Abstract
BackgroundTranscription factors (TFs) co-ordinately regulate target genes that are dispersed throughout the genome. This co-ordinate regulation is achieved, in part, through the interaction of transcription factors with conserved cis-regulatory motifs that are in close proximity to the target genes. While much is known about the families of transcription factors that regulate gene expression in plants, there are few well characterised cis-regulatory motifs.In Arabidopsis, over-expression of the MYB transcription factor PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT 1) leads to transgenic plants with elevated anthocyanin levels due to the co-ordinated up-regulation of genes in the anthocyanin biosynthetic pathway. In addition to the anthocyanin biosynthetic genes, there are a number of un-associated genes that also change in expression level. This may be a direct or indirect consequence of the over-expression of PAP1.ResultsOligo array analysis of PAP1 over-expression Arabidopsis plants identified genes co-ordinately up-regulated in response to the elevated expression of this transcription factor. Transient assays on the promoter regions of 33 of these up-regulated genes identified eight promoter fragments that were transactivated by PAP1. Bioinformatic analysis on these promoters revealed a common cis-regulatory motif that we showed is required for PAP1 dependent transactivation.ConclusionCo-ordinated gene regulation by individual transcription factors is a complex collection of both direct and indirect effects. Transient transactivation assays provide a rapid method to identify direct target genes from indirect target genes. Bioinformatic analysis of the promoters of these direct target genes is able to locate motifs that are common to this sub-set of promoters, which is impossible to identify with the larger set of direct and indirect target genes. While this type of analysis does not prove a direct interaction between protein and DNA, it does provide a tool to characterise cis-regulatory sequences that are necessary for transcription activation in a complex list of co-ordinately regulated genes.
Highlights
Transcription factors (TFs) co-ordinately regulate target genes that are dispersed throughout the genome
Bioinformatic analysis of the promoters of these direct target genes is able to locate motifs that are common to this sub-set of promoters, which is impossible to identify with the larger set of direct and indirect target genes
While this type of analysis does not prove a direct interaction between protein and DNA, it does provide a tool to characterise cis-regulatory sequences that are necessary for transcription activation in a complex list of co-ordinately regulated genes
Summary
Transcription factors (TFs) co-ordinately regulate target genes that are dispersed throughout the genome. While much is known about the families of transcription factors that regulate gene expression in plants, there are few well characterised cis-regulatory motifs. In plants with annotated whole genome sequence such as Arabidopsis [2], flanking DNA sequences upstream of the coding region can be defined Such sequences are commonly referred to as the promoter and while they can be difficult to delineate in the absence of experimental characterisation, they can be defined as the intergenic sequence upstream of the ATG, and often limited to a defined length eg. DNAse I footprinting [4] and electrophoretic or gel mobility shift assays [5] have been extensively used to characterise cis-regulatory elements Both methods rely on the direct interaction between DNA fragments that contain the DNA-binding region and the corresponding transcription factor. Studies which have used ChIP to identify TF binding sites include the analysis of the AGAMOUS [8], AGL15, [9] and the FLOWERING LOCUS C PROTEIN (FLC) [10] MADS box genes from Arabidopsis, all of which have been shown to bind to a CArG box contained in the promoter of the target gene
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