Abstract:The anti-silencing function protein 1 (Asf1) is a chaperone that forms a complex with histones H3 and H4 facilitating dimer deposition and removal from chromatin. Most eukaryotes possess two different Asf1 chaperones but their specific functions are still unknown. Trypanosomes, a group of early-diverged eukaryotes, also have two, but more divergent Asf1 paralogs than Asf1 of higher eukaryotes. To unravel possible different functions, we characterized the two Asf1 proteins in Trypanosoma brucei. Asf1A is mainly… Show more
“…For example, we noticed the downregulation in the MT of the T. cruzi homolog of the Anti-Silencing Function protein (ASF1) (Additional file 4 ). This protein acts as a nucleosome assembly factor and as such would not be necessary in the non replicative infective stage [ 38 ]. Figure 7 Ribosomal proteins (RP) genes decrease their TE upon differentiation.…”
BackgroundDue to the absence of transcription initiation regulation of protein coding genes transcribed by RNA polymerase II, posttranscriptional regulation is responsible for the majority of gene expression changes in trypanosomatids. Therefore, cataloging the abundance of mRNAs (transcriptome) and the level of their translation (translatome) is a key step to understand control of gene expression in these organisms.ResultsHere we assess the extent of regulation of the transcriptome and the translatome in the Chagas disease causing agent, Trypanosoma cruzi, in both the non-infective (epimastigote) and infective (metacyclic trypomastigote) insect’s life stages using RNA-seq and ribosome profiling. The observed steady state transcript levels support constitutive transcription and maturation implying the existence of distinctive posttranscriptional regulatory mechanisms controlling gene expression levels at those parasite stages. Meanwhile, the downregulation of a large proportion of the translatome indicates a key role of translation control in differentiation into the infective form. The previously described proteomic data correlate better with the translatomes than with the transcriptomes and translational efficiency analysis shows a wide dynamic range, reinforcing the importance of translatability as a regulatory step. Translation efficiencies for protein families like ribosomal components are diminished while translation of the transialidase virulence factors is upregulated in the quiescent infective metacyclic trypomastigote stage.ConclusionsA large subset of genes is modulated at the translation level in two different stages of Trypanosoma cruzi life cycle. Translation upregulation of virulence factors and downregulation of ribosomal proteins indicates different degrees of control operating to prepare the parasite for an infective life form. Taking together our results show that translational regulation, in addition to regulation of steady state level of mRNA, is a major factor playing a role during the parasite differentiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1563-8) contains supplementary material, which is available to authorized users.
“…For example, we noticed the downregulation in the MT of the T. cruzi homolog of the Anti-Silencing Function protein (ASF1) (Additional file 4 ). This protein acts as a nucleosome assembly factor and as such would not be necessary in the non replicative infective stage [ 38 ]. Figure 7 Ribosomal proteins (RP) genes decrease their TE upon differentiation.…”
BackgroundDue to the absence of transcription initiation regulation of protein coding genes transcribed by RNA polymerase II, posttranscriptional regulation is responsible for the majority of gene expression changes in trypanosomatids. Therefore, cataloging the abundance of mRNAs (transcriptome) and the level of their translation (translatome) is a key step to understand control of gene expression in these organisms.ResultsHere we assess the extent of regulation of the transcriptome and the translatome in the Chagas disease causing agent, Trypanosoma cruzi, in both the non-infective (epimastigote) and infective (metacyclic trypomastigote) insect’s life stages using RNA-seq and ribosome profiling. The observed steady state transcript levels support constitutive transcription and maturation implying the existence of distinctive posttranscriptional regulatory mechanisms controlling gene expression levels at those parasite stages. Meanwhile, the downregulation of a large proportion of the translatome indicates a key role of translation control in differentiation into the infective form. The previously described proteomic data correlate better with the translatomes than with the transcriptomes and translational efficiency analysis shows a wide dynamic range, reinforcing the importance of translatability as a regulatory step. Translation efficiencies for protein families like ribosomal components are diminished while translation of the transialidase virulence factors is upregulated in the quiescent infective metacyclic trypomastigote stage.ConclusionsA large subset of genes is modulated at the translation level in two different stages of Trypanosoma cruzi life cycle. Translation upregulation of virulence factors and downregulation of ribosomal proteins indicates different degrees of control operating to prepare the parasite for an infective life form. Taking together our results show that translational regulation, in addition to regulation of steady state level of mRNA, is a major factor playing a role during the parasite differentiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1563-8) contains supplementary material, which is available to authorized users.
“…Histone H2A and H3 antibodies were generated by immunization with full-length recombinant proteins. For detection of DOT1 enzymes via the MBP tag, a monoclonal rat antibody (anti-MBP IgG2a: 1:10,000, gift from E. Kremmer, Helmholtz Center Munich) 47 was used. Secondary antibody signals (goat anti-rabbit IRDye 800 CW: 1:50,000; donkey anti-guinea pig IRDye 800CW: 1:50,000; goat anti-rat IRDye 680LT: 1:50,000; and goat anti-rabbit 680LT: 1:50,000, all from LI-COR Biosciences) were detected with an Odyssey infrared imaging system (LI-COR Biosciences).…”
DOT1 enzymes are conserved methyltransferases that catalyse the methylation of lysine 79 on histone H3 (H3K79). Most eukaryotes contain one DOT1 enzyme, whereas African trypanosomes have two homologues, DOT1A and DOT1B, with different enzymatic activities. DOT1A mediates mono-and dimethylation of H3K76, the homologue of H3K79 in other organisms, whereas DOT1B additionally catalyses H3K76 trimethylation. However, it is unclear how these different enzymatic activities are achieved. Here we employ a trypanosomal nucleosome reconstitution system and structure-guided homology modelling to identify critical residues within and outside the catalytic centre that modulate product specificity. Exchange of these residues transfers the product specificity from one enzyme to the other, and reveals the existence of distinct regulatory domains adjacent to the catalytic centre. Our study provides the first evidence that a few crucial residues in DOT1 enzymes are sufficient to catalyse methyl-state-specific reactions. These results might also have far-reaching consequences for the functional understanding of homologous enzymes in higher eukaryotes.
“…Interestingly there is considerable divergence of PfAsf1 compared to its closely related protozoan parasites belonging to genus Trypanosoma, although they share the same node. Remarkably, trypanosomes harbour two isoforms of Asf1 [46], which is a characteristic of higher vertebrates, whereas PfAsf1, like its yeast homologue (yAsf1) harbours a…”
Section: Evolutionary Analysis Of Pfasf1mentioning
Asf1 is a highly-conserved histone chaperone that regulates tightly-coupled nucleosome assembly/disassembly process. We observed that Plasmodium falciparum Asf1 (PfAsf1) is ubiquitously expressed in different stages of the life cycle of the parasite. To gain further insight into its biological activity, we solved the structure of N-terminal histone chaperone domain of PfAsf1(1-159 amino acids) by X-ray crystallography to a resolution of 2.4 Å. The structure is composed of two beta-sheet to form a beta-sandwich, which resembles an immunoglobulin-like fold. The surface-charge distribution of PfAsf1 is distinct from yAsf1 and hAsf1 although the core-structure shows significant similarity. The crystal-structure indicated that PfAsf1 may exist in a dimeric-state which was further confirmed by solution cross-linking experiment. PfAsf1 was found to specifically interact with Plasmodium histone H3 and H4 and was able to deposit H3/H4 dimer onto DNA-template to form disomes, showing its characteristic histone chaperone activity. We mapped the critical residues of PfAsf1 involved in histone H3/H4 interaction and confirmed by site directed mutagenesis. Further analysis indicates that histone interacting surface of Asf1 is highly conserved while the dimerization interface is variable. Our results identify the role of PfAsf1 as mediator of chromatin assembly in Plasmodium falciparum, which is the causative agent of malignant malaria in humans.
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