Multi-level regulation of even-skipped stripes by the ubiquitous factor Zelda.

The zinc-finger protein Zelda (Zld) is a key activator of zygotic transcription in early Drosophila embryos. Here, we study Zld-dependent regulation of the seven-striped pattern of the pair-rule gene even-skipped (eve). Individual stripes are regulated by discrete enhancers that respond to broadly distributed activators; stripe boundaries are formed by localized repressors encoded by the gap genes. The strongest effects of Zld are on stripes 2, 3 and 7, which are regulated by two enhancers in a 3.8 kb genomic fragment that includes the eve basal promoter. We show that Zld facilitates binding of the activator Bicoid and the gap repressors to this fragment, consistent with its proposed role as a pioneer protein. To test whether the effects of Zld are direct, we mutated all canonical Zld sites in the 3.8 kb fragment, which reduced expression but failed to phenocopy the abolishment of stripes caused by removing Zld in trans. We show that Zld also indirectly regulates the eve stripes by establishing specific gap gene expression boundaries, which provides the embryonic spacing required for proper stripe activation.

Suboptimal Intermediates Underlie Evolution of the Bicoid Homeodomain.

Changes in regulatory networks generate materials for evolution to create phenotypic diversity. For transcription networks, multiple studies have shown that alterations in binding sites of cis-regulatory elements correlate well with the gain or loss of specific features of the body plan. Less is known about alterations in the amino acid sequences of the transcription factors (TFs) that bind these elements. Here we study the evolution of Bicoid (Bcd), a homeodomain (HD) protein that is critical for anterior embryo patterning in Drosophila. The ancestor of Bcd (AncBcd) emerged after a duplication of a Zerknullt (Zen)-like ancestral protein (AncZB) in a suborder of flies. AncBcd diverged from AncZB, gaining novel transcriptional and translational activities. We focus on the evolution of the HD of AncBcd, which binds to DNA and RNA, and is comprised of four subdomains: an N-terminal arm (NT) and three helices; H1, H2, and Recognition Helix (RH). Using chimeras of subdomains and gene rescue assays in Drosophila, we show that robust patterning activity of the Bcd HD (high frequency rescue to adulthood) is achieved only when amino acid substitutions in three separate subdomains (NT, H1, and RH) are combined. Other combinations of subdomains also yield full rescue, but with lower penetrance, suggesting alternative suboptimal activities. Our results suggest a multistep pathway for the evolution of the Bcd HD that involved intermediate HD sequences with suboptimal activities, which constrained and enabled further evolutionary changes. They also demonstrate critical epistatic forces that contribute to the robust function of a DNA-binding domain.