Recruitment Dynamics and Suggested Role of SPT6 at the Drosophila Histone Locus Body during Early Embryogenesis

Histones are essential for packaging DNA and maintaining genomic stability, with their expression tightly controlled and coupled to the cell cycle. In Drosophila, replication-dependent (RD) histone mRNAs are transcribed from a single locus at which an evolutionarily conserved, phase-separated nuclear body forms — the histone locus body (HLB). Previous work has demonstrated that components are recruited to the HLB in a cell-cycle dependent, hierarchical manner, with SPT6 established as a dynamic component. Highly-conserved SPT6 is generally considered as a nucleosome remodeler and transcription elongation factor known to bind RNA Pol II, though its roles are not fully understood. Notably, recent work has indicated that SPT6 is not necessary for elongation at short genes, a classification including the RD histone genes. Given the known presence of SPT6 at the HLB, we were curious about its role in HLB formation and transcription of the RD histone genes. Here, using antibodies and fluorescently-tagged proteins we visualized the spatiotemporal localization of SPT6 at the HLB in developing Drosophila embryos via confocal microscopy. Combining this approach with sensitive FISH probes to detect nascent RD histone transcripts, we examined the relationship between the localization of SPT6 to the HLB and active transcription. From imaging both live and fixed embryos, we found that SPT6 is continuously enriched at the HLB throughout S phase of the syncytial nuclear cycles, yet only transiently present at the HLB during the beginning of nuclear cycle 14. In contrast, unpaused RNA Pol II and nascent histone mRNA remain present at the HLB throughout all of cycle 14, showing an unexpected uncoupling of the association between SPT6 and RNA Pol II during active transcription. These data also suggest that SPT6 may be functioning at the HLB primarily as a nucleosome remodeler rather than a transcription elongation factor.Bachelor of Scienc