The Current State of Chromatin Immunoprecipitation (ChIP) from FFPE Tissues.

Stefano Amatori,Mirco Fanelli

doi:10.3390/ijms23031103

Abstract

Cancer cells accumulate epigenomic aberrations that contribute to cancer initiation and progression by altering both the genomic stability and the expression of genes. The awareness of such alterations could improve our understanding of cancer dynamics and the identification of new therapeutic strategies and biomarkers to refine tumor classification and treatment. Formalin fixation and paraffin embedding (FFPE) is the gold standard to preserve both tissue integrity and organization, and, in the last decades, a huge number of biological samples have been archived all over the world following this procedure. Recently, new chromatin immunoprecipitation (ChIP) techniques have been developed to allow the analysis of histone post-translational modifications (PTMs) and transcription factor (TF) distribution in FFPE tissues. The application of ChIP to genome-wide chromatin studies using real archival samples represents an unprecedented opportunity to conduct retrospective clinical studies thanks to the possibility of accessing large cohorts of samples and their associated diagnostic records. However, although recent attempts to standardize have been made, fixation and storage conditions of clinical specimens are still extremely variable and can affect the success of chromatin studies. The procedures introduced in the last few years dealt with this problem proponing successful strategies to obtain high-resolution ChIP profiles from FFPE archival samples. In this review, we compare the different FFPE-ChIP techniques, highlighting their strengths, limitations, common features, and peculiarities, as well as pitfalls and caveats related to ChIP studies in FFPE samples, in order to facilitate their application.

Highlights

Chromatin in eukaryotes is a finely organized nuclear complex of genomic DNA, histones, and non-histone proteins
Chromatin function is mainly regulated by histone post-translational modifications (PTMs), which consist of enzyme-mediated chemical modifications of specific histone residues, among which those targeting the N-terminal tail of histones seem to play a major regulatory role
We aim to examine the ways through which different research groups and companies have dealt with chromatin extraction and immunoprecipitation from Formalin fixation and paraffin embedding (FFPE) tissues, while providing recommendations/guidelines and practical examples to improve the success of chromatin studies in such intriguing samples

Summary

Introduction

Chromatin in eukaryotes is a finely organized nuclear complex of genomic DNA, histones, and non-histone proteins. ChIP consists of the isolation of chromatin fragments from a biological matrix and the consequent immunoselection of a protein of interest to identify the genomic loci associated with it [9,10] In this technique, the starting material (cultured cells of fresh/frozen tissues) is normally fixed by formaldehyde, and chromatin is extracted and fragmented by controlled sonication. Formalin fixation followed by paraffin embedding (FFPE) is the standard method for long-term preservation of most archived pathological specimens These samples allow the pathological evaluation of tissue histology by immunohistochemistry (IHC) and immunofluorescence (IF), while maintaining the possibility to isolate high-quality DNA and RNA [17,18,19,20,21]. We aim to examine the ways through which different research groups and companies have dealt with chromatin extraction and immunoprecipitation from FFPE tissues, while providing recommendations/guidelines and practical examples to improve the success of chromatin studies in such intriguing samples

The Development of FFPE-ChIP Techniques over Time

Chromatin Extraction from FFPE Tissues

Chromatin Immunoselection

DNA Analysis and Experimental Controls

A Novel Enzyme-Tethering Strategy

Conclusions and Future Prospects