Cracking chromatin with proteomics: From chromatome to histone modifications

Abstract

Chromatin is the assembly of genomic DNA and proteins packaged in the nucleus of eukaryotic cells, which together are crucial in regulating a plethora of cellular processes. Histones may be the best known class of protein constituents in chromatin, which are decorated by a range of post-translational modifications to recruit accessory proteins and protein complexes to execute specific functions, ranging from DNA compaction, repair, transcription, and duplication, all in a dynamic fashion and depending on the cellular state. The key role of chromatin in cellular fitness is emphasized by the deregulation of chromatin determinants predisposing to different diseases, including cancer. For this reason, deep investigation of chromatin composition is fundamental to better understand cellular physiology. Proteomic approaches have played a crucial role to understand critical aspects of this complex interplay, benefiting from the ability to identify and quantify proteins and their modifications in an unbiased manner. This review gives an overview of the proteomic approaches that have been developed by combining mass spectrometry-based with tailored biochemical and genetic methods to examine overall protein make-up of chromatin, to characterize chromatin domains, to determine protein interactions, and to decipher the broad spectrum of histone modifications that represent the quintessence of chromatin function.

Isolation of proteins on chromatin (iPOC) reveals signaling pathway-dependent alterations in the DNA-bound proteome

“…In recent years, several methods have been established to capture, identify, and quantify DNA-binding proteins in order to investigate chromatin dynamics in time and space [19]. Among these, incorporation of EdU has been efficiently exploited to characterize the chromatin composition at defined genomic regions[21, 22, 42].…”

“…In addition, separation of chromatin from other cellular organelles is difficult, often resulting in contamination with cytosolic proteins [3, 18]. Yet, several biochemical strategies have been recently developed that effectively combine different means of chromatin purification strategies with mass spectrometry-based protein identification [19]. In this regard, crosslinking strategies to stabilize protein-DNA interactions allow for stringent washing conditions and resulting in higher chromatin purity, e.g.…”

“…To further boost the sensitivity of DNA-bound proteome identification, a variety of strategies have been developed that take advantage of nucleoside analogue integration and click chemistry-based enrichment of DNA. Successful examples are the isolation of proteins on nascent DNA (iPOND) and DNA-mediated chromatin pulldown (Dm-ChP) [19, 21, 22], where alkyne-containing thymidine analogs (e.g. 5-ethynyl-2′-deoxyuridine, EdU) are incorporated into replicating DNA.…”

A chromatin-associated regulator of RNA Polymerase III assembly at tRNA genes revealed by locus-specific proteomics

“…In Epi-Decoder, the local chromatin abundance of each protein in the cell at the barcoded locus of interest can be measured by chromatin immunoprecipitation followed by DNA-barcode sequencing and counting [41][42][43]. This method is sensitive and quantitative, and overcomes common hurdles associated with other proteomics approaches such as capture of a locus combined with mass spectrometry [44][45][46].…”

Unnatural Amino Acid Crosslinking for Increased Spatiotemporal Resolution of Chromatin Dynamics

“…Surveying all crosslinks within a single sample reveals how multiple proteins interact at a defined nucleosomal residue along a chromosomal fiber that is under distinctively different regulation at multiple locally specialized chromatin structures and becomes a notable tool for the identification of histone–protein interactions. A recent review of MS approaches to chromatin studies elegantly details various advances in methodologies specific to histone and PTM dynamics [ 49 ], yet the use of pBPA in these approaches is absent. Utilizing pBPA for protein–protein studies, across numerous species, has been well documented [ 29 , 36 , 50 , 51 , 52 , 53 , 54 ], with a myriad of protein–protein interaction studies explicitly aimed at chromatin dynamics [ 55 ].…”