Company	Catalog Number	Comments
Bullet Blender Tissue Homogenizer	Next Advence	BBX24
Stainless Steel Beads 3.2mm Diameter	Next Advence	SSB32
Bioruptor Sonicator	Diagenode
1.5 ml Micro Tube TPX Plastic	Diagenode	C30010010-5
Complete-Protease inhibitor	Roche	11836145001
Protein A Agarose Slurry	Invitrogen	101041
GPS2 antibody	In house		Rabbit polyclonal, Ct antibody (Cardamone et al., Mol Cell 2018)
Pol2 antibody	Diagenode	C15100055
h3K9me3 antibody	Millipore	05-1242
Fast Syber Green Master Mix	Aplied Biosytem	4385612
ViiA7	Aplied Biosytem
TruSeq ChIP Library Preparation Kit	Illumina	IP-202-1012
HiSeq 2000	Illumina