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Sex differences in the genome-wide DNA methylation pattern and impact on gene expression, microRNA levels and insulin secretion in human pancreatic islets.

Hall E, Volkov P, Dayeh T, Esguerra JL, Salö S, Eliasson L, Rönn T, Bacos K, Ling C - Genome Biol. (2014)

Bottom Line: While the chromosome-wide DNA methylation level on the X-chromosome is higher in female versus male islets, the autosomes do not display a global methylation difference between sexes.Silencing of Nkap or Apln in clonal beta-cells results in increased insulin secretion.Differential methylation between sexes is associated with altered levels of microRNAs miR-660 and miR-532 and related target genes.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Epigenetic factors regulate tissue-specific expression and X-chromosome inactivation. Previous studies have identified epigenetic differences between sexes in some human tissues. However, it is unclear whether epigenetic modifications contribute to sex-specific differences in insulin secretion and metabolism. Here, we investigate the impact of sex on the genome-wide DNA methylation pattern in human pancreatic islets from 53 males and 34 females, and relate the methylome to changes in expression and insulin secretion.

Results: Glucose-stimulated insulin secretion is higher in female versus male islets. Genome-wide DNA methylation data in human islets clusters based on sex. While the chromosome-wide DNA methylation level on the X-chromosome is higher in female versus male islets, the autosomes do not display a global methylation difference between sexes. Methylation of 8,140 individual X-chromosome sites and 470 autosomal sites shows sex-specific differences in human islets. These include sites in/near AR, DUSP9, HNF4A, BCL11A and CDKN2B. 61 X-chromosome genes and 18 autosomal genes display sex-specific differences in both DNA methylation and expression. These include NKAP, SPESP1 and APLN, which exhibited lower expression in females. Functional analyses demonstrate that methylation of NKAP and SPESP1 promoters in vitro suppresses their transcriptional activity. Silencing of Nkap or Apln in clonal beta-cells results in increased insulin secretion. Differential methylation between sexes is associated with altered levels of microRNAs miR-660 and miR-532 and related target genes.

Conclusions: Chromosome-wide and gene-specific sex differences in DNA methylation associate with altered expression and insulin secretion in human islets. Our data demonstrate that epigenetics contribute to sex-specific metabolic phenotypes.

Show MeSH
In vitromethylation of theNKAPandSPESP1promoters decreased reporter gene activity. (A,B) The 1,500 bp promoter regions of NKAP(A) and SPESP1(B) were inserted into the pCpGL basic vector. Promoter constructs were then methylated (gray and black bars) with HhaI or SssI or mock-methylated (white bar) before transfection into clonal β-cells for 48 h. After the 48 h transfection, the luciferase assay was run. The data were normalized with co-transfected renilla luciferase control vector and are the mean of five (NKAP) or nine (SPESP1) separate experiments of five replicates in each. Cells transfected with an empty pCpGL vector were used as background control for firefly luciferase results, and untransfected cells were used as a background for renilla luciferase results. Data were log-transformed and statistical tests calculated using ANOVA followed by paired t-tests with Bonferroni correction post hoc. Data are presented as mean ± standard error of the mean. *P <0.05 versus control; ¤P <0.05 versus SssI.
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Fig9: In vitromethylation of theNKAPandSPESP1promoters decreased reporter gene activity. (A,B) The 1,500 bp promoter regions of NKAP(A) and SPESP1(B) were inserted into the pCpGL basic vector. Promoter constructs were then methylated (gray and black bars) with HhaI or SssI or mock-methylated (white bar) before transfection into clonal β-cells for 48 h. After the 48 h transfection, the luciferase assay was run. The data were normalized with co-transfected renilla luciferase control vector and are the mean of five (NKAP) or nine (SPESP1) separate experiments of five replicates in each. Cells transfected with an empty pCpGL vector were used as background control for firefly luciferase results, and untransfected cells were used as a background for renilla luciferase results. Data were log-transformed and statistical tests calculated using ANOVA followed by paired t-tests with Bonferroni correction post hoc. Data are presented as mean ± standard error of the mean. *P <0.05 versus control; ¤P <0.05 versus SssI.

Mentions: DNA methylation in the proximal promoter region is generally associated with decreased transcriptional activity [30,31]. We therefore functionally tested if promoter methylation affects expression of two selected genes that exhibited both differential DNA methylation and expression in islets due to sex (Figure 4B,D). While NKAP is located on the X chromosome, SPESP1 is an autosomal gene located on chromosome 15. The promoter region of NKAP contains several differentially methylated CpG sites (q <0.05) and the mRNA expression of NKAP was lower in females compared with males (P <0.05) (Figure 4B). SPESP1 has a higher degree of DNA methylation in the promoter and the first exon, in parallel with lower expression in female compared with male islets (Figure 4D). To functionally test if DNA methylation of the NKAP and SPESP1 promoters could influence their gene expression, we used a luciferase reporter assay. A 1,500 bp DNA sequence upstream of their respective TSSs was inserted into a CpG free vector containing the firefly luciferase gene. The constructs were then either mock-methylated or methylated using two different enzymes, HhaI and SssI, where HhaI methylates the internal CpG site in the GCGC sequence, and SssI methylates all CpG sites in the sequence. The numbers of CpG sites methylated by these enzymes in the 1,500 bp NKAP and SPESP1 promoter sequences are shown in Figure 9. SssI methylation of the NKAP promoter almost completely repressed the transcriptional activity of the reporter gene, while HhaI methylation did not significantly affect the transcriptional activity (P = 0.065), probably due to the low number of GCGC sequences in the promoter sequence of NKAP (Figure 9A). For the SPESP1 promoter, methylation with either enzyme caused a significant reduction in transcriptional activity, with methylation by HhaI having a stronger repressive effect on transcription than SssI (Figure 9B). It may seem surprising that methylation of fewer CpG sites by HhaI decreased the transcriptional activity of the SPESP1 promoter more than methylation by SssI. To further resolve why methylation by HhaI had a stronger repressive effect than SssI, we analyzed which transcription factors and repressive factors may bind to the SPESP1 promoter and tested if their binding motif may overlap with CpG sites methylated by HhaI or SssI. Using TFSearch [54] we found numerous putative binding sites for transcription factors with previously known repressive function in the SPESP1 promoter (Additional file 22). While the binding sites for many of these repressive factors overlap with or are in the immediate vicinity of CpG sites methylated by SssI, only three of these binding sites overlap with the GCGC sequence methylated by HhaI (Additional file 22). This may be an explanation for why methylation by HhaI had a stronger repressive effect on the transcriptional activity of SPESP1 than SssI - that is, methylation of CpG sites where repressive factors bind may result in higher gene transcription.Figure 9


Sex differences in the genome-wide DNA methylation pattern and impact on gene expression, microRNA levels and insulin secretion in human pancreatic islets.

Hall E, Volkov P, Dayeh T, Esguerra JL, Salö S, Eliasson L, Rönn T, Bacos K, Ling C - Genome Biol. (2014)

In vitromethylation of theNKAPandSPESP1promoters decreased reporter gene activity. (A,B) The 1,500 bp promoter regions of NKAP(A) and SPESP1(B) were inserted into the pCpGL basic vector. Promoter constructs were then methylated (gray and black bars) with HhaI or SssI or mock-methylated (white bar) before transfection into clonal β-cells for 48 h. After the 48 h transfection, the luciferase assay was run. The data were normalized with co-transfected renilla luciferase control vector and are the mean of five (NKAP) or nine (SPESP1) separate experiments of five replicates in each. Cells transfected with an empty pCpGL vector were used as background control for firefly luciferase results, and untransfected cells were used as a background for renilla luciferase results. Data were log-transformed and statistical tests calculated using ANOVA followed by paired t-tests with Bonferroni correction post hoc. Data are presented as mean ± standard error of the mean. *P <0.05 versus control; ¤P <0.05 versus SssI.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4256841&req=5

Fig9: In vitromethylation of theNKAPandSPESP1promoters decreased reporter gene activity. (A,B) The 1,500 bp promoter regions of NKAP(A) and SPESP1(B) were inserted into the pCpGL basic vector. Promoter constructs were then methylated (gray and black bars) with HhaI or SssI or mock-methylated (white bar) before transfection into clonal β-cells for 48 h. After the 48 h transfection, the luciferase assay was run. The data were normalized with co-transfected renilla luciferase control vector and are the mean of five (NKAP) or nine (SPESP1) separate experiments of five replicates in each. Cells transfected with an empty pCpGL vector were used as background control for firefly luciferase results, and untransfected cells were used as a background for renilla luciferase results. Data were log-transformed and statistical tests calculated using ANOVA followed by paired t-tests with Bonferroni correction post hoc. Data are presented as mean ± standard error of the mean. *P <0.05 versus control; ¤P <0.05 versus SssI.
Mentions: DNA methylation in the proximal promoter region is generally associated with decreased transcriptional activity [30,31]. We therefore functionally tested if promoter methylation affects expression of two selected genes that exhibited both differential DNA methylation and expression in islets due to sex (Figure 4B,D). While NKAP is located on the X chromosome, SPESP1 is an autosomal gene located on chromosome 15. The promoter region of NKAP contains several differentially methylated CpG sites (q <0.05) and the mRNA expression of NKAP was lower in females compared with males (P <0.05) (Figure 4B). SPESP1 has a higher degree of DNA methylation in the promoter and the first exon, in parallel with lower expression in female compared with male islets (Figure 4D). To functionally test if DNA methylation of the NKAP and SPESP1 promoters could influence their gene expression, we used a luciferase reporter assay. A 1,500 bp DNA sequence upstream of their respective TSSs was inserted into a CpG free vector containing the firefly luciferase gene. The constructs were then either mock-methylated or methylated using two different enzymes, HhaI and SssI, where HhaI methylates the internal CpG site in the GCGC sequence, and SssI methylates all CpG sites in the sequence. The numbers of CpG sites methylated by these enzymes in the 1,500 bp NKAP and SPESP1 promoter sequences are shown in Figure 9. SssI methylation of the NKAP promoter almost completely repressed the transcriptional activity of the reporter gene, while HhaI methylation did not significantly affect the transcriptional activity (P = 0.065), probably due to the low number of GCGC sequences in the promoter sequence of NKAP (Figure 9A). For the SPESP1 promoter, methylation with either enzyme caused a significant reduction in transcriptional activity, with methylation by HhaI having a stronger repressive effect on transcription than SssI (Figure 9B). It may seem surprising that methylation of fewer CpG sites by HhaI decreased the transcriptional activity of the SPESP1 promoter more than methylation by SssI. To further resolve why methylation by HhaI had a stronger repressive effect than SssI, we analyzed which transcription factors and repressive factors may bind to the SPESP1 promoter and tested if their binding motif may overlap with CpG sites methylated by HhaI or SssI. Using TFSearch [54] we found numerous putative binding sites for transcription factors with previously known repressive function in the SPESP1 promoter (Additional file 22). While the binding sites for many of these repressive factors overlap with or are in the immediate vicinity of CpG sites methylated by SssI, only three of these binding sites overlap with the GCGC sequence methylated by HhaI (Additional file 22). This may be an explanation for why methylation by HhaI had a stronger repressive effect on the transcriptional activity of SPESP1 than SssI - that is, methylation of CpG sites where repressive factors bind may result in higher gene transcription.Figure 9

Bottom Line: While the chromosome-wide DNA methylation level on the X-chromosome is higher in female versus male islets, the autosomes do not display a global methylation difference between sexes.Silencing of Nkap or Apln in clonal beta-cells results in increased insulin secretion.Differential methylation between sexes is associated with altered levels of microRNAs miR-660 and miR-532 and related target genes.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Epigenetic factors regulate tissue-specific expression and X-chromosome inactivation. Previous studies have identified epigenetic differences between sexes in some human tissues. However, it is unclear whether epigenetic modifications contribute to sex-specific differences in insulin secretion and metabolism. Here, we investigate the impact of sex on the genome-wide DNA methylation pattern in human pancreatic islets from 53 males and 34 females, and relate the methylome to changes in expression and insulin secretion.

Results: Glucose-stimulated insulin secretion is higher in female versus male islets. Genome-wide DNA methylation data in human islets clusters based on sex. While the chromosome-wide DNA methylation level on the X-chromosome is higher in female versus male islets, the autosomes do not display a global methylation difference between sexes. Methylation of 8,140 individual X-chromosome sites and 470 autosomal sites shows sex-specific differences in human islets. These include sites in/near AR, DUSP9, HNF4A, BCL11A and CDKN2B. 61 X-chromosome genes and 18 autosomal genes display sex-specific differences in both DNA methylation and expression. These include NKAP, SPESP1 and APLN, which exhibited lower expression in females. Functional analyses demonstrate that methylation of NKAP and SPESP1 promoters in vitro suppresses their transcriptional activity. Silencing of Nkap or Apln in clonal beta-cells results in increased insulin secretion. Differential methylation between sexes is associated with altered levels of microRNAs miR-660 and miR-532 and related target genes.

Conclusions: Chromosome-wide and gene-specific sex differences in DNA methylation associate with altered expression and insulin secretion in human islets. Our data demonstrate that epigenetics contribute to sex-specific metabolic phenotypes.

Show MeSH