Abstract
BackgroundDifferent approaches have been developed to dissect the interplay between transcription factors (TFs) and their cis-acting sequences on DNA in order to identify TF target genes. Here we used a combination of computational and experimental approaches to identify novel direct targets of TFAP2A, a key TF for a variety of physiological and pathological cellular processes. Gene expression profiles of HeLa cells either silenced for TFAP2A by RNA interference or not were previously compared and a set of differentially expressed genes was revealed.ResultsThe regulatory regions of 494 TFAP2A-modulated genes were analyzed for the presence of TFAP2A binding sites, employing the canonical TFAP2A Positional Weight Matrix (PWM) reported in Jaspar http://jaspar.genereg.net/. 264 genes containing at least 2 high score TFAP2A binding sites were identified, showing a central role in "Cellular Movement" and "Cellular Development". In an attempt to identify TFs that could cooperate with TFAP2A, a statistically significant enrichment for SP1 binding sites was found for TFAP2A-activated but not repressed genes. The direct binding of TFAP2A or SP1 to a random subset of TFAP2A-modulated genes was demonstrated by Chromatin ImmunoPrecipitation (ChIP) assay and the TFAP2A-driven regulation of DCBLD2/ESDN/CLCP1 gene studied in details.ConclusionsWe proved that our computational approaches applied to microarray selected genes are valid tools to identify functional TF binding sites in gene regulatory regions as confirmed by experimental validations. In addition, we demonstrated a fine-tuned regulation of DCBLD2/ESDN transcription by TFAP2A.
Highlights
Different approaches have been developed to dissect the interplay between transcription factors (TFs) and their cis-acting sequences on DNA in order to identify Transcription Factor (TF) target genes
Functional TF binding sites (TFBSs) can be identified in the genome by computational approaches or experimentally by Chromatin ImmunoPrecipitation and hybridization on a genomic microarray (ChIP on Chip) [2] or by high-throughput selection procedures (SELEX) in which pools of random DNA sequences are mixed with a TF and those that are preferentially bound are recovered and sequenced [3,4]
Affinity scores were assigned using standard log-likelihood ratios [40] and a binding site defined as an oligonucleotide with log-likelihood ratio higher than 66% of the maximum score possibly associated to the Positional Weight Matrix (PWM)
Summary
Different approaches have been developed to dissect the interplay between transcription factors (TFs) and their cis-acting sequences on DNA in order to identify TF target genes. We used a combination of computational and experimental approaches to identify novel direct targets of TFAP2A, a key TF for a variety of physiological and pathological cellular processes. Transcription is the initial step of gene expression and it involves a multitude of transcription factors (TFs), their corresponding cis-acting elements on DNA, additional co-factors and the influence of chromatin structure [1]. Functional TF binding sites (TFBSs) can be identified in the genome by computational approaches or experimentally by Chromatin ImmunoPrecipitation and hybridization on a genomic microarray (ChIP on Chip) [2] or by high-throughput selection procedures (SELEX) in which pools of random DNA sequences are mixed with a TF and those that are preferentially bound are recovered and sequenced [3,4]. The microarray assays help to narrow down the number of genes to be analyzed, focusing on those more likely to be regulated by the same TFs, reducing the false positive and negative rates
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