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

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to preserve both tissue integrity and organization, and, in the last decades, a huge number of biological samples have been archived all over the world following this procedure.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.

Keywords: FFPE tissues; archival samples; cancer epigenetics; chromatin; chromatin immunoprecipitation (ChIP).

Figure 1

Timeline of the FFPE-ChIP procedures and kits introduced over time. Procedures are in rectangles while kits are in ovals. Specific colors are used for each research group and company.

Figure 2

Schematic overview of the main steps shared by the different procedures described in this review. FFPE tissue sections can be collected from the block as a whole or dissected (macro- or micro-) to enrich the starting material of specific tissue components. The sections are deparaffinized and rehydrated prior to be subjected to limited reversal of crosslinking by heat and chromatin extraction and shearing by focused ultrasonication or canonical probe sonication. Then, chromatin should be checked prior to proceed with immunoselection: an aliquot of chromatin is taken and decrosslinked, and the DNA is purified, fluorimetrically quantified, and separated by electrophoresis to evaluate the size of fragments. Chromatin is then immunoselected using antibodies directed against the protein or histone modification of interest. After capture of chromatin–antibody complexes and their washing to remove the nonspecific chromatin binding, selected chromatin is decrosslinked and purified using spin-column strategies. Finally, a preliminary evaluation of the specificity of the immunoselection by real-time qPCR should be performed prior to library preparation and NGS sequencing.

Figure 3

Identification of H3K4me3 control regions for the evaluation of the specificity of the immunoselection by real-time qPCR. Regions known to be enriched or not enriched can be used for antibody titration and to evaluate the specificity of the immunoselected DNA prior to library preparation and sequencing. Snapshots of mouse H3K4me3-enriched (ACTB) and not enriched (COL2A1) gene promoters are shown as example. ChIP-Seq data are from the ENCODE project and were taken from UCSC Genome Browser (http://genome.ucsc.edu (accessed on 7 November 2021)). The sequences we used to amplify as a control of specificity of H3K4me3 experiments on mouse tissues are indicated by the light-red vertical bars.