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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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Fold enrichment of transcription factor binding to the region of the -1607 MMP1 promoter polymorphism. (a) Fold enrichment of c-Fos, ETS1 and ETS2 binding at the region of the -1607 promoter polymorphism relative to a region 5600 bases upstream of the promoter polymorphism. (b) Complete replication of experiment in 5a using cells from different 1G and 2G individuals. (c) Fold enrichment of GATA3 binding at the region of the -1607 promoter polymorphism relative to a region 5600 bases upstream of the promoter polymorphism. (d) Replication of experiment 5c. Input control refers to enrichment in the absence of any precipitating antibody.
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Figure 8: Fold enrichment of transcription factor binding to the region of the -1607 MMP1 promoter polymorphism. (a) Fold enrichment of c-Fos, ETS1 and ETS2 binding at the region of the -1607 promoter polymorphism relative to a region 5600 bases upstream of the promoter polymorphism. (b) Complete replication of experiment in 5a using cells from different 1G and 2G individuals. (c) Fold enrichment of GATA3 binding at the region of the -1607 promoter polymorphism relative to a region 5600 bases upstream of the promoter polymorphism. (d) Replication of experiment 5c. Input control refers to enrichment in the absence of any precipitating antibody.

Mentions: To determine whether differential binding of transcription factors to the MMP1 promoter region containing the -1607 polymorphism is a potential mechanism for the differential response of MMP1 to IM treatment, the TFSEARCH algorithm [31] was used to identify putative transcription factor binding sites in this region. This analysis revealed in addition to the ETS binding domain spanning the polymorphic region, AP-1 and GATA3 binding sites were located 44 bp and 5 bp respectively downstream from the polymorphism. To determine whether these TFs actually bind to this region of the MMP1 gene promoter, immunoprecipitation using antibodies against c-fos, ETS1, ETS2 and GATA3 was carried out in HUVECs isolated from two individuals of 1G genotype and in HUVECs isolated from two individuals of 2G genotype cultured in both IM and UT conditions. Quantitative PCR was used to measure the enrichment of the immunoprecipitated region (157 bp region, adjacent to an ETS, GATA3, AP-1 and NFKB binding site), relative to a 173 bp region positioned 5600 bases upstream of the polymorphism that contained no relevant motifs. DNA precipitated by anti-c-fos, anti-ETS1, anti-ETS2 and anti-GATA3 antibodies was enriched for the MMP1 promoter region containing the polymorphism, relative to the control upstream region and relative to the un-immunoprecipitated material in all four individuals and in cells cultured in UT and IM conditions (Figure 8). The aim of this experiment was to identify potential TF binding around the polymorphism, not to compare the degree of enrichment between IG and 2G individuals or between UT or IM cultured cells, which was impossible due to the small study size and variable degree of enrichment between individuals (Figure 8). Nevertheless, it was interesting to observe that, consistent with the previously published role of ETS1 in MMP-1 induction [16], ETS1 binding was reproducibly enriched in the 1G isolates treated with IM.


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)

Fold enrichment of transcription factor binding to the region of the -1607 MMP1 promoter polymorphism. (a) Fold enrichment of c-Fos, ETS1 and ETS2 binding at the region of the -1607 promoter polymorphism relative to a region 5600 bases upstream of the promoter polymorphism. (b) Complete replication of experiment in 5a using cells from different 1G and 2G individuals. (c) Fold enrichment of GATA3 binding at the region of the -1607 promoter polymorphism relative to a region 5600 bases upstream of the promoter polymorphism. (d) Replication of experiment 5c. Input control refers to enrichment in the absence of any precipitating antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Fold enrichment of transcription factor binding to the region of the -1607 MMP1 promoter polymorphism. (a) Fold enrichment of c-Fos, ETS1 and ETS2 binding at the region of the -1607 promoter polymorphism relative to a region 5600 bases upstream of the promoter polymorphism. (b) Complete replication of experiment in 5a using cells from different 1G and 2G individuals. (c) Fold enrichment of GATA3 binding at the region of the -1607 promoter polymorphism relative to a region 5600 bases upstream of the promoter polymorphism. (d) Replication of experiment 5c. Input control refers to enrichment in the absence of any precipitating antibody.
Mentions: To determine whether differential binding of transcription factors to the MMP1 promoter region containing the -1607 polymorphism is a potential mechanism for the differential response of MMP1 to IM treatment, the TFSEARCH algorithm [31] was used to identify putative transcription factor binding sites in this region. This analysis revealed in addition to the ETS binding domain spanning the polymorphic region, AP-1 and GATA3 binding sites were located 44 bp and 5 bp respectively downstream from the polymorphism. To determine whether these TFs actually bind to this region of the MMP1 gene promoter, immunoprecipitation using antibodies against c-fos, ETS1, ETS2 and GATA3 was carried out in HUVECs isolated from two individuals of 1G genotype and in HUVECs isolated from two individuals of 2G genotype cultured in both IM and UT conditions. Quantitative PCR was used to measure the enrichment of the immunoprecipitated region (157 bp region, adjacent to an ETS, GATA3, AP-1 and NFKB binding site), relative to a 173 bp region positioned 5600 bases upstream of the polymorphism that contained no relevant motifs. DNA precipitated by anti-c-fos, anti-ETS1, anti-ETS2 and anti-GATA3 antibodies was enriched for the MMP1 promoter region containing the polymorphism, relative to the control upstream region and relative to the un-immunoprecipitated material in all four individuals and in cells cultured in UT and IM conditions (Figure 8). The aim of this experiment was to identify potential TF binding around the polymorphism, not to compare the degree of enrichment between IG and 2G individuals or between UT or IM cultured cells, which was impossible due to the small study size and variable degree of enrichment between individuals (Figure 8). Nevertheless, it was interesting to observe that, consistent with the previously published role of ETS1 in MMP-1 induction [16], ETS1 binding was reproducibly enriched in the 1G isolates treated with IM.

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