Genomic Selection Identifies Vertebrate Transcription Factor Fezf2 Binding Sites and Target Genes

Nan Yang,Su Guo,Hao Li,Lishan Chen,Jiashun Zheng

doi:10.1074/jbc.m111.236471

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.

Highlights

Multiple technologies have been developed to define binding sites for transcription factor (TF) with unknown DNA binding profiles
One well established in vitro method for identifying TF binding sites is based on systematic evolution of ligands by exponential enrichment (SELEX), which allows extraction of oligomers from an initially random pool of oligonucleotides based on their binding affinity for the transcription factor of interest [3,4,5,6,7]
SELEX is highly effective in recovering DNA sequences that interact with the transcription factor of interest in vitro, it remains a difficult task to uncover target genes using the DNA motifs identified through SELEX because they are often short, highly degenerate, and can be found in numerous locations throughout the genome

Summary

EXPERIMENTAL PROCEDURES

Zebrafish Strains and Maintenance—Zebrafish (wild type and the tof mutant) were maintained and bred following standard procedures [40]. Third, or fourth rounds of PCR/binding/elution, purified PCR DNA products were digested with NotI restriction endonuclease (NEB) and subcloned into the NotI site of pBluescript II KSϩ vector (Stratagene) for sequencing. For the analyses of unique Fezf2-bound selected genomic fragments, we first determined whether one SELEX fragment contain the core binding motif by calculating the matrix similarity score using the derived Fezf positional weight matrix from BioProspector. Motifs with scores larger than the cutoff (0.8469) were regarded as a match to the core binding site of Fezf2 This cutoff is the smallest Matrix similarity score of the motifs on the SELEX fragments that were sequenced multiple times. The affinity-purified antibody was tested on zebrafish embryonic extracts, and the HEK293 cell lysates were transfected with or without Fezf through Western blotting analysis. In Situ Hybridization and Immunostaining—RNA in situ hybridization and immunohistochemistry were performed as described [45]

RESULTS

Number of total sequences

DISCUSSION