D1 is a novel enhancer blocker protein that functions via multiple AT-rich simple sequence repeats

Simple sequence repeats (SSRs) are tandem repetitions of 1-6 bp DNA motifs at least 12 bp long. Certain length-enriched SSRs function as enhancers/silencers, enhancer blockers and barriers in a mammalian cell line. However, whether SSRs have similar cis-regulatory functions in vivo and their underlying mechanisms are unknown. To address this, we looked for SSR-binding proteins and investigated if SSRs function as enhancer blockers in vivo. We developed a novel Drosophila assay to assess enhancer blocker activity in vivo and to circumvent the low throughput and position effects seen in traditional enhancer blocker assays. Our assay uses endogenous vestigial gene enhancers, which, when blocked, result in easily scorable wing phenotypes increasing throughput. The attP-attB-based recombination system to integrate test fragments at specific site avoids positional effects. Furthermore, using EMSAs and DNA pull-downs followed by LC-MS/MS, we show that SSRs bind to proteins in a sequence-specific manner and identify 33 unique SSR-binding proteins. One of these proteins, D1, was enriched in several SSRs, viz., AAT14, AAAT10, AAAAT8, AATAT9, AAAG13 and AAAAG11. Using our novel vestigial in vivo enhancer blocker assay, we show for the first time that AT-rich SSRs function as enhancer blockers in a D1-dependent manner in Drosophila.