Genome-Wide Chromatin Analysis of FFPE Tissues Using a Dual-Arm Robot with Clinical Potential

Abstract

Although chromatin immunoprecipitation and next-generation sequencing (ChIP-seq) using formalin-fixed paraffin-embedded tissue (FFPE) has been reported, it remained elusive whether they retained accurate transcription factor binding. Here, we developed a method to identify the binding sites of the insulator transcription factor CTCF and the genome-wide distribution of histone modifications involved in transcriptional activation. Importantly, we provide evidence that the ChIP-seq datasets obtained from FFPE samples are similar to or even better than the data for corresponding fresh-frozen samples, indicating that FFPE samples are compatible with ChIP-seq analysis. H3K27ac ChIP-seq analyses of 69 FFPE samples using a dual-arm robot revealed that driver mutations in EGFR were distinguishable from pan-negative cases and were relatively homogeneous as a group in lung adenocarcinomas. Thus, our results demonstrate that FFPE samples are an important source for epigenomic research, enabling the study of histone modifications, nuclear chromatin structure, and clinical data.

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

“…A further procedure, named RCRA ChIP-seq, was published a few months ago by Kaneko and colleagues (Figure 1 and Table 1) [27]; again, a heating step (1 h at 65 • C followed by 30 min at 90 • C) was proposed to efficiently extract, through a focused ultrasonicator, high-quality chromatin from archival lung adenocarcinoma samples. Interestingly, the procedure was applied through a dual-arm robot to 69 samples, being the first attempt to automate ChIP-seq studies from FFPE samples.…”

“…When the tissue component of interest is not homogeneous, tissue sections can be macrodissected or subjected to laser capture microdissection (LCM). Most procedures make use of slides of 5-10 µm thickness, and they have been demonstrated to also be compatible with LCM [23,24,26,27,29]. Histological analysis plays an important role in the success of a study since high and constant purity allows obtaining robust data for final computational analysis.…”

“…Best results are obtained when most of the fragments range between 200 and 1000 bp, since lower-size fragments normally indicate high degradation of the sample (due to excessive sonication and/or poor quality of the starting material), while higher-size fragments are normally lost during library preparation and could reduce the final resolution of the assay. Many of the FFPE-ChIP procedures herein described agree to evaluate the fragmentation of the extracted chromatin before proceeding with the immunoselection [22,24,25,27].…”

“…In 2010, our research group was the first to propose a technique that allows epigenomic studies in FFPE tissues [22]. From that date on, several other techniques (including an upgrade of our first procedure) have been developed with the intent of improving chromatin profiling in FFPE tissues, underlining the importance of demonstrating the applicability of the ChIP procedure by using FFPE samples, paving the way to the development of this new area of research [23][24][25][26][27][28]. In particular, the main hurdle to overcome is the difficulty to reproducibly extract high-quality chromatin from FFPE samples, mainly as a result of the extended and variable level of formalin fixation, as well as the different conditions and timing of sample storage.…”

Epigenetic Mechanisms Underlying COVID-19 Pathogenesis

“…The three basic systems of epigenetic regulation are DNA methylation of gene regulatory regions; histone protein modifications such as methylation, acetylation, phosphorylation, and sumoylation; and non-coding RNAs [ 15 ]. Many techniques for epigenetics analysis have already been developed, and this field is steadily undergoing technological innovation [ 15 , 28 , 29 ]. In this review, we present the latest findings on the importance of epigenetics in the mechanisms of human infection with SARS-CoV-2 and pathogenesis of COVID-19, and discuss future diagnostic and therapeutic strategies for COVID-19 targeting epigenetics.…”