Grainyhead and Zelda compete for binding to the promoters of the earliest-expressed Drosophila genes

Abstract

Maternally contributed mRNAs and proteins control the initial stages of development following fertilization. During this time, most of the zygotic genome remains transcriptionally silent. The initiation of widespread zygotic transcription is coordinated with the degradation of maternally provided mRNAs at the maternal-to-zygotic transition (MZT). While most of the genome is silenced prior to the MZT, a small subset of zygotic genes essential for the future development of the organism is transcribed. Previous work in our laboratory and others identified the TAGteam element, a set of related heptameric DNA-sequences in the promoters of many early-expressed Drosophila genes required to drive their unusually early transcription. To understand how this unique subset of genes is regulated, we identified a TAGteam-binding factor Grainyhead (Grh). We demonstrated that Grh and the previously characterized transcriptional activator Zelda (Zld) bind to different TAGteam sequences with varying affinities, and that Grh competes with Zld for TAGteam occupancy. Moreover, overexpression of Grh in the early embryo causes defects in cell division, phenocopying Zld depletion. Our findings indicate that during early embryonic development the precise timing of gene expression is regulated by both the sequence of the TAGteam elements in the promoter and the relative levels of the transcription factors Grh and Zld.

Fig. 1

Purification and identification of the Sxl_Pe and zen VRE-binding factor Grainyhead. (A) Purification scheme used to identify factor(s) binding to Sxl_Pe and the zen VRE. (B) DNase I protection by Mono S fractions (indicated above each lane) using a Sxl_Pe DNA fragment. (C) Analysis of proteins eluting from the DNA-affinity column by SDS-PAGE and silver staining (top). The two polypeptides eluting from the column at 0.25 M and 0.5 M KCl were identified as Grh by mass spectrometry. Asterisks denote keratin. 15% of each elution was loaded. IN, 1% of input. FT, 1% of flow through. W, 1.5% of wash. Shown below is an immunoblot using αGrh antibodies on protein from the DNA-affinity column. 5% of each elution was loaded. (D) SDS-PAGE and silver stain analysis of the purified recombinant Grh. Molecular weight (left) is indicated in kD. (E) DNase I protection by increasing amounts of recombinant Grh (rGrh) (left) or purified protein from nuclear extract (right) of a zen VRE DNA fragment. +, protein from embryonic nuclear extract. −, no protein.

Fig. 2

Grh binds to TAGteam elements. (A) DNase I protection by protein purified from embryonic nuclear extract of a fragment of Sxl_Pe containing either wild-type (WT) or mutated (MUT) overlapping CAGGCAG elements. +, protein from Mono S peak. −, no protein. (B) DNase I protection by rGrh using portions of the sc promoter containing either a CAGGCAG element (left) or multiple CAGGTAG elements (right). +, rGrh. −, no protein. (C) Quantitation of EMSAs using rGrh and probes containing different TAGteam elements. Data are the mean +/− s.d. from two independent EMSAs. A representative EMSA is shown below. −, no protein.

Fig. 3

Grh is expressed in the pre-cellular blastoderm embryo. (A) RT-PCR, detecting all possible grh mRNAs, on RNA extracted from wild-type egg chambers and embryos at the stages of development indicated above each lane. The asterisk indicates a primer dimer as evidenced by a reaction containing no cDNA (not shown). AEL, after egg laying. (B) An immunoblot using αGrh 25 antibodies on protein extracted from equal volumes of egg chambers and embryos at various stages. Tubulin was included as a loading control.

Fig. 4

Divergent TAGteam elements are bound by Grh and Zld with different affinities. (A) SDS-PAGE and silver stain analysis of the purified recombinant Zld. Molecular weight (left) is indicated in kD. The asterisk indicates a copurifying degradation product. (B) DNase I protection by rGrh or increasing amounts rZld on a fragment of the zen VRE. −, no protein. (C) Quantitation of EMSAs using rZld and probes containing different TAGteam elements. Data are the mean +/− s.d. from two independent EMSAs. A representative EMSA is shown below. −, no protein.

Fig. 5

Grh and Zld compete for binding to TAGteam elements. (A) EMSAs using (Grh(603–1032), rZld, or both proteins combined (as indicated above each lane). The sequence of the probe used in the assays is shown below with the TAGteam elements bolded. Arrows indicate the two overlapping CAGGCAG elements. (B) Immunoblots using the antibodies indicated on either full-length recombinant protein (left; Zld, upper panel; Grh, middle panel) or protein extract from the equivalent of five embryos (right). Tubulin was included as a loading control. AEL, after egg laying. (C, D, E, F) DAPI-stained cycle 13 embryos. (C) A wild-type embryo. (D) An embryo with overexpressed Grh. Arrowheads indicate anaphase bridges, and arrows indicate perpendicular cell divisions that have resulted in nuclei below the plane of focus. (E, F) Two different focal planes of a single embryo overexpressing Grh. The dotted circle encompasses the identical area in each focal plane highlighting two nuclei resulting from a perpendicular cell division. Scale bars, 5 µm.