Abstract: FR-PO383
Smad3 Regulates Cell Fate Through Silencing of Enhancer and Superenhancer Elements During the Specification of iPSC Derived Kidney Organoids
Session Information
- Diabetic Kidney Disease: Basic - II
October 26, 2018 | Location: Exhibit Hall, San Diego Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Diabetic Kidney Disease
- 601 Diabetic Kidney Disease: Basic
Authors
- Davis, Jessica L., University College Dublin, Dublin, Ireland
- Dodd, Thomas K., University College Dublin, Dublin, Ireland
- Kennedy, Ciarán, University College Dublin, Dublin, Ireland
- Clerkin, Shane, University College Dublin, Dublin, Ireland
- Andrews, Darrell C., University College Dublin, Dublin, Ireland
- Godson, Catherine, University College Dublin, Dublin, Ireland
- Crean, John, University College Dublin, Dublin, Ireland
Background
Critical pathological features of diabetic nephropathy are now accepted to include dysregulation of epigenetic processes as evidenced by the observed differential methylation in patients with or without progressive disease. We recently demonstrated a novel direct interaction between Smad3 and EZH2, the enzymatic component of the PRC2 complex, during cell fate specification and TGFβ mediated epithelial dedifferentiation. Targeting this interaction in iPSC derived renal organoids protected against TGFβ mediated tubular epithelial dedifferentiation. Here we further delineate the molecular mechanism underlying this interaction.
Methods
Co-localisation of Smad3 and EZH2 was analysed and visualised using ChIP-Atlas (www.chip-atlas.org). Putative superenhancers (SE) were identified using the Illumina BaseSpace platform. Differential expression of SE target genes in healthy and CKD kidney samples were analysed using Nephroseq (www.nephroseq.org). Target genes were further analysed during the specification of iPSC derived kidney organoids and during TGFβ mediated epithelial dedifferentiation using quantitative real-time PCR.
Results
ChIP-seq identified Smad3 and EZH2 co-localisation at specific loci in human ESCs, and newly occupied enhancers in ESCs and iPSCs treated with activin A. Bioinformatic analysis of these sites identified 243 putative SEs potentially regulated by Smad3/EZH2. Further analysis identified a number of these putative SE target genes that are silenced in normal adult kidney but whose expression is significantly increased during disease. These included critical determinants of cell fate including ZIC3, ZIC5, and DPPA4. Further analysis by RT-PCR indicated a TGFβ dependent downregulation of genes within this cohort suggesting a novel mechanism of gene repression during both development and disease. Functional analysis of enhancer reporter activity verified a Smad3/EZH2 dependent mechanism.
Conclusion
We propose that this complex forms a molecular switch that regulates enhancer and/or promoter access through epigenetic mechanisms and controls gene silencing, informing the fundamental mechanisms through which subsets of genes are switched on and off during fate specification and during the pathogenesis of diabetic nephropathy.