Genome-wide promoter analysis of the SOX4 transcriptional network in prostate cancer cells.

SOX4 is a critical developmental transcription factor in vertebrates and is required for precise differentiation and proliferation in multiple tissues. In addition, SOX4 is overexpressed in many human malignancies, but the exact role of SOX4 in cancer progression is not well understood. Here, we have identified the direct transcriptional targets of SOX4 using a combination of genome-wide localization chromatin immunoprecipitation-chip analysis and transient overexpression followed by expression profiling in a prostate cancer model cell line. We have also used protein-binding microarrays to derive a novel SOX4-specific position-weight matrix and determined that SOX4 binding sites are enriched in SOX4-bound promoter regions. Direct transcriptional targets of SOX4 include several key cellular regulators, such as EGFR, HSP70, Tenascin C, Frizzled-5, Patched-1, and Delta-like 1. We also show that SOX4 targets 23 transcription factors, such as MLL, FOXA1, ZNF281, and NKX3-1. In addition, SOX4 directly regulates expression of three components of the RNA-induced silencing complex, namely Dicer, Argonaute 1, and RNA Helicase A. These data provide new insights into how SOX4 affects developmental signaling pathways and how these changes may influence cancer progression via regulation of gene networks involved in microRNA processing, transcriptional regulation, the TGFbeta, Wnt, Hedgehog, and Notch pathways, growth factor signaling, and tumor metastasis.

Genome-wide analysis of the homeobox C6 transcriptional network in prostate cancer.

Homeobox transcription factors are developmentally regulated genes that play crucial roles in tissue patterning. Homeobox C6 (HOXC6) is overexpressed in prostate cancers and correlated with cancer progression, but the downstream targets of HOXC6 are largely unknown. We have performed genome-wide localization analysis to identify promoters bound by HOXC6 in prostate cancer cells. This analysis identified 468 reproducibly bound promoters whose associated genes are involved in functions such as cell proliferation and apoptosis. We have complemented these data with expression profiling of prostates from mice with homozygous disruption of the Hoxc6 gene to identify 31 direct regulatory target genes of HOXC6. We show that HOXC6 directly regulates expression of bone morphogenic protein 7, fibroblast growth factor receptor 2, insulin-like growth factor binding protein 3, and platelet-derived growth factor receptor alpha (PDGFRA) in prostate cells and indirectly influences the Notch and Wnt signaling pathways in vivo. We further show that inhibition of PDGFRA reduces proliferation of prostate cancer cells, and that overexpression of HOXC6 can overcome the effects of PDGFRA inhibition. HOXC6 regulates genes with both oncogenic and tumor suppressor activities as well as several genes such as CD44 that are important for prostate branching morphogenesis and metastasis to the bone microenvironment.

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.

Molecular characterization of HOXA13 polyalanine expansion proteins in hand-foot-genital syndrome.

We report on a father and daughter with hand-foot-genital syndrome (HFGS) with typical skeletal and genitourinary anomalies due to a 14-residue polyalanine expansion in HOXA13. This is the largest (32 residues) polyalanine tract so far described for any polyalanine mutant protein. Polyalanine expansion results in protein misfolding, cytoplasmic aggregation and degradation; however, HOXA13 polyalanine expansions appear to act as loss of function mutations in contrast to gain of function for HOXD13 polyalanine expansions. To address this paradox we examined the cellular consequences of polyalanine expansions on HOXA13 protein using COS cell transfection and immunocytochemistry. HOXA13 polyalanine expansion proteins form cytoplasmic aggregates, and distribution between cytoplasmic aggregates or the nucleus is polyalanine tract size-dependent. Geldanamycin, an Hsp90 inhibitor, reduces the steady-state abundance of all polyalanine-expanded proteins in transfected cells. We also found that wild-type HOXA13 or HOXD13 proteins are sequestered in HOXA13 polyalanine expansion cytoplasmic aggregates. Thus, the difference between HOXA13 polyalanine expansion loss-of-function and HOXD13 polyalanine expansion dominant-negative effect is not the ability to aggregate wild-type group 13 paralogs but perhaps to variation in activities associated with refolding, aggregation or degradation of the proteins.

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.