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.

The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted

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For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

April 2020
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The datasets supporting the conclusions of this article are available in the ProteomeXchange Consortium via Pride repository, PXD019241 in http://www.ebi.ac.uk/ pride/archive/projects/PXD019241; and in the SRA, BioProject PRJNA630690 in https://www.ncbi.nlm.nih.gov/bioproject/PRJNA630690.

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Life sciences study design
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Sample size
Sample sizes for mass spectrometry experiments were established as quadruplicates (LFQ) or duplicates (DML), as is well-accepted in the proteomics field.
-results in Fig. 1a/b were derived from technical replicates -results for all other IP-qMS experiments were derived from biological replicates (4 separate extract preparations per condition) Sample sizes for the worm experiments were not based on statistical methods but on previously published similar experiments yielding consistent and reproducible results.
-IP experiments followed by Western blot: Embryo samples were prepared from bleaching one high density plate of gravid adults per sample, young adult samples were prepared from a pool of grown young adult worms aliquoted to 100 μl per sample.
-Colocalization was checked in separate animals (gravid adults, embryos) on different days and each showed similar results. Representative images from two life stages per strain are shown in the manuscript. A total of n>10 individuals was imaged for both strains.
-Telomere Southern Blot: per sample and strain gDNA derived from 100 μl mixed stage worms was used -for qFISH sample size was not predetermined, images were taken to be around n=10-15 individual germlines/embryos per strain and selected before analysis according to pre-established criteria.
-for double mutant cross analysis >100 F2 were singled before genotyping and subsequent analysis (Fig. 4) -brood sizes were determined from n=15 worms for Supplementary Fig 5 d/e, lower n resulting from excluding worms due to pre-established criteria -brood sizes were determined blindly from n>50 worms for Fig. 4 d/e, the total n was divided after determination of the respective genotypes -germline health (Fig. 4 g/h) was determined for n>320 individual worms over three generations and divided into the separate strains after genotyping -the Mortal Germline experiment was conducted on n=15 plates per strain Data exclusions We excluded qFISH images when one of the pre-established criteria was met: -additional telomeric signal stemming from other cell types -aggregation of the probe leading to high intensity signal We excluded worms from broodsize assays and mortal germline when one of the pre-established criteria was met: -contamination of the plates leading to sickness of the worms -early death of the worm due to morphological defects or crawling up the plate walls Replication -Results from initial MS screens were verified by DNA binding immunoprecipitation of recombinant proteins and C. elegans extract.
-Experiments for western blots from Fig. 1,5,6 and Supplementary Fig. 6 were performed at least twice with similar results. Exceptions are Fig.  1 c, Fig. 2a, Fig. 5b and Fig. S6h, which were not replicated. Fig. 1c depicts an expected result from a previous publication, Fig. 2a is supported by similar results from the RNAseq of Fig. S3a-c.
-Colocalization was checked at least twice by fluorescence microscopy, representative images of two individuals per strain from one experiment are shown in the manuscript.
-The qFISH experiment was repeated four independent times with different individual worms imaged, yielding similar results.
-The Telomere Southern Blot was repeated three independent times with genomic DNA from independent worm populations, yielding similar results.
-IP-qMS experiments were not repeated as the significant enrichments stem from four separate biological replicates used in the IP.
-The initial Y2H screen (Fig. 6a, Fig. S6) was performed twice independently with the same results. The other Y2H experiments were performed once each with elements from the other screens included as positive controls.
-Mortal germline and broodsize assays were performed once as n>15 of individual animals is sufficient and the positive controls used were previously published with similar generation times.
-Fluorescence polarization was performed at least twice for Fig. 1g/h and once for Fig.S2, yielding the overall same result.

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April 2020 -Gel filtration assays were performed twice (Fig. 5a & Supplementary Fig. 6a) with the exeption of the run including treatment with Sm nuclease (Fig. 5b). This run was performed only once.
Randomization In the establishment of the double mutants by crossing and the subsequent follow up, the worms for each generation were picked randomly from a parent with a known genotype. Worms for longevity assays and mortal germline were selected randomly from a maintenance plate containing worms of the same ages and genotypes. Images of germlines and embryos for qFISH analysis were taken randomly throughout the slides from randomly picked and fixed worms.

Blinding
Germline categories in the double mutant experiments were assigned without knowledge of the genotype. Otherwise blinding was not relevant for this study, as for the majority of experiments the strains analyzed needed to be assigned before to be able to perform the respective experiment and was therefore not part of the experimental design.
Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Validation anti-GFP antibody quality control by Sigma-Aldrich: -Western Blot: specific band from E.coli extract containing a recombinant GFP fusion protein detected at a 0.4 ug/ml antibody concentration. No non-specific binding was seen for a negative control lysate without GFP fusion protein.

Materials
-IP: Capture of GFP fusion protein from E. coli extract by antibody, vizualized by western blot anti-FLAG: no validation statement on manufacturer's website, western blot and IP functionality were confirmed using C. elegans extract containing a FLAG fusion protein.
anti-actin quality control by Sigma-Aldrich: working dilutions for western blot of at least 1:100 were determined using chicken gizzard extract. Anti-His HRP: no documentation on manufacturer's website, validation by western blot with E. coli lysate containing His-tagged proteins

Animals and other organisms
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Laboratory animals
Caenorhabditis elegans N2 Bristol -all mutants or tagged alleles were derived from this strain -> hermaphrodite worms used for all experiments, developmental stages: embryos, larval stages L1-L4, young adults, gravid adults -> male worms used for crosses Caenorhabditis briggsae AF16 (hemaphrodite worms, gravid adults used for extract preparation) Wild animals the study did not involve wild animals Field-collected samples the study did not involve samples collected from the field

Ethics oversight
No ethical approval or guidance was required.
Note that full information on the approval of the study protocol must also be provided in the manuscript.