title: Quantitative Proteomic Analysis of Replicative and Nonreplicative Forms Reveals Important Insights into Chromatin Biology of Trypanosoma cruzi.
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.
authors.affiliation: From the ‡Laboratório Especial de Ciclo Celular - Center of Toxins, Immune-Response and Cell Signaling - CeTICS, Instituto Butantan, São Paulo, SP, 05503-900, Brazil.|§Departamento de Física e Informática, Instituto de Física de São Carlos, Universidade de São Paulo - USP, São Carlos, SP, 13563-120, Brazil.
authors.forename: Teresa Cristina
authors.initials: TC
authors.lastname: Leandro de Jesus
authors.affiliation: From the ‡Laboratório Especial de Ciclo Celular - Center of Toxins, Immune-Response and Cell Signaling - CeTICS, Instituto Butantan, São Paulo, SP, 05503-900, Brazil.|¶Laboratório de Parasitologia, Instituto Butantan, São Paulo, 05503-900, Brazil.
authors.forename: Simone Guedes
authors.initials: SG
authors.lastname: Calderano
authors.affiliation: From the ‡Laboratório Especial de Ciclo Celular - Center of Toxins, Immune-Response and Cell Signaling - CeTICS, Instituto Butantan, São Paulo, SP, 05503-900, Brazil.
authors.forename: Francisca Nathalia de Luna
authors.initials: FN
authors.lastname: Vitorino
authors.affiliation: From the ‡Laboratório Especial de Ciclo Celular - Center of Toxins, Immune-Response and Cell Signaling - CeTICS, Instituto Butantan, São Paulo, SP, 05503-900, Brazil.
authors.forename: Ricardo Pariona
authors.initials: RP
authors.lastname: Llanos
authors.affiliation: From the ‡Laboratório Especial de Ciclo Celular - Center of Toxins, Immune-Response and Cell Signaling - CeTICS, Instituto Butantan, São Paulo, SP, 05503-900, Brazil.
authors.forename: Mariana de Camargo
authors.initials: MC
authors.lastname: Lopes
authors.affiliation: From the ‡Laboratório Especial de Ciclo Celular - Center of Toxins, Immune-Response and Cell Signaling - CeTICS, Instituto Butantan, São Paulo, SP, 05503-900, Brazil.
authors.forename: Christiane Bezerra
authors.initials: CB
authors.lastname: de Araújo
authors.affiliation: §Departamento de Física e Informática, Instituto de Física de São Carlos, Universidade de São Paulo - USP, São Carlos, SP, 13563-120, Brazil.
authors.forename: Otavio Henrique
authors.initials: OH
authors.lastname: Thiemann
authors.affiliation: From the ‡Laboratório Especial de Ciclo Celular - Center of Toxins, Immune-Response and Cell Signaling - CeTICS, Instituto Butantan, São Paulo, SP, 05503-900, Brazil.
authors.forename: Marcelo da Silva
authors.initials: MD
authors.lastname: Reis
authors.affiliation: From the ‡Laboratório Especial de Ciclo Celular - Center of Toxins, Immune-Response and Cell Signaling - CeTICS, Instituto Butantan, São Paulo, SP, 05503-900, Brazil.
authors.forename: Maria Carolina
authors.initials: MC
authors.lastname: Elias
authors.affiliation: From the ‡Laboratório Especial de Ciclo Celular - Center of Toxins, Immune-Response and Cell Signaling - CeTICS, Instituto Butantan, São Paulo, SP, 05503-900, Brazil; julia.cunha@butantan.gov.br.
authors.forename: Julia Pinheiro
authors.identifier: 0000-0003-0466-8879
authors.initials: JP
authors.lastname: Chagas da Cunha
mesh_terms: D002460:Cell Line; D002843:Chromatin / Q000378:metabolism*; D002853:Chromatography, Liquid; D006801:Humans; D008018:Life Cycle Stages; D040901:Proteomics / Q000379:methods*; D015800:Protozoan Proteins / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D014349:Trypanosoma cruzi / Q000378:metabolism / Q000502:physiology*
chemical_list: D002843:Chromatin; D015800:Protozoan Proteins