A genomic approach to the identification and characterization of HOXA13 functional binding elements

HOX proteins are important transcriptional regulators in mammalian embryonic development and are dysregulated in human cancers. However, there are few known direct HOX target genes and their mechanisms of regulation are incompletely understood. To isolate and characterize gene segments through which HOX proteins regulate transcription we used cesium chloride centrifugation-based chromatin purification and immunoprecipitation (ChIP). From NIH 3T3-derived HOXA13-FLAG expressing cells, 33% of randomly selected, ChIP clones were reproducibly enriched. Hox-enriched fragments (HEFs) were more AT-rich compared with cloned fragments that failed reproducible ChIP. All HEFs augmented transcription of a heterologous promoter upon coexpression with HOXA13. One HEF was from intron 2 of Enpp2, a gene highly upregulated in these cells and has been implicated in cell motility. Using Enpp2 as a candidate direct target, we identified three additional HEFs upstream of the transcription start site. HOXA13 upregulated transcription from an Enpp2 promoter construct containing these sites, and each site was necessary for full HOXA13-induced expression. Lastly, given that HOX proteins have been demonstrated to interact with histone deacetylases and/or CBP, we explored whether histone acetylation changed at Enpp2 upon HOXA13-induced activation. No change in the general histone acetylation state was observed. Our results support models in which occupation of multiple HOX binding sites is associated with highly activated genes

Genomic Promoter Analysis Predicts Functional Transcription Factor Binding

Background. The computational identification of functional transcription factor binding sites (TFBSs) remains a major challenge of computational biology. Results. We have analyzed the conserved promoter sequences for the complete set of human RefSeq genes using our conserved transcription factor binding site (CONFAC) software. CONFAC identified 16296 human-mouse ortholog gene pairs, and of those pairs, 9107 genes contained conserved TFBS in the 3 kb proximal promoter and first intron. To attempt to predict in vivo occupancy of transcription factor binding sites, we developed a novel marginal effect isolator algorithm that builds upon Bayesian methods for multigroup TFBS filtering and predicted the in vivo occupancy of two transcription factors with an overall accuracy of 84%. Conclusion. Our analyses show that integration of chromatin immunoprecipitation data with conserved TFBS analysis can be used to generate accurate predictions of functional TFBS. They also show that TFBS cooccurrence can be used to predict transcription factor binding to promoters in vivo

HEF enrichment without protein–protein crosslinking and chromatin purification

<p><b>Copyright information:</b></p><p>Taken from "A genomic approach to the identification and characterization of HOXA13 functional binding elements"</p><p>Nucleic Acids Research 2005;33(21):6782-6794.</p><p>Published online 30 Nov 2005</p><p>PMCID:PMC1301594.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () ChIP was performed in HOXA13-FLAG and HOX (−) cells using anti-FLAG agarose. Elimination of crosslinking with DMA preceding formaldehyde crosslinking is represented in indicated lanes [DMA(−)]. HEF1 and HEF2 are enriched upon addition of DMA as well as without DMA in the HOXA13-FLAG versus HOX (−) cells. HEF1 demonstrated a visibly higher signal with DMA versus DMA (−) in the HOXA13-FLAG cells (1.9-fold by BioRad Quantity One analysis) while HEF2 recovery was equal between the samples. () ChIP was performed in HOXA13-FLAG and HOX (−) cell lines using anti-FLAG agarose. CsCl purification of chromatin was eliminated in the indicated samples (−). HEF1 and HEF2 are both enriched in the HOXA13-FLAG cells versus the HOX (−) cells both with and without chromatin purification. There was consistently no product in the HOX (−) sample for HEF1; however, there is a product present in the CsCl purified HEF2 sample