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MMP1 bimodal expression and differential response to inflammatory mediators is linked to promoter polymorphisms.

Affara M, Dunmore BJ, Sanders DA, Johnson N, Print CG, Charnock-Jones DS - BMC Genomics (2011)

Bottom Line: Identifying the functional importance of the millions of single nucleotide polymorphisms (SNPs) in the human genome is a difficult challenge.In this study, we used a novel but straightforward method based on agglomerative hierarchical clustering to identify bimodally expressed transcripts in human umbilical vein endothelial cell (HUVEC) microarray data from 15 individuals.We describe a simple method to identify putative bimodally expressed RNAs from transcriptome data that is effective yet easy for non-statisticians to understand and use.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK.

ABSTRACT

Background: Identifying the functional importance of the millions of single nucleotide polymorphisms (SNPs) in the human genome is a difficult challenge. Therefore, a reverse strategy, which identifies functionally important SNPs by virtue of the bimodal abundance across the human population of the SNP-related mRNAs will be useful. Those mRNA transcripts that are expressed at two distinct abundances in proportion to SNP allele frequency may warrant further study. Matrix metalloproteinase 1 (MMP1) is important in both normal development and in numerous pathologies. Although much research has been conducted to investigate the expression of MMP1 in many different cell types and conditions, the regulation of its expression is still not fully understood.

Results: In this study, we used a novel but straightforward method based on agglomerative hierarchical clustering to identify bimodally expressed transcripts in human umbilical vein endothelial cell (HUVEC) microarray data from 15 individuals. We found that MMP1 mRNA abundance was bimodally distributed in un-treated HUVECs and showed a bimodal response to inflammatory mediator treatment. RT-PCR and MMP1 activity assays confirmed the bimodal regulation and DNA sequencing of 69 individuals identified an MMP1 gene promoter polymorphism that segregated precisely with the MMP1 bimodal expression. Chromatin immunoprecipitation (ChIP) experiments indicated that the transcription factors (TFs) ETS1, ETS2 and GATA3, bind to the MMP1 promoter in the region of this polymorphism and may contribute to the bimodal expression.

Conclusions: We describe a simple method to identify putative bimodally expressed RNAs from transcriptome data that is effective yet easy for non-statisticians to understand and use. This method identified bimodal endothelial cell expression of MMP1, which appears to be biologically significant with implications for inflammatory disease. (271 Words).

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Quantitative PCR of transcripts DDX3Y and MMP1 in 29 donor isolates under untreated (UT) and inflammatory mediator treated (IM) conditions. Donors of female gender are shown in red and donors of male gender are shown in black. Only DDX3Y appears to segregate with gender. Delta Ct values are calculated as Ct of target gene of interest (DDX3Y or MMP1) relative to the Ct value of the internal control (18S ribosomal RNA). Low delta Ct values represent high basal levels of expression and vice versa.
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Figure 3: Quantitative PCR of transcripts DDX3Y and MMP1 in 29 donor isolates under untreated (UT) and inflammatory mediator treated (IM) conditions. Donors of female gender are shown in red and donors of male gender are shown in black. Only DDX3Y appears to segregate with gender. Delta Ct values are calculated as Ct of target gene of interest (DDX3Y or MMP1) relative to the Ct value of the internal control (18S ribosomal RNA). Low delta Ct values represent high basal levels of expression and vice versa.

Mentions: The pool of individuals examined was expanded and the abundance of DDX3Y, MMP1 and SLC2A11 mRNA in HUVECs from 29 additional individuals cultured in both UT and IM conditions was analysed using quantitative Reverse Transcription-PCR (qRT-PCR). We confirmed in this new group of individuals a bimodal expression pattern for DDX3Y using q RT-PCR. DDX3Y is encoded by a Y-chromosome gene and, as expected, its expression segregated with the gender of the individual from which the HUVEC were isolated in both the IM and UT data (Figure 3).


MMP1 bimodal expression and differential response to inflammatory mediators is linked to promoter polymorphisms.

Affara M, Dunmore BJ, Sanders DA, Johnson N, Print CG, Charnock-Jones DS - BMC Genomics (2011)

Quantitative PCR of transcripts DDX3Y and MMP1 in 29 donor isolates under untreated (UT) and inflammatory mediator treated (IM) conditions. Donors of female gender are shown in red and donors of male gender are shown in black. Only DDX3Y appears to segregate with gender. Delta Ct values are calculated as Ct of target gene of interest (DDX3Y or MMP1) relative to the Ct value of the internal control (18S ribosomal RNA). Low delta Ct values represent high basal levels of expression and vice versa.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3040839&req=5

Figure 3: Quantitative PCR of transcripts DDX3Y and MMP1 in 29 donor isolates under untreated (UT) and inflammatory mediator treated (IM) conditions. Donors of female gender are shown in red and donors of male gender are shown in black. Only DDX3Y appears to segregate with gender. Delta Ct values are calculated as Ct of target gene of interest (DDX3Y or MMP1) relative to the Ct value of the internal control (18S ribosomal RNA). Low delta Ct values represent high basal levels of expression and vice versa.
Mentions: The pool of individuals examined was expanded and the abundance of DDX3Y, MMP1 and SLC2A11 mRNA in HUVECs from 29 additional individuals cultured in both UT and IM conditions was analysed using quantitative Reverse Transcription-PCR (qRT-PCR). We confirmed in this new group of individuals a bimodal expression pattern for DDX3Y using q RT-PCR. DDX3Y is encoded by a Y-chromosome gene and, as expected, its expression segregated with the gender of the individual from which the HUVEC were isolated in both the IM and UT data (Figure 3).

Bottom Line: Identifying the functional importance of the millions of single nucleotide polymorphisms (SNPs) in the human genome is a difficult challenge.In this study, we used a novel but straightforward method based on agglomerative hierarchical clustering to identify bimodally expressed transcripts in human umbilical vein endothelial cell (HUVEC) microarray data from 15 individuals.We describe a simple method to identify putative bimodally expressed RNAs from transcriptome data that is effective yet easy for non-statisticians to understand and use.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK.

ABSTRACT

Background: Identifying the functional importance of the millions of single nucleotide polymorphisms (SNPs) in the human genome is a difficult challenge. Therefore, a reverse strategy, which identifies functionally important SNPs by virtue of the bimodal abundance across the human population of the SNP-related mRNAs will be useful. Those mRNA transcripts that are expressed at two distinct abundances in proportion to SNP allele frequency may warrant further study. Matrix metalloproteinase 1 (MMP1) is important in both normal development and in numerous pathologies. Although much research has been conducted to investigate the expression of MMP1 in many different cell types and conditions, the regulation of its expression is still not fully understood.

Results: In this study, we used a novel but straightforward method based on agglomerative hierarchical clustering to identify bimodally expressed transcripts in human umbilical vein endothelial cell (HUVEC) microarray data from 15 individuals. We found that MMP1 mRNA abundance was bimodally distributed in un-treated HUVECs and showed a bimodal response to inflammatory mediator treatment. RT-PCR and MMP1 activity assays confirmed the bimodal regulation and DNA sequencing of 69 individuals identified an MMP1 gene promoter polymorphism that segregated precisely with the MMP1 bimodal expression. Chromatin immunoprecipitation (ChIP) experiments indicated that the transcription factors (TFs) ETS1, ETS2 and GATA3, bind to the MMP1 promoter in the region of this polymorphism and may contribute to the bimodal expression.

Conclusions: We describe a simple method to identify putative bimodally expressed RNAs from transcriptome data that is effective yet easy for non-statisticians to understand and use. This method identified bimodal endothelial cell expression of MMP1, which appears to be biologically significant with implications for inflammatory disease. (271 Words).

Show MeSH
Related in: MedlinePlus