Long-range transcriptional regulation by the p110 CUX1 homeodomain protein on theENCODE array

Charles Vadnais,Ryoko Harada,Alain Nepveu,Pier-Luc Clermont,Lam Leduy,Arif A Awan,Ginette Bérubé

doi:10.1186/1471-2164-14-258

Charles Vadnais, Ryoko Harada + Show 5 more

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https://doi.org/10.1186/1471-2164-14-258

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Abstract

BackgroundOverexpression of the Cut homeobox 1 gene, CUX1, inversely correlates with patient survival in breast cancers. Cell-based assays and molecular studies have revealed that transcriptional regulation by CUX1 involves mostly the proteolytically processed p110 isoform. As there is no antibody specific to p110 CUX1 only, an alternate strategy must be employed to identify its targets.ResultsWe expressed physiological levels of a tagged-p110 CUX1 protein and performed chromatin affinity purification followed by hybridization on ENCODE and promoter arrays. Targets were validated by chromatin immunoprecipitation and transcriptional regulation by CUX1 was analyzed in expression profiling and RT-qPCR assays following CUX1 knockdown or p110 CUX1 overexpression. Approximately 47% and 14% of CUX1 binding sites were respectively mapped less than 4 Kbp, or more than 40 Kbp, away from a transcription start site. More genes exhibited changes in expression following CUX1 knockdown than p110 CUX1 overexpression. CUX1 directly activated or repressed 7.4% and 8.4% of putative targets identified on the ENCODE and promoter arrays respectively. This proportion increased to 11.2% for targets with 2 binding sites or more. Transcriptional repression was observed in a slightly higher proportion of target genes. The CUX1 consensus binding motif, ATCRAT, was found at 47.2% of the CUX1 binding sites, yet only 8.3% of the CUX1 consensus motifs present on the array were bound in vivo. The presence of a consensus binding motif did not have an impact on whether a target gene was repressed or activated. Interestingly, the distance between a binding site and a transcription start site did not significantly reduced the ability of CUX1 to regulate a target gene. Moreover, CUX1 not only was able to regulate the next adjacent gene, but also regulated the gene located beyond this one as well as the gene located further away in the opposite direction.ConclusionOur results demonstrate that p110 CUX1 can activate or repress transcription when bound at a distance and can regulate more than one gene on certain genomic loci.

Highlights

Overexpression of the Cut homeobox 1 gene, CUX1, inversely correlates with patient survival in breast cancers
Since p110 CUX1 is generated by proteolytic processing, its primary sequence is included in the full-length CUX1 protein sequence
When we considered all 513 CUX1 binding sites and 445 adjacent ENCyclopedia of DNA Elements (ENCODE) genes, we found that 92 genes (21%) were identified in the promoter array (Table 7, third column)

Summary

Introduction

Overexpression of the Cut homeobox 1 gene, CUX1, inversely correlates with patient survival in breast cancers. We know that c-MYC binds to approximately 20% of gene promoters and is capable of regulating genes at a distance [14,15,16] Another major conceptual advance concerns the criteria to define a transcriptional target. Experimental evidence typically included the presence of a consensus binding motif within a core promoter, in vitro binding assays and luciferase reporter assays While these assays are still employed, it is clear that they cannot provide definitive evidence that a transcription factor regulates a specific gene. Additional evidence must include chromatin immunoprecipitation assays to demonstrate "in vivo" DNA binding, and change in expression of the endogenous gene target in response to the knockdown and/or overexpression of the transcription factor

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