Histone genes are amongst the most evolutionary conserved in eukaryotic genomes, yet cis-regulatory mechanisms of histone gene regulation differ considerably amongst species. In Drosophila melanogaster, an interaction between GA-rich cis elements in the H3/H4 promoter and the GA-binding transcription factor CLAMP is important for promoting histone gene regulation and factor recruitment to the locus. CLAMP also participates in male dosage compensation by recruiting the Male Specific Lethal Complex (MSLc) to the X-chromosome. We discovered that the male-specific protein of MSLc, MSL2, is recruited to the autosomal major histone locus in D. virilis but not to the minor locus or to the single histone locus in other species. While the histone coding sequences are well conserved between species, the critical GA-rich cis elements in the H3/H4 promoter are poorly conserved between D. melanogaster and D. virilis. We show that CLAMP still targets the two D. virilis histone loci in vivo. Further, CLAMP interacts with the D. virilis H3/H4 promoter in vitro, even when the poorly-conserved GA-rich cis elements are deleted, indicating that the protein interacts differently with the D. virilis promoter than it does with the D. melanogaster promoter. Since CLAMP and MSL2 directly interact in D. melanogaster, we propose that D. virilis CLAMP recruits MSL2 to an ectopic autosomal site through interaction with X-like cis elements. Further, localization of MSL2 to one of the D. virilis histone loci suggests that the loci are regulated differently and that males and females have different requirements for histone gene regulation.

The histone locus body (HLB) is a conserved nuclear body that regulates histone mRNA production in metazoans. While some HLB components are known, there are likely uncharacterized factors that target the histone locus. We identified the Drosophila melanogaster protein Max, which interacts with known HLB member Myc, as an HLB candidate. We mapped Max ChIP-seq and ChIP-nexus datasets, which revealed encouraging signal over the histone gene array. However, we discovered that Max does not colocalize with HLB components on polytene chromosomes. Therefore, we conclude that Max is likely not at the D. melanogaster histone locus.

Nuclear bodies are membraneless structures containing concentrated regulatory factors that coordinate nuclear processes such as gene expression. The histone locus body (HLB) is a discrete nuclear body that is the main site of histone mRNA production. While many factors of the HLB are known, there are many unknown factors. In addition, while HLB function is highly conserved, it is also unknown how the HLB has changed and evolved in different species. The histone locus (HL) of the model organism Drosophila melanogaster contains ~100 tandem arrays of the five histone genes. While D. melanogaster has one HL, the related species Drosophila virilis, has two HL. We observe localization of the male‐specific dosage compensation protein MSL2 to one of the two D. virilishistone loci using polytene chromosome immunofluorescence, which we do not observe in other Drosophila species. In order to confirm our immunofluorescence observations, we mapped existing MSL2 ChIP‐seq data and discovered that when MSL2 from either species is overexpressed, it targets the H2a‐H2b promoter. We immunostained transgenic flies carrying an H2a‐H2b promoter transgene to determine if MSL2 targets this region when outside the context of the histone locus. We do not see the same HL localization when MSL2 is expressed at wild‐type levels. Finally, we performed RNA isolation and qPCR analysis on four Drosophila species to compare expression levels of msl2. Our results indicate that MSL2 may be playing a different role at the HL in different Drosophila species. Overall, this gives us better insight on histone gene regulation.