Investigating pathological epigenetic aberrations by epi-proteomics

(2022) 14:145 Robusti et al. Clinical Epigenetics https://doi.org/10.1186/s13148-022-01371-y Open Access REVIEW Investigating pathological epigenetic aberrations by epi‑proteomics Giulia Robusti1, Alessandro Vai1, Tiziana Bonaldi1,2† and Roberta Noberini1* Abstract Epigenetics includes a complex set of processes that alter gene activity without modifying the DNA sequence, which ultimately determines how the genetic information common to all the cells of an organism is used to generate different cell types. Dysregulation in the deposition and maintenance of epigenetic features, which include histone posttranslational modifications (PTMs) and histone variants, can result in the inappropriate expression or silencing of genes, often leading to diseased states, including cancer. The investigation of histone PTMs and variants in the context of clinical samples has highlighted their importance as biomarkers for patient stratification and as key players in aberrant epigenetic mechanisms potentially targetable for therapy. Mass spectrometry (MS) has emerged as the most powerful and versatile tool for the comprehensive, unbiased and quantitative analysis of histone proteoforms. In recent years, these approaches—which we refer to as “epi-proteomics”—have demonstrated their usefulness for the investigation of epigenetic mechanisms in pathological conditions, offering a number of advantages compared with the antibody-based methods traditionally used to profile clinical samples. In this review article, we will provide a critical overview of the MS-based approaches that can be employed to study histone PTMs and variants in clinical samples, with a strong focus on the latest advances in this area, such as the analysis of uncommon modifications and the integration of epi-proteomics data into multi-OMICs approaches, as well as the challenges to be addressed to fully exploit the potential of this novel field of research. Keywords: Cancer, Epigenetics, Histone-modifying enzyme, Histone posttranslational modification, Histone variant, Mass spectrometry, Proteomics Background Epigenetics includes a complex set of processes that alter gene activity without modifying the DNA sequence, which ultimately defines cell fate by determining how shared genetic information is used to generate different phenotypes. Histones are part of the epigenetic machinery and contribute to two fundamental nuclear functions: DNA compaction and regulation of gene expression. Histones are small, basic proteins characterized by a † Senior author: Tiziana Bonaldi *Correspondence: 1 Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, 20139 Milan, Italy Full list of author information is available at the end of the article C-terminal globular domain and an N-terminal tail. In the nucleus of eukaryotic cells, they are bound to DNA to form the nucleosome, the basic unit of the chromatin. Around 146 bp of DNA are wrapped around the so-called core histone octamer that consists of two copies of histone H2A and H2B, and a dimer of histone H3 and H4, while a linker histone H1 contributes to chromatin stabilization by binding the nucleosome and the linker DNA present between nucleosomes [1]. In addition to the canonical forms, variants of core and linker histones exist and play a role in the regulation of chromatin structure and gene expression [2]. Histones are decorated by a number of posttranslational modifications (PTMs), which occur mainly at their N-terminal tails and include methylation, acylation (the most abundant © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Robusti et al. Clinical Epigenetics (2022) 14:145 of which is mono-acetylation), phosphorylation, ubiquitylation, ADP-ribosylation, SUMOylation, deamination, as well as other less common modifications [3–5]. Histones contribute to DNA packaging within the nucleus, and thanks to the presence of different combinations of PTMs and variants, they contribute to the regulation of gene expression and cell fate. Histone PTMs are deposed and removed by a group of enzymes collectively known as histone-modifying enzymes (HMEs) and exert their downstream effects by binding to effector proteins called “readers” [6]. In addition, histone chaperones influence histone levels by transporting newly synthesized histones to specific sites in the genome [7]. Aberrations in the patterns of histone PTMs and variants can result in the inappropriate expression of genes, which causes altered transcript, protein and metabolite levels, ultimately leading to aberrant phenotypes (Fig. 1). In the last decade, histone PTMs and variants have been investigated in a wide range of human diseases, including cancer, neurodegenerative diseases, heart failure, as well as autoimmune and infectious diseases. While genetic defects, such as mutations, deletions, or copy number changes, have been long considered the major contributors to cancer development and progression, epigenetics has emerged as an important player in various cancer-related processes [8]. For instance, the loss of H3K14ac, H4K20me3 and H4K16ac was reported as a common hallmark of cancer [9, 10], while other modifications—including acetylation, H3K4me2, H3K9me3, H3K27me3—or combinations of modifications, correlate with cancer patient prognosis [11], with effects that are context dependent, and can even go in opposite directions depending on the specific cancer type [12]. Histone PTMs also have diagnostic potential, particularly when measured from circulating nucleosomes, which are released in the blood following cell death and apoptosis [13], as demonstrated by studies detecting histone PTM patterns specific to the cancerous state in pancreatic and colorectal cancers [14, 15]. In addition to cancer, epigenetic modifications are emerging to have a key role in the development of other diseases. Histone PTMs have been described in the development of neurodegenerative disorders, cha (...truncated)