Epigenomic profiling of archived FFPE tissues by enhanced PAT-ChIP technology and in vivo decoding of the aging-associated epigenetic drift and possible role of caloric restriction

In the last 25 years, chromatin immunoprecipitation (ChIP) has become a powerful experimental approach to better understand the role of epigenetic modifications. Developed in the 1980s by Gilmour and Lis, chromatin immunoprecipitation makes possible the study of histone post-translational modifications (HPTMs) or other chromatin-associated proteins (e.g., transcription factors) to better understand their crucial role in reversible chromatin remodelling and regulation of gene expression. Coupled with next-generation sequencing, chromatin-immunoprecipitation (ChIP-sep) allows the mapping of HPTMs over the entire genome unrevealing the so-called “epigenome”. Recent studies indicate that alterations of the epigenome is one of the most important hallmark of aging process, attracting much interest due the potential reversibility of epigenetic marks that makes them promising therapeutic targets to delay or minimize age-related diseases and potentially extend lifespan. Although the majority of ChIP studies have been conducted on cultured cells with several limitations, the main being the alteration of the epigenetic profile in consequence of the adaptation of cells to tissue culture conditions. However, in 2010 Fanelli and colleagues introduced a modified version of ChIP, named pathology tissue-chromatin immunoprecipitation (PAT-ChIP), that allows chromatin extraction and immunoprecipitation from formalin-fixed and paraffin-embedded (FFPE) tissues. Formalin fixation followed by embedding in paraffin is the most cost-effective and simple method used to storage biopsy specimens in different therapeutics areas such as oncology. By extending the application of chromatin studies to clinical patient samples, PAT-ChIP makes possible epigenomic studies in a vast number of clinically annotated tissues stored in pathology archives, providing an unprecedented opportunity to understand the epigenetic mechanisms underlying genome activity in progression of several human pathologies including aging-associated diseases. However, due to the lack of standardization in the formalin fixation procedure, many FFPE tissue specimens stored in hospital archives or in tissue banks result heavily crosslinked and thus not suitable for genome- wide chromatin immunoprecipitation studies. To overcome this problem, part of my Ph.D activity was dedicated to the improvement of PAT-ChIP to allow histone epigenomic studies also using “complex” biological samples. Thanks to these studies, a new procedure, called EPAT - ChIP, has been developed and recently published in “Clinical Epigenetics” journal (Amatori-Persico et al., 2018). At the same time, in collaboration with the European Institute of Oncology of Milan, we exploited the already set PAT-ChIP-seq protocol to characterize four different epigenomic landscapes during aging process in mice livers and the possible effects of caloric restriction on them. The first part of this work provides a detailed overview of all the procedures used to improve the original PAT-ChIP protocol, while the second part describes the global characterization of aging-associated epigenetic landscapes and the effects induced by CR.