Characterising ISWI chromatin remodeler in Trypanosoma cruzi

Abstract

BACKGROUND Imitation SWItch (ISWI) ATPase is the catalytic subunit in diverse chromatin remodeling complexes. These complexes modify histone-DNA interactions and therefore play a pivotal role in different DNA-dependent processes. In Trypanosoma cruzi, a protozoan that controls gene expression principally post-transcriptionally, the transcriptional regulation mechanisms mediated by chromatin remodeling are poorly understood. OBJECTIVE To characterise the ISWI remodeler in T. cruzi (TcISWI). METHODS A new version of pTcGW vectors was constructed to express green fluorescent protein (GFP)-tagged TcISWI. CRISPR-Cas9 system was used to obtain parasites with inactivated TcISWI gene and we determined TcISWI partners by cryomillingaffinity purification-mass spectrometry (MS) assay as an approximation to start to unravel the function of this protein. FINDINGS Our approach identified known ISWI partners [nucleoplasmin-like protein (NLP), regulator of chromosome condensation 1-like protein (RCCP) and phenylalanine/tyrosine-rich protein (FYRP)], previously characterised in T. brucei, and new components in TcISWI complex [DRBD2, DHH1 and proteins containing a domain characteristic of structural maintenance of chromosomes (SMC) proteins]. Data are available via ProteomeXchange with identifier PXD017869. MAIN CONCLUSIONS In addition to its participation in transcriptional silencing, as it was reported in T. brucei, the data generated here provide a framework that suggests a role for TcISWI chromatin remodeler in different nuclear processes in T. cruzi, including mRNA nuclear export control and chromatin compaction. Further work is necessary to clarify the TcISWI functional diversity that arises from this protein interaction study.

Histone Modifications and Other Facets of Epigenetic Regulation in Trypanosomatids: Leaving Their Mark

“…In budding yeast, the acetylation of H4 tails promotes deposition of H2A.Z into nucleosomes by the bromodomain-containing SWR1 complex ( 98 ). While a four-protein ISWI complex has been characterized in T. brucei and a multiprotein ISWI complex has been identified in T. cruzi also, none of the components carry an apparent bromodomain ( 99 , 100 ). Of the six T. brucei bromodomain factors (BDFs), TbBDF3 was the only one to colocalize with H4acetylK10, though direct binding to H4acetylK10 was not demonstrated ( 63 ).…”

Characterization of novel and essential kinetoplast-associated proteins inTrypanosoma brucei

“…KP86 represents the most recent kinetoplast innovation identified in this study, being confined to the genus Trypanosoma. In T. cruzi , an ortholog to KP86 has been identified as an interacting partner of chromatin remodeling imitation switch ATPase (Díaz-Olmos, Batista, Ludwig, & Marchini, 2020). The abundance of aberrant RNAi cells exhibiting two nuclei and a dividing kinetoplast, accompanied by no reduction in probed maxicircles or minicircles, resembles the phenotype observed in a recently characterized segregation factor TbNAB70 (Tb927.11.7580) (Cadena et al, 2024).…”

Two distinctTrypanosomaeIF4F complexes co-exist, bind different mRNAs and are regulated during nutritional stress

“…pDEST17 TM (Invitrogen) was used for eIF4E3 1-447 , eIF4E4 1-410 and eIF4A1 1-404 expression with a 6xhis tag. eIF4E3 1-447 , eIF4E4 1-410 were subcloned into pTcGW version 2.0 for episomal expression in T. cruzi of the proteins with a GFP tag at the C-terminus (pTcGFPN-CO) [49]. eIF4E3 1-447 , eIF4E4 1-410 , PABP1 1-566 , PABP2 1-550 and eIF4G4 1-678 were subcloned into pDEST22 TM and pDEST32 TM (Invitrogen) for N-terminal tagging with DNA binding and activation domains used in yeast two hybrid assays.…”