C-Terminal Extended Domain-Independent Telomere Maintenance: Modeling the Function of TIN2 Isoforms in Mus musculus

Abstract

TIN2 (TERF1 interacting nuclear factor 2) is a telomeric shelterin complex component, essential for telomere protection and early embryonic development in mammals. In humans, TIN2 isoforms arise from alternative splicing, but their specific roles in vivo remain unclear. Here, we explore TIN2 isoform functions in the laboratory mouse Mus musculus. Our comparative analysis of TIN2 protein sequences reveals that mouse TIN2 (TINF2) closely resembles the human TIN2L isoform, both of which harbor a C-terminal extended domain (CTED) absent from the human TIN2 small (TIN2S) isoform. To further characterize the functions of TIN2 isoforms, we generated a Tinf2 LD (long-form deficiency) allele in M. musculus encoding a short form of TINF2 lacking the CTED. Mice heterozygous or homozygous for this Tinf2 LD allele were viable, fertile, and showed no tissue abnormalities. Furthermore, protein product of Tinf2 LD allele localized to telomeres and maintained telomere integrity in mouse embryonic fibroblasts, demonstrating that the CTED is dispensable for telomere protection and normal development in mice. These findings indicate functional redundancy among TIN2 isoforms and underscore the utility of the Tinf2 LD model for uncovering isoform-specific mechanisms of telomere regulation.

Keywords: DNA damage; alternative splicing; telomere.

Figure 1

Characterization of human TIN2 and mouse Tinf2 mRNA expression. (A) Schematic of the structural organization of human TIN2 (hTIN2) and mouse Tinf2 (mTINF2) genomic sequences. Exons are represented as numbered rectangular shapes, and connecting lines indicate introns splicing between exons. Open rectangular shapes represent the untranslated region (UTR), while closed rectangular shapes (black rectangles) indicate the coding sequences in exons. Sequences encoding the CTED of human TIN2L and mouse TINF2 are marked. Red arrows indicate the genomic sequence used as a primer for the PCR-based amplification shown in (B). (B) Detection of hTIN2 isoform expression in adult tissues. cDNAs from various human adult tissues were used for PCR primers indicated in (A). The arrowheads labeled S, M, L represent the detected signals corresponding to TIN2S, TIN2M, and TIN2L, respectively. (C) Human and mouse TIN2 protein sequence alignment results in the regions corresponding to the putative CK2 phosphorylation sites. The putative binding site is highlighted in the open rectangular shapes, and the potential phosphorylation sites are underlined. Full-length sequence alignment results are provided in Supplementary Figure S1. Full-length gels and blots are provided in Supplementary Figure S4.

Figure 2

Generation of the Tinf2 long-form deficiency (LD) allele in mouse. (A) Schematics illustrating the gene structure of the wildtype (WT) and long-form deficiency (LD) alleles. Two guide RNAs (red arrows), flanking mouse Tinf2 intron 6 and partial exon 9, along with Cas9-expressing constructs, were designed to generate a modified Tinf2 allele in mice, replicating human TIN2S. Blue arrows denote PCR genotyping primers. (B) Representative genotyping results of mouse embryonic fibroblasts (MEFs). MEFs were isolated from embryos on embryonic day 14.5 (E14.5) for genomic DNA extraction. PCRs were carried out with the primers indicated in (A), and the resulting oligonucleotides were subjected to agarose gel electrophoresis. (C) Representative RT-PCR results obtained from MEFs. mRNA samples were extracted from the indicated MEFs and subjected to reverse transcription to generate cDNA samples for PCRs with the primers indicated in (A). PCR products were analyzed by agarose gel electrophoresis. (D) Western blotting for TINF2 and actin protein expression in MEFs of different genotypes. Red arrowheads highlight bands corresponding to the wildtype (WT) or LD allele. Full-length gels and blots are provided in Supplementary Figure S6.

Figure 3

Telomeric localization and protection function of wildtype TINF2 and TINF2LD in MEFs. (A) Representative images of IF-FISH illustrating expression of mouse TINF2 protein and telomere signal in MEFs. Telomere signals were visualized using a telomere probe (red), whereas endogenous mouse TINF2 signals were detected using mouse anti-Tinf2 antibodies (green). Representative telomere-localized mouse TINF2 (colocalized signals) is indicated by white arrowheads. Scale bar: 2 μm. (B) Representative images of control and siRNA-treated MEFs of different Tinf2 genotypes (Tinf2^+/+, Tinf2^LD/+, and Tinf2^LD/LD). White arrows indicate colocalized signals of γH2AX and telomere puncta, indicative of telomere damage-induced foci (TIFs). Cells displaying more than five TIFs were defined as TIF+ cells. Scale bar: 2 μm. (C) Western blotting analysis for TINF2 and GAPDH in siRNA-treated primary MEFs with different genotypes. WT bands correspond to wild-type protein, while LD bands represent TINF2LD. (D) Percentage of telomeres with mouse TINF2 signals for different genotypes with Tinf2 or control knockdown treatment. Data are presented as mean ± SD. More than 50 cells were counted for each condition. Statistical analysis was conducted using one-way ANOVA. ****, p-value < 0.0001. Representative images are shown in Supplementary Figure S3. (E) Numbers of TIFs in each cell. Data represent three primary MEF lines, with over 300 cells assessed in each group. Data are presented as mean ± SD, with longer lines denoting the mean value and shorter lines representing standard deviation. Statistical analysis was conducted using one-way ANOVA. n.s., not significant; ****, p-value < 0.0001. (F) Percentages of TIF+ cells for various genotypes. For each genotype, analysis was performed on three independent primary MEF lines, with the percentage of TIF+ cells determined from more than 100 cells of each line. Data are presented as mean ± SD. Statistical analysis was conducted using one-way ANOVA. n.s., not significant; ****, p-value < 0.0001. Further statistical details are shown in Supplementary Table S2.

Figure 4

Phenotypic comparison of adult animals of different genotypes. (A) Gross views of mice representing different genotypes. (B) Gross views of mouse brain, spleen, and kidney. (C,D) Body weight trajectories over time (4–14 weeks) of male and female mice. For the male body weight, the analysis comprised 4 Tinf2^+/+, 6 Tinf2^LD/+, and 5 Tinf2^LD/LD animals. For the female body weight, the analysis comprised 3 Tinf2^+/+, 4 Tinf2^LD/+, and 6 Tinf2^LD/LD animals. Data are represented as mean ± SD. A Chi-square-based statistical analysis was conducted for male body weight: Tinf2^+/+ vs. Tinf2^LD/+: p-value = 0.19; Tinf2^+/+ vs. Tinf2^LD/LD: p-value = 0.94; and Tinf2^LD/+ vs. Tinf2^LD/LD: p-value = 0.0006. The same statistical analysis was conducted on the data of female body weight: Tinf2^+/+ vs. Tinf2^LD/+: p-value = 0.45; Tinf2^+/+ vs. Tinf2^LD/LD: p-value = 0.91; and Tinf2^LD/+ vs. Tinf2^LD/LD: p-value = 0.20. (E) Survival curves for mice of the indicated genotypes were tracked over a period of up to 4 years. The analysis comprised 10 Tinf2^+/+, 12 Tinf2^LD/+, and 15 Tinf2^LD/LD animals. Survival probability is depicted as a Kaplan–Meier plot. An overall log-rank test revealed an χ² value of 2.02 and a p-value of 0.64 [for independent comparisons: Tinf2^+/+ vs. Tinf2^LD/+: χ² = 1.22, p-value = 0.73; Tinf2^+/+ vs. Tinf2^LD/LD: χ² = 0.17, p-value = 0.32; and Tinf2^LD/+ vs. Tinf2^LD/LD: χ² = 1.36, p-value = 0.76].