Bicoid-Dependent Activation of the Target Gene hunchback Requires a Two-Motif Sequence Code in a Specific Basal Promoter

Abstract

In complex genetic loci, individual enhancers interact most often with specific basal promoters. Here we investigate the activation of the Bicoid target gene hunchback (hb), which contains two basal promoters (P1 and P2). Early in embryogenesis, P1 is silent, while P2 is strongly activated. In vivo deletion of P2 does not cause activation of P1, suggesting that P2 contains intrinsic sequence motifs required for activation. We show that a two-motif code (a Zelda binding site plus TATA) is required and sufficient for P2 activation. Zelda sites are present in the promoters of many embryonically expressed genes, but the combination of Zelda plus TATA does not seem to be a general code for early activation or Bicoid-specific activation per se. Because Zelda sites are also found in Bicoid-dependent enhancers, we propose that simultaneous binding to both enhancers and promoters independently synchronizes chromatin accessibility and facilitates correct enhancer-promoter interactions.

Keywords: enhancer; gene regulation; promoter; promoter choice; transcription.

Figure 1.. Testing Intrinsic Enhancer-Promoter Compatibilities at hb

(A) Schematic of a 5.6 kb fragment from the hb locus showing two Bcd-dependent enhancers (gray boxes labeled Dist and Prox), approximate positions of Bcd and Zld binding sites in the two enhancers, and the P1 and P2 basal promoters (shown as green and red arrows, respectively). These conventions are used throughout this paper. See Data S1 for a detailed annotation of the hb locus. (B) Read profiles of ATAC sequencing (ATAC-seq) in embryos at 18 min into NC13 (Blythe and Wieschaus, 2016). (C) Binding profile of Bcd in NC9–NC14 embryos at the hb locus (Xu et al., 2014). (D) Binding profile of Zld in NC13–NC14 embryos (Nien et al., 2011; Sun et al., 2015). (E and F) Expression patterns of P1 transcripts (E) and hb transcripts (F) early in NC14. (G–K) lacZ expression patterns driven by five enhancer-promoter combinations. All embryos in this paper are oriented with anterior to the left and dorsal up.

Figure 2.. Testing Whether Deletion of P2 Causes the Activation of P1

(A) Schematic of the two-step strategy for CRISPR/Cas9-based deletion of P2 core (P2C) sequences. (B–G) Analysis of mutant hb alleles carrying P2 and P1 deletions (for precise breakpoint sequences, see Table S1). Expression patterns of hb mRNA are shown for a wild-type embryo (B), a ΔP2C homozygote (D), and a ΔP1C homozygote (F). hb mRNA levels are also quantified in Figure S1. Cuticle preparations of first instar larvae (C, E, and G) are shown to the right. Each cuticle image is a merged composite of photographs of the anterior and posterior regions of a single larva. Thoracic segments (T1–T3) are labeled when present. (H and I) Analysis of a wild-type dual reporter gene (H) and an identical reporter containing a 517 bp deletion of the P2 promoter (I). See STAR Methods, Figures S2 and S3, Table S2, and Data S2. Schematics of reporter genes are shown on the left and individually labeled. All reporter genes in this paper contain the 5′ and 3′ halves of the lacZ transcription unit cloned immediately downstream of the P1 and P2 basal promoters, respectively. Green and red bars on the right represent levels of 5′ and 3′ lacZ transcripts, respectively, driven by each construct. The levels are shown as number of transcripts per nucleus (see STAR Methods). Error bars represent SEM. The number of embryos measured for each construct is shown below each reporter schematic.

Figure 3.. Mapping Sequences Required and Sufficient for Bcd-Dependent Activation

(A F) Analysis of a wild type dual reporter gene (A) and identical reporter genes containing replacements of P1 sequences with P2 sequences (B, C, and D), and vice versa (E and F). Reporter gene schematics are shown on the left and individually labeled (see also Table S2). P2L and P1L refer to long promoter fragments (~500 bp), while P2C and P1C refer to core (120 bp) promoter fragments. Green and red bars on the right represent levels of 5′ and 3′ lacZ transcripts (number per nucleus), respectively, driven by each construct. Error bars represent SEM. The number of embryos measured for each construct is shown below each reporter schematic.

Figure 4.. Testing Specific Promoter Motifs

(A–L) Analysis of a wild type dual reporter gene (A and B) and identical reporter genes containing replacements of P1 core sequences (B) with P2 sequences (C–E), and conversions of P1 core sequences to insert specific promoter motifs from P2 (F–L). Reporter gene schematics are shown on the left and individually labeled. Complete sequences of each construct are shown in Table S3. Green bars on the right represent levels of 5′ lacZ transcripts (number per nucleus) driven by each construct. All of these constructs drive similar levels of 3′ lacZ transcripts, and we have omitted them from this figure for clarity. Error bars represent SEM. The number of embryos measured for each construct is shown below each reporter schematic.

Figure 5.. Testing the Roles of the Prox and Dist Enhancers in Activating P2

(A) Schematic of the 5.6 kb hb locus showing approximate positions of Bcd and Zld binding sites in the Dist and Prox enhancers (for precise positions, see Data S1). (B–F) Analysis of dual reporter genes containing deletions of the Prox enhancer alone (B), both Prox and Dist enhancers (C), the Dist enhancer alone (D), the Zld site in P2 alone (E), and the Prox enhancer plus the Zld site in P2 (F). Sequences of deletion breakpoints are shown in Table S2. Green and red bars on the right represent levels of 5′ and 3′ lacZ transcripts (number per nucleus), respectively, driven by each construct. Error bars represent SEM. The number of embryos measured for each construct is shown below each reporter schematic. (G–O) Analysis of mutant hb alleles carrying deletions of the Prox enhancer alone (G–I), the Dist enhancer alone (J–L), and the Prox enhancer plus the Zld site in P2 (M–O) (see deletion breakpoints in Table S1). Expression patterns of hb mRNA (G, J, and M; for quantification, see Figure S4), cuticle preparations of first instar larvae (H, K, and N), and graphical representations of the percentage of larvae containing one, two, or three thoracic segments (I, L, and O) are shown for each construct. As in Figure 2, cuticle images (H, K, and N) are each merged composites of photographs of the anterior and posterior regions of single larvae.