ZNF524 directly interacts with telomeric DNA and supports telomere integrity.

Telomeres are nucleoprotein structures at the ends of linear chromosomes. In humans, they consist of TTAGGG repeats, which are bound by dedicated proteins such as the shelterin complex. This complex blocks unwanted DNA damage repair at telomeres, e.g. by suppressing nonhomologous end joining (NHEJ) through its subunit TRF2. Here, we describe ZNF524, a zinc finger protein that directly binds telomeric repeats with nanomolar affinity, and reveal base-specific sequence recognition by cocrystallization with telomeric DNA. ZNF524 localizes to telomeres and specifically maintains the presence of the TRF2/RAP1 subcomplex at telomeres without affecting other shelterin members. Loss of ZNF524 concomitantly results in an increase in DNA damage signaling and recombination events. Overall, ZNF524 is a direct telomere-binding protein involved in the maintenance of telomere integrity.

Characterization of two novel proteins involved in mitochondrial DNA anchoring in Trypanosoma brucei.

Trypanosoma brucei is a single celled eukaryotic parasite in the group of the Kinetoplastea. The parasite harbors a single mitochondrion with a singular mitochondrial genome that is known as the kinetoplast DNA (kDNA). The kDNA consists of a unique network of thousands of interlocked circular DNA molecules. To ensure proper inheritance of the kDNA to the daughter cells, the genome is physically linked to the basal body, the master organizer of the cell cycle in trypanosomes. The connection that spans, cytoplasm, mitochondrial membranes and the mitochondrial matrix is mediated by the Tripartite Attachment Complex (TAC). Using a combination of proteomics and RNAi we test the current model of hierarchical TAC assembly and identify TbmtHMG44 and TbKAP68 as novel candidates of a complex that connects the TAC to the kDNA. Depletion of TbmtHMG44 or TbKAP68 each leads to a strong kDNA loss but not missegregation phenotype as previously defined for TAC components. We demonstrate that the proteins rely on both the TAC and the kDNA for stable localization to the interface between these two structures. In vitro experiments suggest a direct interaction between TbmtHMG44 and TbKAP68 and that recombinant TbKAP68 is a DNA binding protein. We thus propose that TbmtHMG44 and TbKAP68 are part of a distinct complex connecting the kDNA to the TAC.

DNA damage repair proteins across the Tree of Life.

Genome maintenance is orchestrated by a highly regulated DNA damage response with specific DNA repair pathways. Here, we investigate the phylogenetic diversity in the recognition and repair of three well-established DNA lesions, primarily repaired by base excision repair (BER) and ribonucleotide excision repair (RER): (1) 8-oxoguanine, (2) abasic site, and (3) incorporated ribonucleotide in DNA in 11 species: Escherichia coli, Bacillus subtilis, Halobacterium salinarum, Trypanosoma brucei, Tetrahymena thermophila, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, Homo sapiens, Arabidopsis thaliana, and Zea mays. Using quantitative mass spectrometry, we identified 337 binding proteins across these species. Of these proteins, 99 were previously characterized to be involved in DNA repair. Through orthology, network, and domain analysis, we linked 44 previously unconnected proteins to DNA repair. Our study presents a resource for future study of the crosstalk and evolutionary conservation of DNA damage repair across all domains of life.

RNA-dependent interactome allows network-based assignment of RNA-binding protein function.

RNA-binding proteins (RBPs) form highly diverse and dynamic ribonucleoprotein complexes, whose functions determine the molecular fate of the bound RNA. In the model organism Sacchromyces cerevisiae, the number of proteins identified as RBPs has greatly increased over the last decade. However, the cellular function of most of these novel RBPs remains largely unexplored. We used mass spectrometry-based quantitative proteomics to systematically identify protein-protein interactions (PPIs) and RNA-dependent interactions (RDIs) to create a novel dataset for 40 RBPs that are associated with the mRNA life cycle. Domain, functional and pathway enrichment analyses revealed an over-representation of RNA functionalities among the enriched interactors. Using our extensive PPI and RDI networks, we revealed putative new members of RNA-associated pathways, and highlighted potential new roles for several RBPs. Our RBP interactome resource is available through an online interactive platform as a community tool to guide further in-depth functional studies and RBP network analysis (https://www.butterlab.org/RINE).

A novel SNF2 ATPase complex in Trypanosoma brucei with a role in H2A.Z-mediated chromatin remodelling.

A cascade of histone acetylation events with subsequent incorporation of a histone H2A variant plays an essential part in transcription regulation in various model organisms. A key player in this cascade is the chromatin remodelling complex SWR1, which replaces the canonical histone H2A with its variant H2A.Z. Transcriptional regulation of polycistronic transcription units in the unicellular parasite Trypanosoma brucei has been shown to be highly dependent on acetylation of H2A.Z, which is mediated by the histone-acetyltransferase HAT2. The chromatin remodelling complex which mediates H2A.Z incorporation is not known and an SWR1 orthologue in trypanosomes has not yet been reported. In this study, we identified and characterised an SWR1-like remodeller complex in T. brucei that is responsible for Pol II-dependent transcriptional regulation. Bioinformatic analysis of potential SNF2 DEAD/Box helicases, the key component of SWR1 complexes, identified a 1211 amino acids-long protein that exhibits key structural characteristics of the SWR1 subfamily. Systematic protein-protein interaction analysis revealed the existence of a novel complex exhibiting key features of an SWR1-like chromatin remodeller. RNAi-mediated depletion of the ATPase subunit of this complex resulted in a significant reduction of H2A.Z incorporation at transcription start sites and a subsequent decrease of steady-state mRNA levels. Furthermore, depletion of SWR1 and RNA-polymerase II (Pol II) caused massive chromatin condensation. The potential function of several proteins associated with the SWR1-like complex and with HAT2, the key factor of H2A.Z incorporation, is discussed.

The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1.

Telomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a Mortal Germline. Notably, tebp-1;tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. Furthermore, we show that POT-1 forms a telomeric complex with TEBP-1 and TEBP-2, which bridges TEBP-1/-2 with POT-2/MRT-1. These results provide insights into the composition and organization of a telomeric protein complex in C. elegans.

A comparison of shared patterns of differential gene expression and gene ontologies in response to water-stress in roots and leaves of four diverse genotypes of Lolium and Festuca spp. temperate pasture grasses.

Ryegrasses (Lolium spp.) and fescues (Festuca spp.) are closely related and widely cultivated perennial forage grasses. As such, resilience in the face of abiotic stresses is an important component of their traits. We have compared patterns of differentially expressed genes (DEGs) in roots and leaves of two perennial ryegrass genotypes and a single genotype of each of a festulolium (predominantly Italian ryegrass) and meadow fescue with the onset of water stress, focussing on overall patterns of DEGs and gene ontology terms (GOs) shared by all four genotypes. Plants were established in a growing medium of vermiculite watered with nutrient solution. Leaf and root material were sampled at 35% (saturation) and, as the medium dried, at 15%, 5% and 1% estimated water contents (EWCs) and RNA extracted. Differential gene expression was evaluated comparing the EWC sampling points from RNAseq data using a combination of analysis methods. For all genotypes, the greatest numbers of DEGs were identified in the 35/1 and 5/1 comparisons in both leaves and roots. In total, 566 leaf and 643 root DEGs were common to all 4 genotypes, though a third of these leaf DEGs were not regulated in the same up/down direction in all 4 genotypes. For roots, the equivalent figure was 1% of the DEGs. GO terms shared by all four genotypes were often enriched by both up- and down-regulated DEGs in the leaf, whereas generally, only by either up- or down-regulated DEGs in the root. Overall, up-regulated leaf DEGs tended to be more genotype-specific than down-regulated leaf DEGs or root DEGs and were also associated with fewer GOs. On average, only 5-15% of the DEGs enriching common GO terms were shared by all 4 genotypes, suggesting considerable variation in DEGs between related genotypes in enacting similar biological processes.

Characterization of the novel mitochondrial genome segregation factor TAP110 in Trypanosoma brucei.

Proper mitochondrial genome inheritance is important for eukaryotic cell survival. Trypanosoma brucei, a protozoan parasite, contains a singular mitochondrial genome, the kinetoplast (k)DNA. The kDNA is anchored to the basal body via the tripartite attachment complex (TAC) to ensure proper segregation. Several components of the TAC have been described; however, the connection of the TAC to the kDNA remains elusive. Here, we characterize the TAC-associated protein TAP110. We find that both depletion and overexpression of TAP110 leads to a delay in the separation of the replicated kDNA networks. Proteome analysis after TAP110 overexpression identified several kDNA-associated proteins that changed in abundance, including a TEX-like protein that dually localizes to the nucleus and the kDNA, potentially linking replication and segregation in the two compartments. The assembly of TAP110 into the TAC region seems to require the TAC but not the kDNA itself; however, once TAP110 has been assembled, it also interacts with the kDNA. Finally, we use ultrastructure expansion microscopy in trypanosomes for the first time, and reveal the precise position of TAP110 between TAC102 and the kDNA, showcasing the potential of this approach.This article has an associated First Person interview with the first author of the paper.

PAR-TERRA is the main contributor to telomeric repeat-containing RNA transcripts in normal and cancer mouse cells.

Telomeric repeat-containing RNA (TERRA) molecules play important roles at telomeres, from heterochromatin regulation to telomerase activity control. In human cells, TERRA is transcribed from subtelomeric promoters located on most chromosome ends and associates with telomeres. The origin of mouse TERRA molecules is, however, unclear, as transcription from the pseudoautosomal PAR locus was recently suggested to account for the vast majority of TERRA in embryonic stem cells (ESC). Here, we confirm the production of TERRA from both the chromosome 18q telomere and the PAR locus in mouse embryonic fibroblasts, ESC, and various mouse cancer and immortalized cell lines, and we identify two novel sources of TERRA on mouse chromosome 2 and X. Using various approaches, we show that PAR-TERRA molecules account for the majority of TERRA transcripts, displaying an increase of two to four orders of magnitude compared to the telomeric 18q transcript. Finally, we present a SILAC-based pull-down screen revealing a large overlap between TERRA-interacting proteins in human and mouse cells, including PRC2 complex subunits, chromatin remodeling factors, DNA replication proteins, Aurora kinases, shelterin complex subunits, Bloom helicase, Coilin, and paraspeckle proteins. Hence, despite originating from distinct genomic regions, mouse and human TERRA are likely to play similar functions in cells.

Differential gene expression and gene ontologies associated with increasing water-stress in leaf and root transcriptomes of perennial ryegrass (Lolium perenne).

Perennial ryegrass (Lolium perenne) is a forage and amenity grass species widely cultivated in temperate regions worldwide. As such, perennial ryegrass populations are exposed to a range of environmental conditions and stresses on a seasonal basis and from year to year. One source of potential stress is limitation on water availability. The ability of these perennial grasses to be able to withstand and recover after periods of water limitation or drought can be a key component of grassland performance. Thus, we were interested in looking at changes in patterns of gene expression associated with increasing water stress. Clones of a single genotype of perennial ryegrass were grown under non-flowering growth room conditions in vermiculite supplemented with nutrient solution. Leaf and root tissue was sampled at 4 times in quadruplicate relating to estimated water contents of 35%, 15%, 5% and 1%. RNA was extracted and RNAseq used to generate transcriptome profiles at each sampling point. Transcriptomes were assembled using the published reference genome sequence and differential gene expression analysed using 3 different programmes, DESeq2, edgeR and limma (with the voom transformation), individually and in combination, deriving Early, Middle and Late stage comparisons. Identified differentially expressed genes were then associated with enriched GO terms using BLAST2GO. For the leaf, up-regulated differentially expressed genes were strongly associated with GO terms only during the Early stage and the majority of GO terms were associated with only down-regulated genes at the Middle or Late stages. For the roots, few differentially expressed genes were identified at either Early or Middle stages. Only one replicate at 1% estimated water content produced high quality data for the root, however, this indicated a high level of differential expression. Again the majority of enriched GO terms were associated with down-regulated genes. The performance of the different analysis programmes and the annotations associated with identified differentially expressed genes is discussed.