Genomic selection identifies vertebrate transcription factor Fezf2 binding sites and target genes

Abstract

Identification of transcription factor targets is critical to understanding gene regulatory networks. Here, we uncover transcription factor binding sites and target genes employing systematic evolution of ligands by exponential enrichment (SELEX). Instead of selecting randomly synthesized DNA oligonucleotides as in most SELEX studies, we utilized zebrafish genomic DNA to isolate fragments bound by Fezf2, an evolutionarily conserved gene critical for vertebrate forebrain development. This is, to our knowledge, the first time that SELEX is applied to a vertebrate genome. Computational analysis of bound genomic fragments predicted a core consensus binding site, which identified response elements that mediated Fezf2-dependent transcription both in vitro and in vivo. Fezf2-bound fragments were enriched for conserved sequences. Surprisingly, ∼20% of these fragments overlapped well annotated protein-coding exons. Through loss of function, gain of function, and chromatin immunoprecipitation, we further identified and validated eomesa/tbr2 and lhx2b as biologically relevant target genes of Fezf2. Mutations in eomesa/tbr2 cause microcephaly in humans, whereas lhx2b is a critical regulator of cell fate and axonal targeting in the developing forebrain. These results demonstrate the feasibility of employing genomic SELEX to identify vertebrate transcription factor binding sites and target genes and reveal Fezf2 as a transcription activator and a candidate for evaluation in human microcephaly.

FIGURE 1.

In vitro genomic selection and computational prediction uncovers a putative Fezf2 core binding site. A, flow chart of the in vitro genomic selection method. B, schematic diagram of the Fezf2 protein and Coomassie Blue-stained gel image showing the expression of GST-Fezf2 zinc finger domain fusion (GST-ZF) as well as the GST control. C, ethidium bromide-stained gel image showing the Sau3A1-digested zebrafish genomic DNA. D–G, PCR amplification of the genomic fragments after each round of selection. D, first PCR. Prior to the first PCR, 1 pmol of NotI/Sau3A oligonucleotides were ligated to either elution (E, lanes 5 and 6) or 0.5 m salt wash (W, lanes 1-4) fractions from the first selection round. Lanes 1, 3, and 5 are samples from the column coupled with the GST-ZF. Lanes 2, 4, and 6 are samples from the column coupled with GST control. 1–2 ml of the PCR product (as shown in lane 5 or 6) was used for subsequent rounds of selection. Lanes 7 and 8 are PCR negative control (lane 7, PCR with only NotI/Sau3A1 oligonucleotides as templates; lane 8, PCR with only primers). E–G, Second to fourth rounds of PCR. Lanes 1–3 are samples selected with GST-ZF, and lanes 4-6 are those selected with GST control. Lane 7 (E and F) and lane 6 (G) are PCR negative controls. The amount of in vitro selected genomic templates used for PCR is indicated above the gel images. H, schematic diagram showing the procedure for computational prediction of the core motif.

FIGURE 2.

Computationally predicted core motif functions as Fezf2 response elements in vitro. A, fluorescein-labeled double-stranded oligonucleotides carrying the WT or mutated predicted response elements (0.1 nm) from in vitro selected genomic fragments 4.26 and 4.430 are shown. Motifs contained in these sequences are underlined as well as shown on the left. B, binding of GST-ZF to these elements was measured by fluorescent anisotropy, and the anisotropy graphs are shown. K_dd values were calculated from curve fits. For the 4.26 mutant sequence, the curve is drawn by interpolation. C, schematic diagram shows the constructs used for the luciferase assay. D and E, Dual-Luciferase assay of zebrafish Fezf2 (D) and mouse Fezf2 (E) show that both Fezf2 can activate the luciferase reporter driven by WT response elements. Mutating the conserved residues in the response elements significantly impairs the transactivation. Relative luciferase activity is calculated as a ratio of Fezf transfected to control plasmid transfected, after normalization of firefly luciferase to Renilla luciferase activity. ***, p < 0.001. Error bars, S.E.

FIGURE 3.

Computationally predicted core motif functions as Fezf2-response element in vivo. A, schematic diagram shows the in vivo reporter assay construct. B, images of transient transgenic zebrafish embryos show that four response elements residing in in vitro-selected genomic fragments can serve as forebrain enhancers in vivo. All are lateral views except the last panel (ventral view). C, images of transient transgenic zebrafish embryos show that mutating the conserved residues in the core binding site abolishes forebrain enhancer activity (middle column), and impairing Fezf2 activity through morpholino antisense oligonucleotide-mediated knockdown also impairs forebrain enhancer activity (right column).

FIGURE 4.

Analyses of the selected unique genomic fragments. A, percent of unique genomic fragment containing at least one core binding site. B, occurrence of core binding sites in 1, 2, 3, and 4 or greater copies. C, percent of unique fragments that are evolutionarily conserved between zebrafish and human. D, percent of unique genomic fragments that are annotated as protein-coding exons. E, diagram showing the frequency among the selected unique genomic fragments containing the core binding sites, which are located either inside the gene, or 0–5 kb, 5–10 kb, 10–50 kb, or >50 kb from a gene.

FIGURE 5.

Fezf2 directly activates eomesa and lhx2b in vivo. A and B, schematic diagrams show the location of the Fezf2 binding sites (black bars) in selected genomic fragments (green) located upstream of eomesa (A) and lhx2b (B) genes. C, Western blotting shows that the affinity-purified Fezf2 antibody specifically recognizes the Fezf2 protein in zebrafish embryonic extracts and in transfected HEK293 cells. D, ChIP analysis shows that Fezf2 binds to the response elements near eomesa and lhx2b in vivo. ChIP primers are indicated in A and B (arrows). The unrelated genomic fragment is a computationally identified zebrafish genomic fragment that contains no Fezf2 binding site. E, in situ hybridization shows the expression of fezf2, eomesa, and lhx2b at three developmental stages. Fezf2 expression in the forebrain precedes and overlaps with that of eomesa and lhx2b. F, in situ hybridization shows the expression of eomesa (first row) and lhx2b (second row) in WT (left column), the tof mutant (second column), the fezf2 morphant (third column), and the fezf2-overexpressing (hsp-gal4;uas-fezf2) embryos (right column). Fezf2 is necessary and sufficient to regulate the expression of eomesa and lhx2b. G, labeling with the anti-acetylated tubulin antibody shows the defect of commissural axon targeting in the tof mutant and fezf2 morphant.