## 2 Results and Discussion
### Results
#### 2.1.3 High Levels of H3K79 me2 Favor Inclusion of Male-Specific Exon of \(\mathit{Imp}\)  
The above results support the possibility that H3K79 methylation marks are directly or indirectly associated with the regulation of the male-specific splicing of \(\mathit{Imp}\) pre-mRNA. We next performed comparative mapping of H3K79 me2 across the alternatively spliced regions of \(\mathit{Imp}\) in females and males by ChIP-qPCR. As shown in Figure 3B, higher distributions of H3K79 me2 were observed in males than in females across \(\mathit{Imp}\) (Figure 3B, left panel). Although the H3K79 me2 enrichments were not limited to the alternatively spliced exon 8, most of the significant differences between males and females were observed in exon 8. In contrast, no significant differences in the levels of H3K79 me2 over \(\mathit{Bmdsx}\) were observed (Figure 3B, middle panel). To investigate whether the distribution of the other histone marks across \(\mathit{Imp}\) differed between males and females, we performed comparative mapping of H3K36 me3 across \(\mathit{Imp}\) in females and males by ChIP-qPCR. As a result, no significant difference between males and females was observed in the levels of H3K36 me3 over \(\mathit{Imp}\) (Figure 3B, right panel). To examine whether siRNA-mediated knockdown of \(\mathit{DOT1L}\) reduces the level of H3K79 me2 accumulation, \(\mathit{DOT1L}\) siRNA-injected embryos were subjected to ChIP-qPCR analyses. Although higher distributions of H3K79 me2 were observed in males than in females across \(\mathit{Imp}\) in negative control individuals (Figure 3C, left panel), \(DOTIL\) knockdown exerted a marked influence on the accumulation of H3K79 me2, with an at least 10-fold reduction throughout the regions examined in males (Figure 3C, right panel). Western blotting analysis demonstrated that \(DOTIL\) siRNA-injection efficiently reduced the total H3K79 me2 level (Figure 4C). Since Dot1 in yeast and its homologs appear to be solely responsible for H3K79 methylation [31], reduction in the accumulation of H3K79 me2 across _Imp_ by DOT1L depletion could be attributed to a reduction in the amount of DOT1L interacting with _Imp_. These results provide overwhelming support for the specificity of the ChIP data. However, reduced accumulation of H3K79 me2 in females was less than that in males. This difference in the extent of reduction in H3K79 me2 between males and females could be attributed to differences in \(DOTIL\)-knockdown efficiencies between males and females, as shown in Figure 1C and Figure 2E. Mean \(DOTIL\) expression relative to control individuals was 27.6% in \(DOTIL\) knockdown females and 15.1% in \(DOTIL\)-knockdown males. At present we cannot explain the sexual difference in knockdown efficiencies. Although loss of H3K79 me2 may affect indirectly the distribution of H3K4 me1, H3K27 me3, or H3K36 me3, such indirect effect seems unlikely to affect the male-specific splicing of _Imp_ since RNAi knockdown of ASH2, EZH2, and SETD2 had no influence on the expression pattern of _Imp_ mRNA (Figure 1C). Overall we conclude that methylation of histone H3 across _Imp_ involves the HMTase DOT1L and high levels of H3K79 me2 favor inclusion of exon 8. To verify the link between the high distributions of H3K79 me2 and inclusion of alternative exons in genes other than _Imp_, we examined the effects of \(DOTIL\)-knockdown on a _Bombyx_ homolog of _Sex-lethal_ gene (_Bm-Sxl_). The primary transcript of the _Bm-Sxl_ gene is alternatively spliced to yield two isoforms, _Bm-Sxl-L_ and _Bm-Sxl-S_[22]. _Bm-Sxl-L_ consists of eight exons, while _Bm-Sxl-S_ is a splice variant that lacks the second exon (Figure 4A). qRT-PCR analysis demonstrated that _Bm-Sxl-L/Bm-Sxl-S_ ratio was more than twofold higher in \(DOTIL\)-knockdown embryos than in negative control embryos (Figure 4B). This result indicated that the \(DOTIL\)-knockdown relatively increased the inclusion of alternatively spliced exon (exon 2). In contrast, _SETD2_-knockdown caused no influence on the _Bm-Sxl-L/Bm-Sxl-S_ ratio.