Kidney Week

Abstract: SA-PO444

Whole-Genome Bisulfite Sequencing Identifies Key Roles for Dnmt3a and Dnmt3b in Renal Tubular Cell Development

Session Information

• Development and Regenerative Medicine
  November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Development, Stem Cells, and Regenerative Medicine

• 500 Development, Stem Cells, and Regenerative Medicine

Authors

• Guan, Yuting, University of Pennsylvania, Philadelphia, Pennsylvania, United States

Yuting Guan,

• Liu, Hongbo, University of Pennsylvania, Philadelphia, Pennsylvania, United States

Hongbo Liu,

• Susztak, Katalin, University of Pennsylvania, Philadelphia, Pennsylvania, United States

Katalin Susztak,

Background

Cytosine methylation is an epigenetic mark that can stably repress gene expression. De novo DNA methyltransferases 3a (Dnmt3a) and 3b (Dnmt3b) play key roles in establishing cell type specific methylation patterns. However, their roles in kidney development are poorly understood.

Methods

We generated mice with genetic deletion Dnmt3a and Dnmt3b in nephron progenitor cells using Six2^Cre and tubule cell specific cells using the Ksp^Cre (Six2^CreDnmt3a/3b and Ksp^CreDnmt3a/3b, DKO). Next generation sequencing techniques, such as reduced representation bisulfite sequencing (RRBS), whole genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) were performed on whole kidney samples and on isolated and sorted renal tubule cells from 3-week-old mice. We induced kidney disease by folic acid injection at 8 weeks of age.

Results

Compared with littermate controls, no obvious developmental defect was identified in Ksp^Cre and Six2^CreDnmt3a^f/f and Dnmt3b^f/f double knock-out mice. RRBS data showed significant methylation changes in both DKO mice consistent with Dnmt3a/b role in establishing de novo methylation pattern. More hypo-methylated regions (Hypo-DMR) were identified in Six2^Cre DKO mice, suggesting the key role of Dnmt3a/b in establishing cell type specific methylation in development. To explore the genome wide effect of Dnmt3a and Dnmt3b, WGBS was performed on isolated Ksp positive renal tubule epithelial cells. Remarkable decreased in cytosine methylation was observed at genome wide level, especially affecting fetal enhancers which gain methylation in normal development. Motif enrichment analysis showed the significant enrichment of hypo-methylated regions in binding sites of kidney developmental transcription factors including Six2. Despite the broad changes in cytosine methylation, the effect of Dnmt3a and Dnmt3b loss on gene expression was less pronounced. DKO mice showed no differences when compared to control following kidney injury.

Conclusion

Our results indicate Dnmt3a and Dnmt3b are necessary for de novo methylation of enhancers which are active in fetal kidney and closed in adult kidney. Despite the significant methylation changes on enhancer regions, the effect of de novo methylation on gene expression regulation and phenotype was less pronounced.