Drosophila Histone Locus Body assembly and function involves multiple interactions

The histone locus body (HLB) assembles at replication-dependent (RD) histone loci and concentrates factors required for RD histone mRNA biosynthesis. The D. melanogaster genome has a single locus comprised of ~100 copies of a tandemly arrayed 5 kB repeat unit containing one copy of each of the 5 RD histone genes. To determine sequence elements required for D. melanogaster HLB formation and histone gene expression, we used transgenic gene arrays containing 12 copies of the histone repeat unit that functionally complement loss of the ~200 endogenous RD histone genes. A 12x histone gene array in which all H3-H4 promoters were replaced with H2a-H2b promoters (12xPR) does not form an HLB or express high levels of RD histone mRNA in the presence of the endogenous histone genes. In contrast, this same transgenic array is active in HLB assembly and RD histone gene expression in the absence of the endogenous RD histone genes and rescues the lethality caused by homozygous deletion of the RD histone locus. The HLB formed in the absence of endogenous RD histone genes on the mutant 12x array contains all known factors present in the wild type HLB including CLAMP, which normally binds to GAGA repeats in the H3-H4 promoter. These data suggest that multiple protein-protein and/or protein-DNA interactions contribute to HLB formation, and that the large number of endogenous RD histone gene copies sequester available factor(s) from attenuated transgenic arrays, thereby preventing HLB formation and gene expression. ChIP-qPCR and ChIP-Seq experiments were done on homozygous histone locus (HisC) deletion embryos rescued by the 12 copy promoter replacement (PR) or designer wild type (DWT) arrays on the third chromosome (VK33) to determine CLAMP's interaction with the locus ChIP was performed according to (Blythe and Wieschaus, 2015) with the following particulars. 2-4 hours old embryos (~200 embryos for each of three biological replicates) from HisC/HisC; 12xDWT/12xDWT or 12xPR/12xPR strains were collected, fixed and stored at −80°C until further processing. Immunoprecipitation of chromatin preparations was performed using rabbit anti-CLAMP antibody (Rieder et al., 2017) and a polyclonal rabbit IgG (Millipore-Sigma, 12-370). qPCR was performed as described in (Urban et al., 2017). Three biological replicates and two technical replicates were used for each genotype. We synthesized libraries using 4ul of inputs and 36ul of each ChIP sample (CLAMP or IgG) according to Blythe and Wieschaus (2015). We made libraries using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs), pooled 12 samples per lane, and sequenced libraries on an Illumina HiSeq 2500 in 2×150-bp mode (Genewiz).