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 and primary pre-adipocytes, with the exception of several in vivo studies2,3,4,5.
Regulation of specific gene expression programs through transcriptional regulation is achieved by coordinated changes in chromatin structure via various transcription factors and co-factors actions. Chromatin immunoprecipitation (ChIP) is a valuable molecular biology approach for investigating the recruitment of these factors to DNA and for profiling the associated changes in the chromatin landscape. Key factors for the success of ChIP experiments include optimizations of crosslinking conditions and chromatin shearing consistency throughout different samples, availability of adequate starting material, and, most notably, quality of the antibodies. When performing ChIP from whole tissues, it is also important to consider heterogeneity of the samples and optimize the protocol to improve efficiency of nuclei isolation, with the latter being a particularly sensitive step when working with adipose tissue due to the elevated lipid content. In fact, molecular isolation techniques from whole adipose depots are complicated by the presence of high levels of triglycerides, and protocols must be optimized to increase the amount of chromatin isolation. Finally, when high-throughput sequencing is performed after ChIP-DNA isolation, the sequencing depth is critical for determining the number of peaks that are confidently detected.
Here, we refer to the working standards and general guidelines for ChIP-seq experiments recommended by the ENCODE and modENCODE consortia6 for best practices, and we focus on a step-by-step description of a protocol optimized for ChIP-seq from BAT. The described protocol allows for efficient isolation of chromatin from adipose tissue to perform genome-wide sequencing for DNA-binding factors with well-defined peaks as well as histone marks with more diffuse signals.
Protocol
Representative Results
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
| 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 |
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