Ascl1 and Neurog2 form novel complexes and regulate Delta-like3 (Dll3) expression in the neural tube

Abstract

Delta-like 3 (Dll3) is a Delta family member expressed broadly in the developing nervous system as neural progenitor cells initiate differentiation. A proximal promoter sequence for Dll3 is conserved across multiple species and is sufficient to direct GFP expression in a Dll3-like pattern in the neural tube of transgenic mice. This promoter contains multiple E-boxes, the consensus binding site for bHLH factors. Dll3 expression and the activity of the Dll3-promoter in the dorsal neural tube depends on the basic helix-loop-helix (bHLH) transcription factors Ascl1 (Mash1) and Neurog2 (Ngn2). Mutations in each E-box identified in the Dll3-promoter allowed distinct enhancer or repressor properties to be assigned to each site individually or in combination. In addition, each E-box has distinct characteristics relative to binding of bHLH factors Ascl1, Neurog1, and Neurog2. Surprisingly, novel Ascl1 containing DNA binding complexes are identified that interact with specific E-box sites within the Dll3-promoter in vitro. These complexes include Ascl1/Ascl1 homodimers and Ascl1/Neurog2 heterodimers, complexes that in some cases require additional undefined factors for efficient DNA binding. Thus, a complex interplay of E-box binding proteins spatially and temporally regulate Dll3 levels during neural tube development.

Figure 1. A proximal Dll3 promoter conserved between mouse and human directs Dll3 like expression in transgenic mice

(A) Diagram illustrating sequence homology regions A, B, and C in the 5’ proximal sequence of Dll3. Sequence from mouse for each homology region is shown with capital letters indicating conserved nucleotides between human and mouse sequences. Location relative to the start codon (ATG in bold) in mouse and human is given. E0-E6 E-boxes and an N-box are highlighted in red. The predicted transcription start site is indicated by the arrow in Homology C. Oligonucleotide probes used for EMSA experiments are underlined. (B) whole mount GFP fluorescence in a transgenic embryo expressing GFP under the control of the Dll3 proximal regulatory sequence (Dll3-WT). Dashed line indicates location of sections shown in (C,D). (C,D) cross sections of the neural tube with flanking dorsal root ganglia (arrows) showing mRNA in situ hybridization for Dll3 and GFP. (C’,D’) are higher magnification images of the dorsal neural tube highlighting the ventricular zone (VZ). (E) whole mount GFP fluorescence in a transgenic embryo expressing GFP from the Dll3 promoter that has been mutated at all seven E-box sites (Dll3-mET, see Fig. 4 for diagram).

Figure 2. Dll3 expression and Dll3 promoter activity in the dorsal neural tube requires Ascl1 and Neurog2

(A-G) mRNA in situ hybridization on transverse sections of E11.5 mouse neural tube. Dll3 expression in wild-type (A), Ascl1-/- (C), Neurog2-/- (E), and Neurog1-/- (G) embryos showing the requirement for Ascl1 for much of the dorsal Dll3 expression (between arrowheads in C), and Neurog2 for Dll3 expression in dorsolateral domains (dashed line in E compared to A). For reference, mRNA expression domains for Ascl1 (B), Neurog2 (D), and Neurog1 (F) in wild-type embryos are shown. (H-I) GFP fluorescence in Dll3-GFP transgenic embryos at E11.5 in the presence (H,H’) or absence (I,I’) of Ascl1. (H’,I’) are higher magnification images of (H,I) to highlight the loss of GFP cells in the ventricular zone (VZ) of the Ascl1 mutants. Arrows indicate the dorsal root ganglia and the arrowheads indicate the normal dorsal domain of Ascl1 expression.

Figure 3. Ascl1 occupies the regulatory regions of Dll1 and Dll3 in embryonic neural tube

Chromatin from E12.5 neural tubes immunoprecipitated using Ascl1 antibodies (top) is enriched for the Dll3 promoter (Dll3) and a previously identified enhancer in Dll1 (Dll1-M) (Castro et al., 2006). Control DNA regions including the open reading frame of the Dll1 gene (Dll1 ORF) and Gapdh are not enriched. Chromatin immunoprecipitated with Ascl1 antibodies from Ascl1 mutant neural tubes was not enriched for any regions tested. The ChIP efficiency with chromatin immunoprecipitated using antibodies to RNA polymerase II (bottom panel) is comparable or higher from the Ascl1 null tissue than from wild-type tissue, confirming the competence of the Ascl1 null tissue in this assay. ** p value <0.001, * p value <0.05.

Figure 4. E-box sites are required for activity of the 640 bp Dll3 promoter

Transient transgenic embryos with a wild-type or E-box mutant Dll3 promoter driving GFP expression at E11.5 are shown in whole mount or as one half of a cross section through the neural tube. The blue circles represent each E-box, and the X indicates a mutation of the site. The relative expression in the neural tube is indicated by +, and expression in limb or in ectopic locations is indicated by Y (expression seen) or N (no expression seen). Temporal ectopic expression is early expression in the ventricular zone (arrowheads), and tissue ectopic expression is expression detected aberrantly outside the neural tube (arrows). The number of expressing embryos analyzed for each transgene is indicated (# Expressing). The images shown were obtained using identical exposure time for the whole mount embryos and identical imaging parameters on the confocal for the cross sections. Each embryo is representative of those obtained for each transgene. The inset in Dll3-mET was imaged at a higher gain to illustrate the low level GFP expression detected is restricted to Dll3 domains.

Figure 5. EMSA using in vitro translated proteins reveal differences in bHLH complexes binding Dll3 promoter E-boxes

(A) A summary of multiple EMSA experiments with different in vitro translated proteins showing band intensities for each E-box probe (E0-E6) normalized to the lowest measurable Ascl1/E12 heterodimer band (probe E3). No detectable gel shifted band is shown as a box below the X-axis. A white box indicates that condition was not tested. (B) A representative EMSA with E5 probe that generated the data summarized in (A). In each case cold competitor oligonucleotides with wild-type or mutant E-box sequences demonstrate the requirement for the E-box. (C) EMSA demonstrating Ascl1/Ascl1 homodimers (lanes 2-6) and Ascl1/Neurog2 heterodimers (lanes 8-12) can bind E2 probe in an E-box dependent manner. Pretreating lysates with antibodies (ab) to Ascl1 and Neurog2, but not control GFP disrupted formation of the Ascl1 containing complexes.

Figure 6. EMSA using nuclear extracts reveal the formation of Ascl1/Neurog2 DNA binding complexes

Nuclear extracts (NE) from E10.5 mouse neural tube contain DNA binding activities (lane 1) using oligonucleotides probes from E0 (A), E2 (B), and E4 (C). Except for E0, complex formation requires an intact E-box shown using cold competitor oligonucleotides (lanes 2,3). Extracts were preincubated with untreated or heat inactivated (Δ) antibodies (ab) specific to Ascl1, Neurog2, Tcfe2a-E12, or control GFP (lanes 4-13). Arrows indicate the position of the complexes containing Ascl1 and Neurog2. The † indicates the lanes where complexes are lost with addition of specific antibodies to Ascl1 and Neurog2 (B-lanes 6, 8; C-lanes 6, 8, 10). Asterisk in (C) indicates a new band revealed by depleting Ascl1 and Neurog2 from the extract. Models shown on the right depict proposed complexes that bind each E-box.

Figure 7. Ascl1/Ascl1 homodimers and Ascl1/Neurog2 heterodimers function as transcriptional activators

The activity of Firefly luciferase reporters with E-box E2 or mutant E2 were assayed in HEK293 cells expressing various bHLH factors. Firefly luciferase activity for each reporter was normalized to control Renilla luciferase activity, and then represented as the fold activation through the E2 elements versus the mutant E2. pMiWIII is the empty expression vector. Tethered constructs are indicated by ‘t’. All bHLH factors activated expression of the E2 reporter constructs but to varying extents. Mean values are shown for n=6 transfections.