DrosophilaHistone Locus Body assembly and function involves multiple interactions

Abstract

30The histone locus body (HLB) assembles at replication-dependent (RD) histone loci and 31 concentrates factors required for RD histone mRNA biosynthesis. The D. melanogaster genome 32 has a single locus comprised of ~100 copies of a tandemly arrayed repeat unit containing one 33 copy of each of the 5 RD histone genes. To determine sequence elements required for D. 34 melanogaster HLB formation and histone gene expression, we used transgenic gene arrays 35 containing 12 copies of the histone repeat unit that functionally complement loss of the ~200 36 endogenous RD histone genes. A 12x histone gene array in which all H3-H4 promoters were 37 replaced with H2a-H2b promoters does not form an HLB or express high levels of RD histone 38 mRNA in the presence of the endogenous histone genes. In contrast, this same transgenic array is 39 active in HLB assembly and RD histone gene expression in the absence of the endogenous RD 40 histone genes and rescues the lethality caused by homozygous deletion of the RD histone locus. 41 The HLB formed in the absence of endogenous RD histone genes on the mutant 12x array 42 contains all known factors present in the wild type HLB including CLAMP, which normally 43 binds to GAGA repeats in the H3-H4 promoter. These data suggest that multiple protein-protein 44 and/or protein-DNA interactions contribute to HLB formation, and that the large number of 45 endogenous RD histone gene copies sequester available factor(s) from attenuated transgenic 46 arrays, thereby preventing HLB formation and gene expression. 47 48 3 109In this work, we leveraged transgenic histone gene arrays to test whether the H3-H4 110 promoter region is necessary for in vivo function of the RD histone locus. We found that 111 replacement of H3-H4 promoters with H2a-H2b promoters results in an attenuated transgenic 112 histone gene array that does not function in the presence of the intact endogenous RD histone 113 locus, but surprisingly provides full in vivo function, including normal HLB assembly and 114 histone gene expression, when the endogenous RD histone locus is absent. These results suggest 115 6 that multiple elements in the histone genes and core HLB proteins are involved in HLB 116 assembly. 118 Results 119To study histone gene regulation and the role of histone post-translational modifications 120 in chromatin we previously developed an experimental system in which BAC-based, transgenic 121 histone gene arrays containing 12 copies of the 5kb histone repeat unit assemble HLBs and 122 functionally complement homozygous loss of the ~100 gene copies at the endogenous RD 123 histone locus, HisC (McKay et al., 2015). To study histone gene expression, we created a 124 12x HWT (Histone Wild Type) transgenic construct containing a synonymous polymorphism in the 125 H2a gene (i.e. mutation of a XhoI site) that allows us to distinguish transgenic H2a gene 126 expression from endogenous H2a gene expression (McKay et al., 2015). Here, we extended this 127 design and created a "Designer Wild Type" (DWT) 5kb ...

MSL2 targets histone genes inDrosophila virilis

“…It is difficult to assay CLAMP mel recruitment to single histone array transgenes (L. Hodkinson, observation) since CLAMP is present at loci genome-wide ( Figure 1A-B ) (Urban et al .). It is also possible that CLAMP is present at the 1×His vir transgene without interacting directly with DNA sequence, as was previously observed in histone array transgenes lacking GA-repeats (Koreski et al . 2020).…”

“…Hodkinson, observation) since CLAMP is present at loci genome-wide (Figure 1A-B) (Urban et al). It is also possible that CLAMP is present at the 1xHis vir transgene without interacting directly with DNA sequence, as was previously observed in histone array transgenes lacking GA-repeats (Koreski et al 2020). We therefore turned to an in vitro approach to probe the interaction between CLAMP mel and DNA sequence.…”

“…The above observations include both polytene chromosome immunostaining and analysis of sequencing datasets from cultured cells. Although the histone loci of polytene chromosomes appear to recruit all known HLB factors (Salzler et al 2013;Rieder et al 2017;Koreski et al 2020), salivary gland nuclei undergo endoreplication without cell division and therefore might have unusual histone biogenesis requirements (Andreyeva et al 2017). Cultured cells are asynchronous, and MSL2 might target the D.…”

“…At the endogenous locus, CLAMP increases histone locus chromatin accessibility and promotes histone gene expression of all five replication-dependent genes (Rieder et al 2017). While there are likely multiple redundant mechanisms of HLB formation (Koreski et al 2020), CLAMP is the first known factor that directly interacts with histone locus DNA sequence to promote recognition of the histone genes and recruitment of HLBspecific factors. However, CLAMP is not specific to the histone locus; it is also critical for Drosophila dosage compensation (Soruco et al 2013;Soruco and Larschan 2014), which increases X-linked gene expression to equalize gene dosage of males to females and the X-chromosome to the autosomes.…”

“…2017). While there are likely multiple redundant mechanisms of HLB formation (Koreski et al . 2020), CLAMP is the first known factor that directly interacts with histone locus DNA sequence to promote recognition of the histone genes and recruitment of HLB-specific factors.…”