Cis element length variability does not confer differential transcription factor occupancy at the D. melanogaster histone locus.

Histone genes require precise regulation to maintain histone homeostasis and ensure nucleosome stoichiometry. Animal histone genes often have unique clustered genomic organization. However, there is variability of histone gene number and organization as well as differential regulation of the histone genes across species. The Drosophila melanogaster histone locus has unique organizational characteristics as it exists as a series of ∼100 highly regular, tandemly repeated arrays of the 5 replication-dependent histone genes at a single locus. Yet D. melanogaster are viable with only 12 transgenic histone gene arrays. We hypothesized that the histone genes across the locus are differentially regulated. We discovered that the GA-repeat within the H3/H4 promoter is the only variable sequence across the histone gene arrays. The H3/H4 promoter GA-repeat is targeted by CLAMP to promote histone gene expression. We also show two additional GA-binding transcription factors, GAGA Factor and Pipsqueak, target the GA-repeat. When we further examined CLAMP and GAF targeting, we determined that neither CLAMP nor GAF show bias for any GA-repeat lengths. Furthermore, we found that the distribution of GA-repeats targeted by both CLAMP and GAF do not change throughout early development. Together our results suggest that the transcription factors targeting the H3/H4 GA-repeat do not impact differential regulation of the histone genes, but indicate that future studies should interrogate additional cis elements or factors that impact histone gene regulation.