A systems biology approach identifies the role of dysregulated PRDM6 in the development of hypertension

Figures

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  Figure 1. GWAS lead SNPs in PRDM6 intron 3 and their CRISPR deletion. (A) Genetic variants in PRDM6 gene discovered by GWAS of BP traits. (B) LD structure of rs13359291, rs2287696, rs1422279, and rs555625 in the East Asian population. R2 and D’ for each SNP pair is represented inside corresponding boxes. (C) Relationship between lead SNPs, open chromatin regions, and histone marks. (50, 51, 52, 53, 54). (D) Schematic of CRISPR-Cas9–mediated deletion of the approximately 22 kb LD region in intron 3, encompassing all lead SNPs, HEK293T, n = 4 technical replicates (E) Expression of PRDM6 in different human cell lines (www.proteinatlas.org/). (F) Significant reduction of PRDM6 mRNA expression upon deletion of the LD region compared with the WT sequence assayed by RT-qPCR, identifying the intronic region as an enhancer locus for PRDM6. n = 4. Unpaired, 2 tailed, t test, n = 4 technical replicates. ****P < 0.0001. NES, normalized effect size.

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  Figure 2. MPRA of PRDM6 intron 3 variants. (A) The schematic of MPRA of 336 common variants in the third intron of PRDM6 from generation of inert and competent libraries, cloning into MPRA vectors, transfection into HEK293T cells, and RNA & DNA sequencing and analysis. (B) Frequency distribution showing the number of tags associated with each MPRA fragment. Distribution statistics are represented. (C) Manhattan plot showing sixteen single SNPs that significantly altered the gene expression due to a single bp change with MPRA. (D) Violin plots of the 16 single SNPs showing allele-specific expression of the reporter gene. Statistics were carried out using 1-way ANOVA. P < 0.05 was considered significant.

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  Figure 3. CRISPR deletion of SNPs with altered allele-specific expression and their location in different haplotypes. (A) 5 different LD groups (R2 > 0.70) in the East Asian population based on the haplotypes generated from lead GWAS SNPs and SNPs discovered by the MPRA. (B) Violin plots showing PRDM6 expression in the tibial artery and aorta with different allelic combinations pertinent to rs13359291 (GTEx database, https://gtexportal.org/home/). (C) Location of the rs13359291 in relationship to open chromatin region of the aorta and histone markers and the sequence conservation in 100 vertebrates. The SNP region is zoomed in for clarity.

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  Figure 4. Screenings for transcription factor and enhancer identification. (A) Putative binding site for CEBP β and α based on PROMO silico motif finder. (B) Downregulation in PRDM6 mRNA expression in the 52 bp CRISPR-Cas9 deletion encompassing rs13359291 compared with WT cells by RT-qPCR. Blue dots indicate control data points while red dots indicate experimental data points. (C) Downregulation in PRDM6 mRNA expression in the depicted sized CRISPR-Cas9 deletions encompassing the mentioned SNPs, compared with WT cells by RT-qPCR. n > 3 independent replicates. ***P < 0.001, **P < 0.01, *P < 0.05. (D) IFNγ stimulation of HEK293T cells with and without CRISPR deletions encompassing STAT1 binding regions. Unpaired 2-tailed t test or 1-way ANOVA. ****P < 0.0001, ***P < 0.001. n > 3 independent replicates.

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  Figure 5. Invasive measurement of BP indices in Prdm6fl/+ SM22-Cre and WT mice. (A–C) SBP, DBP, and PP of Prdm6fl/+ SM22-Cre and Prdm6fl/+ mice. Mice were on a chow diet (day 4–5) and a high-salt diet (day 6–14). (D–F) Changes of all 3 traits during the resting (daytime) and active (nighttime) phases under different dietary conditions. Multiple, paired, 2-tailed t test. ****adjusted P < 0.0001, ***adjusted P < 0.001. n > 5–11 mice per group.

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  Figure 6. RNA-Seq analysis of Prdm6fl/fl SM22-Cre mice aortic tissue. (A) RNA-Seq–hit map of homozygous SMC-specific knockout (Prdm6fl/fl SM22-Cre) mice aortic tissue and their littermate controls at P0.5. A total of 349 of the genes were upregulated, while 769 genes were downregulated in Prdm6fl/fl SM22-Cre versus WT mouse aortas. (B) Volcano plot demonstrating genes with significantly altered expression in Prdm6fl/fl SM22-Cre mouse aortas versus WT mouse aortas; the plot shows fold change versus P value; a selected number of genes associated with hypertension are annotated for better visibility. (C) A bubble plot representing aforementioned genes associated with different BP traits. (D and E) GSEA show many blood pressure-related processes to be significantly enriched in both up- (D) and down-regulated (E) genes.

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  Figure 7. The increase in renin-producing cells and renin-aldosterone expression in Prdm6fl/+ SM22-Cre mice. (A) Schematic of renin-angiotensin-aldosterone–axis regulation. (B) Comparison of body weight and area of kidney between Prdm6fl/+ SM22-Cre and WT mice. n = 6 mice per group. (C and D) Higher renin mRNA and protein levels in the kidneys and aldosterone synthase mRNA levels in the adrenal glands of adult Prdm6fl/+ SM22-Cre mice versus WT littermates n = 4–6 mice per group. The quantification of the normalized Renin relative to GAPDH is shown in C. n = 4 mice per group. (E) The fraction of sodium excretion (FENA) in Prdm6fl/+ SM22-Cre versus WT mice. n = 4–5 mice per group. (F) Renin protein levels in E18.5 kidney of Prdm6fl/+ SM22-Cre and Prdm6fl/fl SM22-Cre mice and their corresponding WT littermates by immunofluorescence staining. (G) The number of renin-expressing cells per fixed area for all 3 genotypes. n = 7 mice per group (H) Mean renin intensity for the mentioned genotypes. (I) SBP and DBP measurements in Prdm6fl/+ SM22-Cre and WT mice on a high-salt diet with and without treatment with aliskiren. n > 6 mice per group. Data are shown as mean ± SEM. Kruskal-Wallis 1-way ANOVA.…

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  Figure 8. Sox6 is transcriptionally regulated by PRDM6. (A) Sox6 transcript levels in Prdm6fl/fl SM22-Cre and WT littermate mouse aortas (n = 3 per group). (B) The immunofluorescence staining of Sox6 in heterozygous, homozygous Prdm6-knockout mice and their WT littermates. The number of cells expressing Sox6 (left) and mean Sox6 intensity (right) are shown underneath the figures (n = 3 mice per group and 20 fields per mouse). (C) PRDM6 and SOX6 expression level in HEK293T cells upon 22 kb CRISPR deletion of the PRDM6 intronic region. Experiments were done in triplicates. (D) PRDM6 binding sites in SOX6 gene in HEK293T cells according to ENCODE ChIP-Seq data (doi:10.17989/ENCSR892QHR). 2 binding loci are zoomed for clarity. (E) Immunofluorescent images of Prdm6fl/+, Prdm6fl/fl Sm22-Cre, Sox6fl/fl Sm22-Cre and Prdm6fl/fl Sox6fl/fl Sm22-Cre embryonic kidneys at E18.5 labeled with SMA (red) and renin (green) along with nuclear stain (blue). n = 3 mice per group and 10 fields per genotype. The plot on the right shows the quantification of a number of cells producing renin in 4 genotypes. Scale bars: 20 μm. IF figures: Kruskal-Wallis 1-way ANOVA, ****P < 0.0001, ***P < 0.001, *P < 0.05.…

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  Figure 9. Fate mapping of neural crest PRDM6. (A) ZsGreen1 and renin-expressing cells in the kidneys of Prdm6fl/fl Wnt1-Cre and WT littermates. The plot on the right shows the quantification of ZsGreen1 (NCC) and renin-positive cells. (B) Augmented part of panel A demonstrates the close proximity of ZsGreen1 and renin-expressing cells on the vascular wall of in Prdm6fl/fl Wnt1-Cre kidneys marked by arrowheads. Arrows show renin. (C) The localization of Sox6 in relationship to αSMA and ZsGreen1positive cells (NCC) in Prdm6fl/fl Wnt1-Cre kidneys and WT littermates. Scale bars: 20 μm. Unpaired 2-tailed t test; *P < 0.05. n = 6 mice per group in A–C.

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  Figure 10. Fate mapping of smooth muscle cell PRDM6. (A) Fate mapping of SMCs in Prdm6fl/fl Sm22-Cre and WT littermates by crossbreeding with ZsGreen1fl/fl mice. (A) Colocalization of Sox6 and ZsGreen1 positive cells is shown; the integrated ZsGreen intensity per area and percent ZsGreen quantification of Sox6 in ZsGreen1 positive cells is compared between Prdm6fl/fl Sm22-Cre and WT littermates. n = 500 cells per genotype. (B) Colocalization between PDGFRβ and ZsGreen1. n = 4 mice per genotype. (C) Sirius red, PAS, and H&E staining in 12 weeks adult heterozygous Prdm6fl/+ SM22-Cre mice kidneys compared with WT mice, n = 4 per group. (D) Tubular and glomerular diameters in Prdm6fl/fl SM22-Cre mice versus WT littermates n = 6–100 measurements. Scale bars: 20 μm. (E) Creatinine levels in Prdm6fl/fl SM22-Cre mice versus WT littermates on chow and high-salt diets, respectively unpaired 2-tailed t test; **** specifies P < 0.0001, **P < 0.01, *P < 0.05. n = 5 mice per group.