Genome-Wide Mapping of Collier In Vivo Binding Sites Highlights Its Hierarchical Position in Different Transcription Regulatory Networks

Abstract

Collier, the single Drosophila COE (Collier/EBF/Olf-1) transcription factor, is required in several developmental processes, including head patterning and specification of muscle and neuron identity during embryogenesis. To identify direct Collier (Col) targets in different cell types, we used ChIP-seq to map Col binding sites throughout the genome, at mid-embryogenesis. In vivo Col binding peaks were associated to 415 potential direct target genes. Gene Ontology analysis revealed a strong enrichment in proteins with DNA binding and/or transcription-regulatory properties. Characterization of a selection of candidates, using transgenic CRM-reporter assays, identified direct Col targets in dorso-lateral somatic muscles and specific neuron types in the central nervous system. These data brought new evidence that Col direct control of the expression of the transcription regulators apterous and eyes-absent (eya) is critical to specifying neuronal identities. They also showed that cross-regulation between col and eya in muscle progenitor cells is required for specification of muscle identity, revealing a new parallel between the myogenic regulatory networks operating in Drosophila and vertebrates. Col regulation of eya, both in specific muscle and neuronal lineages, may illustrate one mechanism behind the evolutionary diversification of Col biological roles.

Fig 1. Genome-wide mapping of Col binding sites.

(A) Col expression in stage 12 and stage 14 embryos, lateral view. In this and all subsequent figures, embryos are oriented anterior to the left. HL: hypopharyngeal lobe; PCs: dorso-lateral muscle progenitors; vnc: ventral nerve chord; lg: lymph gland; md: class IV multidendritic neuron. (B) Fold enrichment distribution of the 559 Col ChIP peaks selected using SISSRs. (C) Single most enriched sequence motif identified by MEME analysis in the 559 Col binding peaks. (D) Graphical representation of the position of Col-binding motifs relative to the center of Col binding peaks. The axis gives the number of motifs in each cluster. (E) GO clusters enriched in putative Col direct target genes. The p-Value of the top two clusters is given in brackets.

Fig 2. Col control of cnc and Ama expression in the head.

(A) Annotation of the Col peak in cnc, adapted from Gene Browser (GEO submission GSE67805). 39,5 kb of cnc genomic region are shown (Chr3R: 19.009.000–19.048.500) with the Flybase gene annotation indicated by bars (transcribed regions) and intervening blue lines (introns). Black arrows indicate the direction of transcription of cnc and fuzzy onions (fzo), inwardly rectifying potassium channel 1 (Irk1). The cnc transcripts coding for the protein isoforms CncA and CncB are indicated. ChIP-seq data for Col (green) substracted from HA (mock) data (red) are shown on the bottom. The Col Dam-ID binding regions [59] are indicated by yellow bars, top line. The summit of the ChIP-Col peak identified by SISSRs and position of the Col binding site(s) identified by MEME are indicated by blue and violet lines, respectively; the position of cnc_Col is represented by a black box; scale is indicated. (B-D) Ventral anterior views of stage 11 embryos. (B) Overlap between cnc_Col (GFP, green) and Col (red) expression in the HL (white arrow). (C) cnc_Col and (D) cnc_Col^mut mRNA expression, showing down-regulation of cnc_Col^mut in the mandibular segment (open arrow). (E,F) Ama_Col, (G,H) Ama_Col^mut expression in stage 10 (E,G) and 11 (F,H) embryos. HL Ama_Col expression (arrow) is lost in Ama_Col^mut (open arrow). (I, J) Overlap between Ama (red), Col (blue), and col2.6–0.9moeGFP (green) expression in the HL (white arrow) in stage 11 wt (I), and col ¹ mutant embryos (J). Separate signals for Ama, GFP (I, J) and Col (inset in I, left panel) are shown in black and white. Ama expression is specifically lost in the HL in col ¹ mutants. The asterisk in C, D, F, H, J indicates Ama and cnc expression independent on Col.

Fig 3. Col direct control of ap expression in Ap neurons.

(A) Annotation of the Col peak in ap, same representation as in Fig 2A; 35.8 kb of the ap genomic region are shown (Chr2R: 1.593.000–1.628.800); the previously described apC enhancer is represented by a blue box. (B) ap_Col (GFP) expression in the dAp (yellow arrow) and Tv1-Tv4 neurons (white arrow) in stage 15 embryos, ventral view. (C) ap_Col^mut expression is severely reduced in dAP neurons and Tv neurons. (D,D’) Close up view of 4 segments of stage 16 embryos, showing the specific overlap between Col (red) and ap_Col (green) in the Tv1 and dAp neurons. (E) all Tv neurons express ap_Col and Eya. (F,G) ap_Col^mut expression is lost in dAp and strongly reduced in Tv neurons.

Fig 4. Col direct control of eya expression in Ap neurons.

(A) Annotation of the Col peaks in eya, same representation as in Fig 2A; 24 kb of the eya genomic region are shown (Chr2L: 6.524.500–6.548.500); the summits of the two ChIP-Col peaks are numbered 1 and 2. (B, C) eya_Col (GFP) expression in the dAp (yellow arrow) and Tv1-Tv4 neurons (white arrow) in stage 16 embryos, ventral view. (C) Close up view of abdominal segments, showing the specific overlap between Col (red) and eya_Col (green) in the dAp (yellow arrow) and Tv1 neurons (white arrow). (D, E) eya_Col^mut expression is lost in dAP neurons and reduced in TV neurons. (F, G) Mutation of the Col binding site 2 (G), but not site 1 (F) eliminates eya_Col (RFP) expression in dAp neurons (yellow arrow).

Fig 5. Cross-regulation between eya and col is required to specify dorso-lateral muscles.

(A) eya_Col (GFP) expression in the DA3 muscle (arrows) in stage 15 embryos, is not detected for eya_Col^mut (B). (C-E) Triple staining of st11 wt (C), +;col ^eCRM -lacZ (D) and col ¹ ;col ^eCRM -lacZ (E) embryos for Nau (blue), eya transcripts (red), and either Col (C) or β-galactosidase (D,E) (green), shows co-expression of Col, col ^e-CRM-LacZ, Nau and eya in DL muscle PCs (white arrow). (C’-E’) only Nau and eya stainings are shown. eya transcription is specifically lost in DL PCs in col ¹ mutant embryos (E,E’); dorso-lateral view of the T2 and T3 segments is shown. (F) Schematic drawing of the dorsal, dorso-lateral and lateral transverse muscles in a stage 16 wt embryo, with the DA3 muscle in red and the LL1 muscle indicated by an arrow. (G, H) Staining of stage 16 wt (G) and (H), eya ^cIi-IID /Df(2L)BSC354 (null) mutant embryos for Col (red) and β3-tubuliin (green). Lateral view of 3 segments. In absence of eya, Col expression is lost in most segments, the LL1 muscle is missing (white arrow) and the DA3 muscle (asterisk) malformed. White brackets indicate dorsal, unaffected muscles while the lateral transverse and ventral muscles (yellow brackets) are moderately affected. (I-J) Col immunostaining of st.16 wt (I), and (J) eya mutant embryos, showing the loss of Col muscle expression in most segments (see also G, H), in eya mutants. (K, L) promuscular Col expression, early stage 11, is reduced in eya mutant embryos (L), compared to wt (K).

Fig 6. Scheme for Col direct regulation of ap and eya in specific neuron and muscle lineages.

Col directly controls ap_Col and eya_Col CRM activity in the Tv and dAP neurons and eya_Col and col_Col CRM in the DA3 muscle lineage. The two Col binding sites in eya_Col CRM are not functionally equivalent (double arrow). eya and col cross-regulate each other in the DA3 PC. Control of DopR and Nplp1 expression in Tv and dAP neurons (Baumgardt et al., 2007) could be indirect.