Abstract
BackgroundThe binding of transcription factors to specific locations in the genome is integral to the orchestration of transcriptional regulation in cells. To characterize transcription factor binding site function on a large scale, we predicted and mutagenized 455 binding sites in human promoters. We carried out functional tests on these sites in four different immortalized human cell lines using transient transfections with a luciferase reporter assay, primarily for the transcription factors CTCF, GABP, GATA2, E2F, STAT, and YY1.ResultsIn each cell line, between 36% and 49% of binding sites made a functional contribution to the promoter activity; the overall rate for observing function in any of the cell lines was 70%. Transcription factor binding resulted in transcriptional repression in more than a third of functional sites. When compared with predicted binding sites whose function was not experimentally verified, the functional binding sites had higher conservation and were located closer to transcriptional start sites (TSSs). Among functional sites, repressive sites tended to be located further from TSSs than were activating sites. Our data provide significant insight into the functional characteristics of YY1 binding sites, most notably the detection of distinct activating and repressing classes of YY1 binding sites. Repressing sites were located closer to, and often overlapped with, translational start sites and presented a distinctive variation on the canonical YY1 binding motif.ConclusionsThe genomic properties that we found to associate with functional TF binding sites on promoters -- conservation, TSS proximity, motifs and their variations -- point the way to improved accuracy in future TFBS predictions.
Highlights
The binding of transcription factors to specific locations in the genome is integral to the orchestration of transcriptional regulation in cells
We expect that our predicted Yin Yang 1 (YY1) binding sites will predominantly be recognized by YY1, rather than its paralogues. It has been reported from motif analysis of high-throughput DNA binding data (ChIP-chip) that YY1 binding sites may be categorized into two distinct classes: one class with binding sites located downstream of the transcriptional start site (TSS), overlapping with translational start sites and another class upstream, or frequently atop, the TSS [77]; in this work, we find that these two classes map onto functional categories, with the former being associated with transcriptional repression and the latter with activation
As described in the Materials and methods section, highthroughput ChIP-seq data were used in conjunction with known specificities (PWMs) to identify putative transcription factor (TF) binding sites on human promoters
Summary
The binding of transcription factors to specific locations in the genome is integral to the orchestration of transcriptional regulation in cells. The most basic elements used for identifying TF binding sites from sequences are the characteristic binding properties for each TF, comprising the width of DNA binding site and the nucleotide preferences at each position These properties are quantitatively described by a position weight matrix (PWM) [13] and can be deduced from aligning a set of DNA sequences that are experimentally known to bind the TF. We know that the vast majority of these predicted sites do not function in the cell While they can accurately predict in vitro binding [14], position weight matrices represent the in vivo reality more accurately when used in concert with additional knowledge.
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