Research Article: Chromatin immunoprecipitation improvements for the processing of small frozen pieces of adipose tissue

Date Published: February 14, 2018

Publisher: Public Library of Science

Author(s): Daniel Castellano-Castillo, Pierre-Damien Denechaud, Isabel Moreno-Indias, Francisco Tinahones, Lluis Fajas, María Isabel Queipo-Ortuño, Fernando Cardona, Hironori Waki.

http://doi.org/10.1371/journal.pone.0192314

Abstract

Chromatin immunoprecipitation (ChIP) has gained importance to identify links between the genome and the proteome. Adipose tissue has emerged as an active tissue, which secretes a wide range of molecules that have been related to metabolic and obesity-related disorders, such as diabetes, cardiovascular failure, metabolic syndrome, or cancer. In turn, epigenetics has raised the importance in discerning the possible relationship between metabolic disorders, lifestyle and environment. However, ChIP application in human adipose tissue is limited by several factors, such as sample size, frozen sample availability, high lipid content and cellular composition of the tissue. Here, we optimize the standard protocol of ChIP for small pieces of frozen human adipose tissue. In addition, we test ChIP for the histone mark H3K4m3, which is related to active promoters, and validate the performance of the ChIP by analyzing gene promoters for factors usually studied in adipose tissue using qPCR. Our improvements result in a higher performance in chromatin shearing and DNA recovery of adipocytes from the tissue, which may be useful for ChIP-qPCR or ChIP-seq analysis.

Partial Text

Epigenetic regulation, generally histone marks and DNA methylation, is a regulatory process which depends on stochastic and environmental stimuli; while DNA methylation is a process usually associated with gene repression that, in mammals, occurs mainly at cytosine residues located within CpG islands, histone marks can exert an activation or repression effect depending on the type of chemical modification and on which residue is modified [1,2].

Here, we have shown for the first time a complete ChIP method for the study of small pieces of frozen AT. We have introduced crucial changes to the standard ChIP protocol, improving the homogenization, fixation and de-crosslinking steps, allowing enough immunoprecipited material to be obtained to perform further steps, as we demonstrated by testing H3K4me3 modifications. Thus, we have shown that the use of only 100 mg of frozen AT is enough for ChIP tests, which will help to advance knowledge about epigenetic marks of AT and their significance for metabolic homeostasis.

 

Source:

http://doi.org/10.1371/journal.pone.0192314

 

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