Abstract
Calcium-Response Factor (CaRF) was first identified as a transcription factor based on its affinity for a neuronal-selective calcium-response element (CaRE1) in the gene encoding Brain-Derived Neurotrophic Factor (BDNF). However, because CaRF shares no homology with other transcription factors, its properties and gene targets have remained unknown. Here we show that the DNA binding domain of CaRF has been highly conserved across evolution and that CaRF binds DNA directly in a sequence-specific manner in the absence of other eukaryotic cofactors. Using a binding site selection screen we identify a high-affinity consensus CaRF response element (cCaRE) that shares significant homology with the CaRE1 element of Bdnf. In a genome-wide chromatin immunoprecipitation analysis (ChIP-Seq), we identified 176 sites of CaRF-specific binding (peaks) in neuronal genomic DNA. 128 of these peaks are within 10kB of an annotated gene, and 60 are within 1kB of an annotated transcriptional start site. At least 138 of the CaRF peaks contain a common 10-bp motif with strong statistical similarity to the cCaRE, and we provide evidence predicting that CaRF can bind independently to at least 64.5% of these motifs in vitro. Analysis of this set of putative CaRF targets suggests the enrichment of genes that regulate intracellular signaling cascades. Finally we demonstrate that expression of a subset of these target genes is altered in the cortex of Carf knockout (KO) mice. Together these data strongly support the characterization of CaRF as a unique transcription factor and provide the first insight into the program of CaRF-regulated transcription in neurons.
Highlights
The function of any given transcription factor is determined in large part by its DNA binding specificity, which defines its potential target genes
Despite the absence of any gene products with significant similarity to Calcium-Response Factor (CaRF) in the Drosophila melanogaster or Caenorhabitas elegans genomes, a conserved CaRF ortholog is present in the genome of the cnidarian starlet sea anemone Nematostella vectensis, which diverged from the vertebrate lineage over 700 million years ago [29] (Figure 1a)
CaRF was first cloned in a yeast one-hybrid screen based on its ability to bind to the CaRE1 calcium-response element of the Bdnf gene [14]
Summary
The function of any given transcription factor is determined in large part by its DNA binding specificity, which defines its potential target genes. Individual transcription factors within a family often serve related functions [2] and may compensate at least in part for the loss of other family members [3]. By using chromatin immunoprecipitation followed either by hybridization to tiled genomic microarrays (ChIP-chip) or high-throughput sequencing (ChIP-Seq) it is possible to identify a large, unbiased set of transcription factor binding sites, suggesting candidate target genes [6,7]. Studies of this kind have been used to reveal unexpected sequence variation between the individual binding sites selected by a single transcription factor. By elucidating large sets of potential target genes, these data may suggest new functions for a transcription factor in previously unanticipated cellular processes [6,7,8]
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