Gametes deficient for Pot1 telomere binding proteins alter levels of telomeric foci for multiple generations

Abstract

Deficiency for telomerase results in transgenerational shortening of telomeres. However, telomeres have no known role in transgenerational epigenetic inheritance. C. elegans Protection Of Telomeres 1 (Pot1) proteins form foci at the telomeres of germ cells that disappear at fertilization and gradually accumulate during development. We find that gametes from mutants deficient for Pot1 proteins alter levels of telomeric foci for multiple generations. Gametes from pot-2 mutants give rise to progeny with abundant POT-1::mCherry and mNeonGreen::POT-2 foci throughout development, which persists for six generations. In contrast, gametes from pot-1 mutants or pot-1; pot-2 double mutants induce diminished Pot1 foci for several generations. Deficiency for MET-2, SET-25, or SET-32 methyltransferases, which promote heterochromatin formation, results in gametes that induce diminished Pot1 foci for several generations. We propose that C. elegans POT-1 may interact with H3K9 methyltransferases during pot-2 mutant gametogenesis to induce a persistent form of transgenerational epigenetic inheritance that causes constitutively high levels of heterochromatic Pot1 foci.

Fig. 1. Pot1 foci increase during early embryonic development.

a DIC and fluorescent images of 1-cell embryos before and after pronuclear fusion in either wild-type or pot-2 mutant strains. Cyan and yellow dashed lines indicate 1-cell embryos before and after pronuclear fusion, respectively. Scale bar is 10 µm. The portion of the image indicated by the magenta square is shown at 10x higher detail to the right. b POT-1::mCherry foci per nucleus of individual cells of embryos from 1- to 32-cell stage in wild-type (blue circles) and pot-2 mutants (red diamonds). The difference between wild-type and pot-2 mutants is significant at every stage (Wilcox p < 10⁻⁵). Error bars are 95% confidence intervals. Central black dots are means.

Fig. 2. Pot gene mutations affect Pot1 foci in subsequent generations.

a POT-1::mCherry foci per cell in embryos from P0 parental controls and descendants of either pot-2 mutant males or hermaphrodites crossed to POT-1::mCherry worms. Dot color indicates stage of embryo. b F1 early- and late-stage embryos from crosses between pot-1::mCherry mNeonGreen::pot-2 hermaphrodites and either pot-1::mCherry pot-2 males or pot-1::mCherry males. Scale bar is 10 µm. c POT-1::mCherry foci counts per nucleus in embryos from parental controls and descendants of either pot-1 or pot-2; pot-1 mutant males crossed to POT-1::mCherry worms. Error bars are 95% confidence intervals.

Fig. 3. Pot1 foci number is epigenetically affected by genes involved in H3K9 methylation.

a, b Quantification of POT-1::mCherry foci in nuclei of F2 embryos derived from crosses of pot-1::mCherry males to hermaphrodites mutant for small RNA genes (a) or chromatin genes (b). c, d Southern blot using DNA from the genetically wild-type descendents of crosses between wild-type and pot-1 (c) or pot-2 (d) mutant worms. The portion of the pot-1 blot indicated by the magenta square is shown at higher detail to the right. Corresponding colored squares show telomere shortening across three generations. Error bars are 95% confidence intervals.

Fig. 4. Models of Pot1 focus dynamics.

a Descriptive model of the developmental timing of Pot1 foci localization in wild type, pot-2 mutants, and pot-1 mutants. Red circles indicate POT-1::mCherry, green circles indicate mNeonGreen::POT-2, and yellow circles indicate colocalization of both transgenic proteins. b Descriptive model of the heritable phenotypes of progeny of gametes from pot-2 or pot-1 mutants.