Abstract
Chromatin associated proteins are key regulators of many important processes in the cell. Trypanosoma cruzi, a protozoa flagellate that causes Chagas disease, alternates between replicative and nonreplicative forms accompanied by a shift on global transcription levels and by changes in its chromatin architecture. Here, we investigated the T. cruzi chromatin proteome using three different protocols and compared it between replicative (epimastigote) and nonreplicative (trypomastigote) forms by high-resolution mass spectrometry. More than 2000 proteins were identified and quantified both in chromatin and nonchromatin extracts. Besides histones and other known nuclear proteins, trypanosomes chromatin also contains metabolic (mainly from carbohydrate pathway), cytoskeleton and many other proteins with unknown functions. Strikingly, the two parasite forms differ greatly regarding their chromatin-associated factors composition and amount. Although the nucleosome content is the same for both life forms (as seen by MNase digestion), the remaining proteins were much less detected in nonreplicative forms, suggesting that they have a naked chromatin. Proteins associated to DNA proliferation, such as PCNA, RPA, and DNA topoisomerases were exclusively found in the chromatin of replicative stages. On the other hand, the nonreplicative stages have an enrichment of a histone H2B variant. Furthermore, almost 20% of replicative stages chromatin-associated proteins are expressed in nonreplicative forms, but located at nonchromatin space. We identified different classes of proteins including phosphatases and a Ran-binding protein, that may shuttle between chromatin and nonchromatin space during differentiation. Seven proteins, including those with unknown functions, were selected for further validation. We confirmed their location in chromatin and their differential expression, using Western blotting assays and chromatin immunoprecipitation (ChIP). Our results indicate that the replicative state in trypanosomes involves an increase of chromatin associated proteins content. We discuss in details, the qualitative and quantitative implication of this chromatin set in trypanosome chromatin biology. Because trypanosomes are early-branching organisms, this data can boost our understanding of chromatin-associated processes in other cell types.
Highlights
To evaluate the presence of chromatin and nonchromatin proteins, the fractions obtained by the three protocols were submitted to Western blotting using antibodies to histone H3, a known chromatin protein, and the eukaryotic translation initiation factor 5A a predominant cytosolic protein [29] (Fig. 1B)
T. cruzi is a good model for chromatin study as it alternates between replicative and nonreplicative forms accompanied by a shift on their global transcription levels and changes in their chromatin architecture
One of the most surprising findings is that the nonreplicative form has few proteins in its chromatin and a less diverse protein repertoire
Summary
Almost 20% of replicative stages chromatin-associated proteins are expressed in nonreplicative forms, but located at nonchromatin space. We identified different classes of proteins including phosphatases and a Ran-binding protein, that may shuttle between chromatin and nonchromatin space during differentiation. Seven proteins, including those with unknown functions, were selected for further validation. Chromatin can be divided as euchromatin, composed of less condensed and actively transcribed regions, and heterochromatin, comprising more compacted and silent regions This binary classification is becoming obsolete as meta-analysis of the interaction of proteins with DNA, and histone post-translational mod-. Chromatin-associated Factors of T. cruzi Life Forms ifications (PTMs), indicates a more complex pattern with at least five different chromatin subtypes [2]. It is believed that the lack of the histone H1 globular domain may be associated with a more relaxed chromatin structure observed in trypanosomes
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