title: Functional analysis of transcription factor binding sites in human promoters.
abstract: BACKGROUND
  The 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.
  
  
  RESULTS
  In 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Program in Bioinformatics and Integrative Biology and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Troy W
authors.initials: TW
authors.lastname: Whitfield
authors.forename: Jie
authors.initials: J
authors.lastname: Wang
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Collins
authors.forename: E Christopher
authors.initials: EC
authors.lastname: Partridge
authors.forename: Shelley Force
authors.initials: SF
authors.lastname: Aldred
authors.forename: Nathan D
authors.initials: ND
authors.lastname: Trinklein
authors.forename: Richard M
authors.initials: RM
authors.lastname: Myers
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Weng
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D015894:Genome, Human; D006801:Humans; D059372:Nucleotide Motifs; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000378:metabolism*; D024363:Transcription Initiation Site; D050986:YY1 Transcription Factor / Q000378:metabolism*
chemical_list: D014157:Transcription Factors; D050986:YY1 Transcription Factor