507B Poster - 06. Regulation of gene expression
Friday April 08, 2:00 PM - 4:00 PM

Authors:
Pamela Diaz-Saldana ¹; Tsutomo Aoki ²; Paul Schedl ²; Leila E. Rieder ¹

Affiliations:
1) Dept. of Biology, Emory University, Atlanta GA; 2) Dept. of Molecular Biology, Princeton University, Princeton, NJ

Keywords:
p. cis-regulatory logic; d. histone variants and modifications

The timing of the nuclear cycles during early Drosophila embryonic development relies heavily upon the availability of histones. The five canonical histone genes are clustered into an array, and ~100 arrays are arranged in tandem at the single histone locus on chromosome 2L. Prior to zygotic genome activation, the histone locus is activated, which includes recruiting a suite of transcription and processing factors collectively known as the histone locus body (HLB). Even though embryonic HLB formation is an evolutionarily conserved event, we do not yet understand the molecular interactions that mark the histone locus for early and unique regulation. Previous in vivo work using transgenes pinpointed specific cis elements (GA-repeats) in the histone3/histone4 promoter that identify the locus for HLB formation by recruiting the transcription factor CLAMP. CLAMP also participates in male dosage compensation. In order to probe the interaction between histone locus and transcription factors more precisely, we tested histone array sequences in vitro for their ability to bind proteins in embryo extract. We performed gel shift assays using histone array probes, confirming the requirement for the GA-repeat cis elements in the histone3/histone4 promoter. In addition, we performed supershifts and observed that both CLAMP and the GA-repeat binding protein GAGA factor bind to this region in vitro. Interestingly, both of these proteins are members of the late boundary complex (LBC), a giant insulator complex that forms in late embryos and participates in both fab7 boundary function and dosage compensation. In the future, we will test the ability of recombinant CLAMP alone to target cis elements in the histone3/histone4 promoter, which it may do prior to LBC formation.