RESUMO
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.
RESUMO
Prostate cancer is the most commonly diagnosed noncutaneous neoplasm and second most common cause of cancer-related mortality in western men. To investigate the mechanisms of prostate cancer development and progression, we did expression profiling of human prostate cancer and benign tissues. We show that the SOX4 is overexpressed in prostate tumor samples compared with benign tissues by microarray analysis, real-time PCR, and immunohistochemistry. We also show that SOX4 expression is highly correlated with Gleason score at the mRNA and protein level using tissue microarrays. Genes affected by SOX4 expression were also identified, including BCL10, CSF1, and NcoA4/ARA70. TLE-1 and BBC3/PUMA were identified as direct targets of SOX4. Silencing of SOX4 by small interfering RNA transfection induced apoptosis of prostate cancer cells, suggesting that SOX4 could be a therapeutic target for prostate cancer. Stable transfection of SOX4 into nontransformed prostate cells enabled colony formation in soft agar, suggesting that, in the proper cellular context, SOX4 can be a transforming oncogene.
Assuntos
Transformação Celular Neoplásica/genética , Proteínas de Grupo de Alta Mobilidade/genética , Oncogenes , Neoplasias da Próstata/genética , Transativadores/genética , Apoptose/genética , Processos de Crescimento Celular/fisiologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Perfilação da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/biossíntese , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXC , Transativadores/biossíntese , TransfecçãoRESUMO
BACKGROUND: We previously analyzed human embryonic kidney (HEK) cell lines for the effects that simian virus 40 (SV40) small tumor antigen (ST) has on gene expression using Affymetrix U133 GeneChips. To cross-validate and extend our initial findings, we sought to compare the expression profiles of these cell lines using an alternative microarray platform. METHODS: We have analyzed matched cell lines with and without expression of SV40 ST using an Applied Biosystems (AB) microarray platform that uses single 60-mer oligonucleotides and single-color quantitative chemiluminescence for detection. RESULTS: While we were able to previously identify only 456 genes affected by ST with the Affymetrix platform, we identified 1927 individual genes with the AB platform. Additional technical replicates increased the number of identified genes to 3478 genes and confirmed the changes in 278 (61%) of our original set of 456 genes. Among the 3200 genes newly identified as affected by SV40 ST, we confirmed 20 by QRTPCR including several components of the Wnt, Notch, and Hedgehog signaling pathways, consistent with SV40 ST activation of these developmental pathways. While inhibitors of Notch activation had no effect on cell survival, cyclopamine had a potent killing effect on cells expressing SV40 ST. CONCLUSIONS: These data show that SV40 ST expression alters cell survival pathways to sensitize cells to the killing effect of Hedgehog pathway inhibitors.
Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Perfilação da Expressão Gênica/métodos , Receptores Notch/metabolismo , Transativadores/metabolismo , Proteínas Wnt/metabolismo , Linhagem Celular , Sobrevivência Celular , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Proteínas Hedgehog , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase/métodos , Receptores Notch/antagonistas & inibidores , Reprodutibilidade dos Testes , Transdução de Sinais , Transativadores/antagonistas & inibidores , Alcaloides de Veratrum/farmacologiaRESUMO
The advent of DNA microarray technology and the sequencing of multiple vertebrate genomes has provided a unique opportunity for the integration of comparative genomics with high-throughput gene expression analysis. Here we describe the conserved transcription factor binding site (CONFAC) software that enables the high-throughput identification of conserved transcription factor binding sites (TFBSs) in the regulatory regions of hundreds of genes at a time (http://morenolab.whitehead.emory.edu/cgi-bin/confac/login.pl). The CONFAC software compares non-coding regulatory sequences between human and mouse genomes to enable identification of conserved TFBSs that are significantly enriched in promoters of gene clusters from microarray analyses compared to sets of unchanging control genes using a Mann-Whitney U-test. Analysis of random gene sets demonstrated that using our approach, over 98% of TFBSs had false positive rates below 5%. As a proof-of-principle, we have validated the CONFAC software using gene sets from four separate microarray studies and identified TFBSs known to be functionally important for regulation of each of the four gene sets.