Defining the chromatin-associated protein landscapes on Trypanosoma brucei repetitive elements using synthetic TALE proteins

Reviewer #1 (Public review):

Summary:

Carloni et al. comprehensively analyze which proteins bind repetitive genomic elements in Trypanosoma brucei. For this, they perform mass spectrometry on custom-designed, tagged programmable DNA-binding proteins. After extensively verifying their programmable DNA-binding proteins (using bioinformatic analysis to infer target sites, microscopy to measure localization, ChIP-seq to identify binding sites), they present, among others, two major findings: 1) 14 of the 25 known T. brucei kinetochore proteins are enriched at 177bp repeats. As T. brucei's 177bp repeat-containing intermediate-sized and mini-chromosomes lack centromere repeats but are stable over mitosis, Carloni et al. use their data to hypothesize that a 'rudimentary' kinetochore assembles at the 177bp repeats of these chromosomes to segregate them. 2) 70bp repeats are enriched with the Replication Protein A complex, which, notably, is required for homologous recombination. Homologous recombination is the pathway used for recombination-based antigenic variation of the 70bp-repeat-adjacent variant surface glycoproteins.

Strengths and Weaknesses:

The manuscript was previously reviewed through Review Commons. As noted there, the experiments are well controlled, the claims are well supported, and the methods are clearly described. The conclusions are convincing. All concerns I raised have been addressed except one (minor point #8):

"The way the authors mapped the ChIP-seq data is potentially problematic when analyzing the same repeat type in different genomic regions. Reads with multiple equally good mapping positions were assigned randomly. This is fine when analyzing repeats by type, independent of genomic position, which is what the authors do to reach their main conclusions. However, several figures (Fig. 3B, Fig. 4B, Fig. 5B, Fig. 7) show the same repeat type at specific genomic locations." Due to the random assignment, all of these regions merely show the average signal for the given repeat. I find it misleading that this average is plotted out at "specific" genomic regions.
Initially, I suggested a workaround, but the authors clarified why the workaround was not feasible, and their explanation is reasonable to me. That said, the figures still show a signal at positions where they can't be sure it actually exists. If this cannot be corrected analytically, it should at least be noted in the figure legends, Results, or Discussion.

Importantly, the authors' conclusions do not hinge on this point; they are appropriately cautious, and their interpretations remain valid regardless.

Significance:

This work is of high significance for chromosome/centromere biology, parasitology, and the study of antigenic variation. For chromosome/centromere biology, the conceptual advancement of different types of kinetochores for different chromosomes is a novelty, as far as I know. It would certainly be interesting to apply this study as a technical blueprint for other organisms with mini-chromosomes or chromosomes without known centromeric repeats. I can imagine a broad range of labs studying other organisms with comparable chromosomes to take note of and build on this study. For parasitology and the study of antigenic variation, it is crucial to know how intermediate- and mini-chromosomes are stable through cell division, as these chromosomes harbor a large portion of the antigenic repertoire. Moreover, this study also found a novel link between the homologous repair pathway and variant surface glycoproteins, via the 70bp repeats. How and at which stages during the process, 70bp repeats are involved in antigenic variation is an unresolved, and very actively studied, question in the field. Of course, apart from the basic biological research audience, insights into antigenic variation always have the potential for clinical implications, as T. brucei causes sleeping sickness in humans and nagana in cattle. Due to antigenic variation, T. brucei infections can be chronic.

Comments on revised version:

All my recommendations have been addressed.

Reviewer #2 (Public review):

The Trypanosoma brucei genome, like that of other eukaryotes, contains diverse repetitive elements. Yet, the chromatin-associated proteome of these regions remains largely unexplored. This study represents a very important conceptual and technical advancement by employing synthetic TALE DNA-binding proteins fused to YFP to selectively capture proteins associated with specific repetitive sequences in T. brucei chromatin. The data presented here are convincing, supported by appropriate controls and a well-validated methodology, aligned with current state-of-the-art approaches.

The authors used synthetic TALE DNA binding proteins, tagged with YFP, which were designed to target five specific repeat elements in T. brucei genome, including centromere and telomeres-associated repeats and those of a transposon element. This is in order to identify specific proteins that bind to these repetitive sequences in T. brucei chromatin. Validation of the approach was done using a TALE protein designed to target the telomere repeat (TelR-TALE) that detected many of the proteins that were previously implicated with telomeric functions. A TALE protein designed to target the 70 bp repeats that reside adjacent to the VSG genes (70R-TALE) detected proteins that function in DNA repair and a protein designed to target the 177 bp repeat arrays (177R-TALE) identified kinetochore proteins associated T. brucei mega base chromosomes, as well as in intermediate and mini-chromosomes, which imply that kinetochore assembly and segregation mechanisms are similar in all T. brucei chromosomes.

This study represents a significant conceptual and technical advancement. To the best of our knowledge, it is the first report of employing TALE-YFP for affinity-based detection of protein complexes bound to repetitive genomic sequences in T. brucei. This approach enhances our understanding the organization in these important regions of the trypanosomal chromatin and provides the foundation for investigating the functional roles of associated proteins in parasite biology. These findings will be of particular interest to researchers studying the molecular biology of kinetoplastid parasites and other unicellular organisms, as well as to scientists investigating the roles of repetitive genomic elements in chromatin structure and their functional role in higher eukaryotes.

Importantly, any essential or unique interacting partners identified using the approach employed here, could serve as a potential target for therapeutic intervention in severe tropical diseases cause by kinetoplastids.