Proteomics of a fuzzy organelle: interphase chromatin

Abstract

Chromatin proteins mediate replication, regulate expression, and ensure integrity of the genome. So far, a comprehensive inventory of interphase chromatin has not been determined. This is largely due to its heterogeneous and dynamic composition, which makes conclusive biochemical purification difficult, if not impossible. As a fuzzy organelle, it defies classical organellar proteomics and cannot be described by a single and ultimate list of protein components. Instead, we propose a new approach that provides a quantitative assessment of a protein's probability to function in chromatin. We integrate chromatin composition over a range of different biochemical and biological conditions. This resulted in interphase chromatin probabilities for 7635 human proteins, including 1840 previously uncharacterized proteins. We demonstrate the power of our large-scale data-driven annotation during the analysis of cyclin-dependent kinase (CDK) regulation in chromatin. Quantitative protein ontologies may provide a general alternative to list-based investigations of organelles and complement Gene Ontology.

A Quantitative Proteomic Analysis of In Vitro Assembled Chromatin

“…Although TriC/CCT has been suggested to be mainly involved in the folding of newly translated proteins (47), there is increasing evidence that it has additional functions such as the prevention of the aggregation of poly-Q proteins (48) or the formation of macromolecular protein complexes in the nucleus (49,50). The TriC/CCT complex can also be observed on nascent and mature chromatin in vivo (13) and in interphase chromatin (12), which also supports its integral role in chromatin dynamics and metabolism. The function of these chaperones in chromatin has so far been enigmatic and no clear function was assigned to them.…”

“…To do this, we compared our results with the most comprehensive data on the composition of nascent chromatin, which was measured by Alabert et al (13). As the nascent chromatin capture assay was done in a human cell line (HeLa) we first determined the number of chromatin associated Drosophila proteins that contained a unique human orthologue using the BioMart algorithm (12). 216 proteins (out of 480) that specifically assembled onto chromatin had a clear orthologue in the human proteome and 171 of them (79%) were also detected as chromatin associated in either nascent or mature chromatin (13).…”

“…A similar assembly pathway is also observed in an in vitro assembly system where the process of histone deposition and chromatin contraction occurs within 30 s (16,17). Regardless of this apparent rapid compaction, it takes much longer for new chromatin to become indistinguishable from the bulk chromatin in vivo (9, 13).Recent systematic studies revealed that mature chromatin adopts a complex molecular structure containing a large variety of binding factors that go way beyond a simple aggregate of DNA and histones (11,12,18,19). This observation raises the question of how this structure is assembled, in which order individual factors bind to the DNA, whether distinct intermediates during chromatin assembly exist and which key players mediate chromatin maturation.…”

“…Recent systematic studies revealed that mature chromatin adopts a complex molecular structure containing a large variety of binding factors that go way beyond a simple aggregate of DNA and histones (11,12,18,19). This observation raises the question of how this structure is assembled, in which order individual factors bind to the DNA, whether distinct intermediates during chromatin assembly exist and which key players mediate chromatin maturation.…”

La chromatine naissante révélée

“…Filtrage bio-informatique afin d'augmenter la valeur prédictive des résultats générés par NCC Bien que le protocole de NCC ait été optimisé pour limiter au maximum le bruit de fond inhérent aux analyses à grande échelle de spectrométrie de masse [14], une partie des 3 995 facteurs identifiés sont associés à la chromatine de manière non spécifique. matine mature) (Figure 1) [12].…”