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A novel HMM-based method for detecting enriched transcription factor binding sites reveals RUNX3 as a potential target in pancreatic cancer biology.

Levkovitz L, Yosef N, Gershengorn MC, Ruppin E, Sharan R, Oron Y - PLoS ONE (2010)

Bottom Line: Using a novel experimental paradigm to distinguish between normal and PAC cells, we find that RUNX3 mRNA (but not RUNX1 or RUNX2 mRNAs) exhibits time-dependent increases in normal but not in PAC cells.These increases are accompanied by changes in mRNA levels of putative RUNX gene targets.The integrated application of DEMON and a novel differentiation system led to the identification of a single family member, RUNX3, which together with four of its putative targets showed a robust response to a differentiation stimulus in healthy cells, whereas this regulatory mechanism was absent in PAC cells, emphasizing RUNX3 as a promising target for further studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT

Background: Pancreatic adenocarcinoma (PAC) is one of the most intractable malignancies. In order to search for potential new therapeutic targets, we relied on computational methods aimed at identifying transcription factor binding sites (TFBSs) over-represented in the promoter regions of genes differentially expressed in PAC. Though many computational methods have been implemented to accomplish this, none has gained overall acceptance or produced proven novel targets in PAC. To this end we have developed DEMON, a novel method for motif detection.

Methodology: DEMON relies on a hidden Markov model to score the appearance of sequence motifs, taking into account all potential sites in a promoter of potentially varying binding affinities. We demonstrate DEMON's accuracy on simulated and real data sets. Applying DEMON to PAC-related data sets identifies the RUNX family as highly enriched in PAC-related genes. Using a novel experimental paradigm to distinguish between normal and PAC cells, we find that RUNX3 mRNA (but not RUNX1 or RUNX2 mRNAs) exhibits time-dependent increases in normal but not in PAC cells. These increases are accompanied by changes in mRNA levels of putative RUNX gene targets.

Conclusions: The integrated application of DEMON and a novel differentiation system led to the identification of a single family member, RUNX3, which together with four of its putative targets showed a robust response to a differentiation stimulus in healthy cells, whereas this regulatory mechanism was absent in PAC cells, emphasizing RUNX3 as a promising target for further studies.

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Kinetics of changes in expression of RUNX genes.hIPCs and PANC-1 cells were either cultured in serum-containing medium (t = 0) or for the indicated times in serum-free medium. RNA was extracted and qRT-PCR performed as described in Materials and Methods. Results are presented as % change in mRNA levels of the three RUNX genes as a function of time in serum-free medium.
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pone-0014423-g004: Kinetics of changes in expression of RUNX genes.hIPCs and PANC-1 cells were either cultured in serum-containing medium (t = 0) or for the indicated times in serum-free medium. RNA was extracted and qRT-PCR performed as described in Materials and Methods. Results are presented as % change in mRNA levels of the three RUNX genes as a function of time in serum-free medium.

Mentions: To obtain experimental evidence for RUNX distinguishing between normal and PAC cells, we monitored expression of RUNX1, 2 and 3 mRNAs by qRT-PCR as a function of time of serum deprivation of hIPCs and PANC-1 cells (Fig. 4). There was little change in the expression of RUNX1 and 2 transcripts in either cell type. The expression of RUNX3, however, was markedly increased in a time-dependent manner in hIPCs while there was virtually no change in PANC-1 cells. It appears, therefore, that the expression of RUNX3 is regulated in hIPCs during differentiation but fails to respond to the differentiation stimulus in PANC-1 cells.


A novel HMM-based method for detecting enriched transcription factor binding sites reveals RUNX3 as a potential target in pancreatic cancer biology.

Levkovitz L, Yosef N, Gershengorn MC, Ruppin E, Sharan R, Oron Y - PLoS ONE (2010)

Kinetics of changes in expression of RUNX genes.hIPCs and PANC-1 cells were either cultured in serum-containing medium (t = 0) or for the indicated times in serum-free medium. RNA was extracted and qRT-PCR performed as described in Materials and Methods. Results are presented as % change in mRNA levels of the three RUNX genes as a function of time in serum-free medium.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3008686&req=5

pone-0014423-g004: Kinetics of changes in expression of RUNX genes.hIPCs and PANC-1 cells were either cultured in serum-containing medium (t = 0) or for the indicated times in serum-free medium. RNA was extracted and qRT-PCR performed as described in Materials and Methods. Results are presented as % change in mRNA levels of the three RUNX genes as a function of time in serum-free medium.
Mentions: To obtain experimental evidence for RUNX distinguishing between normal and PAC cells, we monitored expression of RUNX1, 2 and 3 mRNAs by qRT-PCR as a function of time of serum deprivation of hIPCs and PANC-1 cells (Fig. 4). There was little change in the expression of RUNX1 and 2 transcripts in either cell type. The expression of RUNX3, however, was markedly increased in a time-dependent manner in hIPCs while there was virtually no change in PANC-1 cells. It appears, therefore, that the expression of RUNX3 is regulated in hIPCs during differentiation but fails to respond to the differentiation stimulus in PANC-1 cells.

Bottom Line: Using a novel experimental paradigm to distinguish between normal and PAC cells, we find that RUNX3 mRNA (but not RUNX1 or RUNX2 mRNAs) exhibits time-dependent increases in normal but not in PAC cells.These increases are accompanied by changes in mRNA levels of putative RUNX gene targets.The integrated application of DEMON and a novel differentiation system led to the identification of a single family member, RUNX3, which together with four of its putative targets showed a robust response to a differentiation stimulus in healthy cells, whereas this regulatory mechanism was absent in PAC cells, emphasizing RUNX3 as a promising target for further studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT

Background: Pancreatic adenocarcinoma (PAC) is one of the most intractable malignancies. In order to search for potential new therapeutic targets, we relied on computational methods aimed at identifying transcription factor binding sites (TFBSs) over-represented in the promoter regions of genes differentially expressed in PAC. Though many computational methods have been implemented to accomplish this, none has gained overall acceptance or produced proven novel targets in PAC. To this end we have developed DEMON, a novel method for motif detection.

Methodology: DEMON relies on a hidden Markov model to score the appearance of sequence motifs, taking into account all potential sites in a promoter of potentially varying binding affinities. We demonstrate DEMON's accuracy on simulated and real data sets. Applying DEMON to PAC-related data sets identifies the RUNX family as highly enriched in PAC-related genes. Using a novel experimental paradigm to distinguish between normal and PAC cells, we find that RUNX3 mRNA (but not RUNX1 or RUNX2 mRNAs) exhibits time-dependent increases in normal but not in PAC cells. These increases are accompanied by changes in mRNA levels of putative RUNX gene targets.

Conclusions: The integrated application of DEMON and a novel differentiation system led to the identification of a single family member, RUNX3, which together with four of its putative targets showed a robust response to a differentiation stimulus in healthy cells, whereas this regulatory mechanism was absent in PAC cells, emphasizing RUNX3 as a promising target for further studies.

Show MeSH
Related in: MedlinePlus