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  • br Results br Discussion To further evaluate

    2022-07-30


    Results
    Discussion To further evaluate the extent of H3T3A and H3T3D roles in the segregation of sister chromatids enriched with different populations of H3 during mitosis (Figure 1B, step two), we modeled all possible segregation patterns in male GSCs and compared these estimates to our experimental results. To simplify our calculations, we made two important assumptions: first, we assume nucleosomal density to be even throughout the genome. This assumption allows us to infer that the overall fluorescent signal contributed by each chromosome is proportional to their respective number of DNA base pairs. Second, by quantifying pre-existing H3-GFP asymmetry in (Z)-4-Hydroxytamoxifen mg and telophase GSCs, we estimate that the establishment of H3-GFP asymmetry is ∼4-fold biased, i.e., 80% on one set of sister chromatids and 20% on the other set of sister chromatids, based on quantification of GFP signal in anaphase (GFP GSC side/GB side = 4.5) and telophase (GFP GSC side/GB side = 3.8) GSCs (Tran et al., 2012). With these two simplifying assumptions, we calculate both GFP and mKO ratios among all 64 possible combinations (Table S2: 2 (for X-ch) × 2 (for Y-ch) × 4 (for 2nd ch) × 4 (for 3rd ch) = 64 combinations in total). If we define asymmetry as a greater than ∼1.5-fold difference in fluorescence intensity, then based on a model of randomized sister chromatid segregation, we estimate that a symmetric pattern should appear for 53.1% (34/64) of GSC-GB pairs whereas both conventional and inverted asymmetric patterns should occur with equal frequencies and account for 18.7% (12/64) of total GSC-GB pairs. The remaining 9.4% (6/64) of GSC-GB pairs should produce histone inheritance patterns with a 1.45- to 1.55-fold difference in signal intensity (predicted ratios in Figure 4K). This estimation is close to our experimental data in both H3T3A- and H3T3D-expressing testes (Figures 4J and 4K; Table S1). Of the 64 quantified post-mitotic GSC-GB pairs in nos>H3T3A testes, ∼71.9% showed symmetric inheritance pattern. Conventional and inverted asymmetric patterns were detected at 9.4% and 12.5%, respectively, and 6.3% at the borderline. Similarly, of the 57 quantified post-mitotic GSC-GB pairs in nos>H3T3D testes, ∼79.0% showed symmetric inheritance pattern. Conventional and inverted asymmetric patterns were detected at 7.0% and 10.5%, respectively with 3.5% of pairs at the borderline. Some differences between predicted ratios and our experimental data could be due to the simplified assumptions, the limited sensitivity of our measurement, and/or some coordinated chromatid segregation modes that bias the eventual read-out (Yadlapalli and Yamashita, 2013). In summary, comparison between the modeling ratios and our experimental data suggest that loss of the tight control of H3T3 phosphorylation in GSCs randomizes segregation of sister chromatids enriched with different populations of H3. If the temporal separation in the phosphorylation of H3T3 on epigenetically distinct sister chromatids facilitates their proper segregation and inheritance during asymmetric cell division, it is likely that mutations of the Haspin kinase will also affect the temporal control of H3T3 phosphorylation. In the context of H3T3A, where the levels of H3T3P are already reduced, a further decrease in H3T3P by reducing Haspin levels should limit the GSC’s ability to distinguish between sister chromatids enriched with distinct H3. Indeed, haspin mutants enhance the phenotypes in nos>H3T3A testes. A different situation appears in the context of H3T3D where sister chromatids experience seemingly elevated levels of H3T3P at the start of mitosis. These elevated H3T3P levels may be exacerbated by the phosphorylation activity of the Haspin kinase. Therefore, it is conceivable that by halving the levels of the Haspin kinase, H3T3 phosphorylation should be reduced to a level more closely resembling wild-type. In this way, some of the temporal specificity that is lost in the H3T3D mutant is restored, resulting in suppression of the phenotypes observed in nos>H3T3D testes. An exciting topic for future study would be to further explore how exactly Haspin phosphorylates H3T3 in the context of chromatin and whether H3T3A and H3T3D mutations act synergistically or antagonistically in regulating asymmetric sister chromatids segregation through differential phosphorylation of a key histone residue.