Enhanced PAT-ChIP (EPAT-ChIP), a new procedure for reliable genome-wide studies in clinical FFPE tissues

Chromatin immunoprecipitation (ChIP) has allowed the study of protein-DNA interactions in chromatin, bringing new insights into the role of histone post-translational modifications (HPTMs) in cancer and other diseases. The coupling of ChIP with next-generation sequencing (NGS) techniques has revealed the distribution of HPTMs over the entire genome allowing the identification of altered epigenetic profiles in pathological conditions. However, until recently, these studies have been limited to cultured cells or fresh/frozen tissues. The development of a new technique named PAT-ChIP has enabled chromatin studies in formalin-fixed paraffin-embedded (FFPE) tissues, opening the doors to a huge number of samples stored in pathology archives and related clinical information. However, chromatin studies in FFPE archival samples can be hindered by the extensive formalin fixation to which these tissues are routinely subjected. Here, we describe enhanced PAT-ChIP (EPAT-ChIP), a new ChIP procedure that facilitates epigenomic studies in archival FFPE tissues. EPAT-ChIP improves the extraction of soluble chromatin, allowing the study of multiple histone marks from limited amounts of starting material. In addition, EPAT-ChIP assures higher yields of final immunoselected DNA, especially when poorly represented histone marks (e.g. narrow peaks of H3K4me3) are investigated, and the consequent production of reliable NGS libraries. In this procedure, FFPE samples are subjected to heat-mediated limited reversal of crosslinking (LRC) which predisposes the tissue to better release soluble chromatin after sonication without altering the antigenic potential of its biomolecules. After investigating differentially-fixed normal colon specimens specifically produced for this study, ePAT-ChIP was successfully used to study three different HPTMs (H3K4me3, K3K27me3, H3K27ac) at genome-wide level in an archival invasive breast carcinoma (IBC) sample. Our newly designed method EPAT-ChIP was compared with standard PAT-ChIP for validation. Both methods were applied in parallel to the genome-wide study of the low abundant histone mark H3K4me3, known to be associated with active gene promoters, in an archival IBC sample. Then, the comparison between standard PAT-ChIP and EPAT-ChIP was extended to other functionally different histone marks, such as H3K27ac, known to be associated with active promoters and enhancers, and H3K27me3, which characterizes silenced genomic regions.