## 2 Results and Discussion
### Results
#### 2.1.3 High Levels of H3K79 me2 Favor Inclusion of Male-Specific Exon of \(\mathit{Imp}\)
Western blotting analysis demonstrated that \(DOTIL\) siRNA-injection and _SETD2_ siRNA-injection efficiently reduced the total H3K79 me2 level and total H3K36 me3 level, respectively (Figure 4C). To investigate whether the high distributions of H3K79 me2 is correlated with DOT1L-dependent inclusion of _Bm-Sxl_ exon 2, we performed mapping of H3K79 me2 around the exon 2 by ChIP-qPCR. The H3K79 me2 mark was specifically enriched on the regions around exon 2 as compared with the H3K36 me3 mark (Figure 4D). These results suggest that enrichment of H3K79 me2 could be correlated with DOT1L-dependent inclusion of alternative exons. Figure 2: Effect of \(DOTIL\) knockdown on male-specific splicing of _Imp_. (**A**) The upper panel shows the location of the reverse transcription (RT)-PCR primers used for detection of _Imp_ mRNA. The arrows above the diagram indicate the primers that were used for amplification of \(Imp^{M}\), and the arrows below the diagram show the primers used to amplify _Imp_ mRNA transcribed from a region common to both sexes (\(Imp^{C}\)). The lower panel indicates a schematic diagram of alternative splicing in the _Bmdsx_ pre-mRNA. The gray boxes indicate the female-specific exons. The arrows point to the approximate locations of the primers used for RT-PCR in E; (**B**) Expression of \(Imp^{M}\) and \(Imp^{C}\) was detected by RT-PCR with primers illustrated in A and analyzed in a 1% agarose gel. The cDNA samples examined in lanes 1, 2, and 3 were identical to those used in lanes 1, 5, and 6, respectively, in Figure 1C. The bottom panel shows amplification of the _Actin3_ transcript, which served as a positive control for RNA extraction and RT-PCR. The expression of \(DOTIL\) relative to the negative control embryos in each individual is indicated above each lane; (**C**) The ratio of \(Imp^{M}\) to \(Imp^{C}\) was analyzed by qRT-PCR; (**D**) Quantification of \(Imp^{C}\) (left panel) or \(Imp^{M}\) mRNA expression (right panel) by qRT-PCR. The _elongation factor 2_ (_EF-2_) served as an internal standard. SD; \(n=5\) individuals. * \(p<0.05\), *** \(p<0.001\), Student’s \(t\)-test; (**E**) Female- or male-specific splicing of _Bmdsx_ pre-mRNA was detected by RT-PCR and analyzed on a 1% agarose gel. The upper panel shows expression of \(Imp^{M}\), and the middle panel indicates the female- and male-specific splicing products of _Bmdsx_ (_Bmdsxf1_, _Bmdsxf2_ and _Bmdsxm_, respectively). The lower panel shows amplification of the _Actin3_ transcript, which served as a positive control for RNA extraction and RT-PCR. The expression levels of \(DOTIL\) and \(Imp^{M}\) relative to those of the negative control embryos in each individual are indicated above each lane. **Figure 3.** Increased H3K79 methylation across _Imp_ in males. (**A**) Schematic representation of _Imp_ and _Bmdsx_ genes showing the distribution of quantitative (q) PCR amplicons used in the analysis; (**B**) Mapping of H3K79 me2 across _Imp_ (left panel) and _Bmdsx_ (middle panel) and of H3K36 me3 across _Imp_ (right panel) in female (black) and male (white) larval tissues by chromatin immunoprecipitation (ChIP)-qPCR. Values represent the means \(\pm\) SE of six qPCR values from one representative of five independent experiments. * \(p<0.05\), **\(p<0.01\), Student's \(t\)-test; (**C**) ChIP assays with antibodies to H3K79 me2 and H3 and chromatin prepared from 60-pooled negative control embryos of each sex (left panel) or 60-pooled _DOTIL_ siRNA-injected embryos of each sex (right panel). The relative enrichment of H3K79 me2 on _EF-2_ exon2 or along _Imp_ was quantified by qPCR using primer sets indicated in A and expressed as a fraction of histone H3. Values represent the means \(\pm\) SE of two independent qPCR assays from one representative of two independent experiments. * \(p<0.05\), Student's \(t\)-test. The percentage of input was normalized to unmodified H3. Figure 3.** Increased H3K79 methylation across _Imp_ in males.