Drosophila melanogaster Zelda and Single-minded collaborate to regulate an evolutionarily dynamic CNS midline cell enhancer

Abstract

The Drosophila Zelda transcription factor plays an important role in regulating transcription at the embryonic maternal-to-zygotic transition. However, expression of zelda continues throughout embryogenesis in cells including the developing CNS and trachea, but little is known about its post-blastoderm functions. In this paper, it is shown that zelda directly controls CNS midline and tracheal expression of the link (CG13333) gene, as well as link blastoderm expression. The link gene contains a 5’ enhancer with multiple Zelda TAGteam binding sites that in vivo mutational studies show are required for link transcription. The link enhancer also has a binding site for the Single-minded: Tango and Trachealess:Tango bHLH-PAS proteins that also influences link midline and tracheal expression. These results provide an example of how a transcription factor (Single-minded or Trachealess) can interact with distinct co-regulatory proteins (Zelda or Sox/POU-homeodomain proteins) to control a similar pattern of expression of different target genes in a mechanistically different manner. While zelda and single-minded midline expression is well-conserved in Drosophila, midline expression of link is not well-conserved. Phylogenetic analysis of link expression suggests that ~60 million years ago, midline expression was nearly or completely absent, and first appeared in the melanogaster group (including D. melanogaster, D. yakuba, and D. erecta) >13 million years ago. The differences in expression are due, in part, to sequence polymorphisms in the link enhancer and likely due to altered binding of multiple transcription factors. Less than 6 million years ago, a second change occurred that resulted in high levels of expression in D. melanogaster. This change may be due to alterations in a putative Zelda binding site. Within the CNS, the zelda gene is alternatively spliced beginning at mid-embryogenesis into transcripts that encode a Zelda isoform missing three zinc fingers from the DNA binding domain. This may result in a protein with altered, possibly non-functional, DNA-binding properties. In summary, Zelda collaborates with bHLH-PAS proteins to directly regulate midline and tracheal expression of an evolutionary dynamic enhancer in the post-blastoderm embryo.

Transcriptional Activation Is a Conserved Feature of the Early Embryonic Factor Zelda That Requires a Cluster of Four Zinc Fingers for DNA Binding and a Low-complexity Activation Domain

“…Are Dominant Negative Inhibitors of ZLD-mediated Activation-At least one of two nearly identical zld splice isoforms that lack the coding region for three of the four clustered zinc fingers is expressed in larvae and late-stage embryos (12,13). The predicted proteins resulting from these two different splice isoforms differ by the number and sequence of amino acids following the single remaining zinc finger: 22 amino acids in ZLD-PD and 17 amino acids in ZLD-PF (Fig.…”

“…These isoforms are predicted to produce protein products of either 1596 (ZLD-PA) or 1373 (ZLD-PD) amino acids. (The 1367 amino acid isoform ZLD-PF is likely not expressed (12,13).) Although ZLD-PA, which contains all four zinc fingers of the DNA-binding domain, activated robust gene expression, neither of the truncated isoforms, ZLD-PD or ZLD-PF, activated transcription.…”

“…Conservation of ZLD and TAGteam Sites in Driving Genome Activation-The coding region for ZLD is conserved in other insects, and zld expression in late-stage embryos of multiple Drosophila species has been observed (13 (25,26). By contrast, A. gambiae and N. vitripennis diverged from D. melanogaster about 250 and 300 million years ago, respectively (27)(28)(29).…”

“…Therefore, the primary sequence of this large protein offers few clues as to how it mediates genome activation. Additional splice isoforms of zld have also been identified, and at least one is expressed both in larvae and in the central nervous system of late-stage embryos (12)(13)(14). The proteins resulting from translation of these isoforms, ZLD-PD and ZLD-PF (Fig.…”

Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition

“…Many genes near the MBT are activated by specific early transcription factors, which include Zelda in Drosophila and Nanog, Pou5f1 (Oct4), and SoxB1 in zebrafish (20)(21)(22)(23)(24)(25)(26). In Drosophila, a proposed site-specific repressor, Grainyhead, might be the titrated factor, because it competes with the transcriptional activator Zelda for the same binding sites (21).…”