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. 2007;35(15):5120-9.
doi: 10.1093/nar/gkm535. Epub 2007 Jul 26.

Dissecting the Action of an Evolutionary Conserved Non-Coding Region on Renin Promoter Activity

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Free PMC article

Dissecting the Action of an Evolutionary Conserved Non-Coding Region on Renin Promoter Activity

Ralf Mrowka et al. Nucleic Acids Res. .
Free PMC article

Abstract

Elucidating the mechanisms of the human transcriptional regulatory network is a major challenge of the post-genomic era. One important aspect is the identification and functional analysis of regulatory elements in non-coding DNA. Genomic sequence comparisons between related species can guide the discovery of cis-regulatory sequences. Using this technique, we identify a conserved region CNSmd of approximately 775 bp in size, approximately 14 kb upstream of the renin gene. Renin plays a pivotal role for mammalian blood pressure regulation and electrolyte balance. To analyse the cis-regulatory role of this region in detail, we perform 132 combinatorial reporter gene assays in an in vitro Calu-6 cell line model. To dissect the role of individual subregions, we fit several mathematical models to the experimental data. We show that a multiplicative switch model fits best the experimental data and that one subregion has a dominant effect on promoter activity. Mapping of the sub-sequences on phylogenetic conservation data reveals that the dominant regulatory region is the one with the highest multi-species conservation score.

Figures

Figure 1.
Figure 1.
(A, B) Percent identity plots (PIP). Each plot shows the position in the human sequence (horizontal axis) and the percent identity (vertical axis) of each aligning sequence of mouse and dog. This plot was used to identify the CNSmd region containing conserved sequences ∼14 kb upstream of REN. (B) shows the region of interest in a 21 kb window and (A) is a zoomed region containing the CNSmd. The plots are modified versions of plots generated using the PIPtool at www.dcode.org. The blue bars correspond to coding, the red bars to non-coding regions. (C) Genomic structure of the renin gene of four species. The conserved regions show different distances to the transcription start site in the four species. (D) Schematic description of vectors used in reporter gene assays. The CNSmd region was divided into four approximately equally sized overlapping parts CNS1 to CNS4. These four parts were tested in 11 different combinatorial constructs with respect to their action on promoter activity in luciferase assay. All possible 11 combinations with natural neighbouring relationships in CNSmd were cloned out of the 15 total possible combinations.
Figure 2.
Figure 2.
Summary of measured reporter gene activity (mean and SD). AM represent the 12 cellular conditions, respectively. Each subplot contains 11 bars corresponding to the 11 constructs which were analysed under the specific cellular condition.
Figure 3.
Figure 3.
(A) Graphical representation of the selected switch model. The sum of the scaled influence of each CNS region is scaled by a condition-specific impact on the promoter. If CNS4 is present its influence becomes dominant. Gene activity from the experiments in comparison with the prediction of the switch model. (B) The reporter gene activity is shown for the 11 constructs and the 12 different cellular conditions. (C) The models prediction after fitting the switch model to the experimental data. The prediction fits well the data obtained in the experiments. The Pearson correlation coefficient between data and model prediction is 0.968.
Figure 4.
Figure 4.
(A) Impact of the CNS regions on reporter gene activity. CNS4 shows the largest impact and this corresponds with a high conservation score. (B) The UCSC conservation score for the corresponding regions on the human chromosome 1. The conservation score was obtained from UCSC and refers to a joined comparison with seven other vertebrates. The dots represent the score and the bold line the moving average of the score.

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