Investigating Mechanisms of D. melanogaster Histone Locus Body (HLB) Initiation and Maintenance

Emory University, Atlanta GA

Investigators

Abstract

PROJECT SUMMARY Coordinated zygotic gene expression is necessary for the rapid cell divisions that lead to proper development. This is especially true for the uniquely regulated replication-dependent histone genes. Histone proteins are essential for compaction and regulation of the eukaryotic genome, and dysregulation of histone gene expression leads to aberrant cell cycle timing, development, and lethality. In animals, genes encoding the histone proteins are commonly clustered at one or several loci. The histone locus body (HLB) is a conserved collection of factors that localize to histone loci and regulate histone gene expression. The HLB must initiate for the first time at the histone genes during early embryogenesis. Then, histone expression must be maintained by the HLB through development and subsequent cell divisions. While we have begun to define HLB initiation mechanisms, we cannot yet describe how the HLB is maintained. Describing HLB initiation and maintenance mechanisms is especially important to the fields of stem cell differentiation and regenerative medicine that require mature cells to adopt embryonic gene regulatory patterns. The histone genes are amongst the most conserved genes in eukaryotes. While many components of the HLB are also conserved between animals, manipulating the repetitive histone genes is prohibitively difficult in most species and can lead to misexpression and developmental lethality. We therefore employ the powerful model system Drosophila melanogaster in which we leverage histone transgenes that recruit HLB factors and express histone genes, allowing us to manipulate cis elements and determine the effects on HLB formation and gene expression. I hypothesize that mechanisms of HLB initiation early in development are the same as those later responsible for HLB maintenance. I will test my hypothesis through two Specific Aims. In Aim 1, I will compare mechanisms of HLB initiation and maintenance by removing important cis elements after initiation and testing for HLB maintenance. I will also perform ChIP-seq in synchronized cells to determine if important HLB factors are associated with the locus throughout the cell cycle. In Aim 2, I will define the latent potential of differentiated tissues to activate HLB initiation mechanisms. First, I will induce mitotic recombination in larval tissues to determine if the HLB can initiate at a new locus in non-embryonic tissues. I will also mentor an undergraduate student who will use a parallel transgenic system, as I designed a transgene that I predict will delay initiation until later in embryogenesis. Overall, these aims allow us to illustrate how and when histone proteins are regulated and in what contexts factors target these crucial genes for regulation. I designed these experiments, in collaboration with my sponsor and co-sponsor, to address critical knowledge gaps in our field, develop expertise in a broad array of techniques, and provide me with the training opportunities required to become an independent researcher.