ASN's Mission

To create a world without kidney diseases, the ASN Alliance for Kidney Health elevates care by educating and informing, driving breakthroughs and innovation, and advocating for policies that create transformative changes in kidney medicine throughout the world.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005

email@asn-online.org

202-640-4660

The Latest on Twitter

Kidney Week

Abstract: FR-PO364

Defining Gene Regulatory Networks in Human Kidney Organoids by Single Cell Multiomic Analysis

Session Information

Category: Development‚ Stem Cells‚ and Regenerative Medicine

  • 500 Development‚ Stem Cells‚ and Regenerative Medicine

Authors

  • Yoshimura, Yasuhiro, Washington University School of Medicine, St. Louis, Missouri, United States
  • Muto, Yoshiharu, Washington University School of Medicine, St. Louis, Missouri, United States
  • Omachi, Kohei, Washington University School of Medicine, St. Louis, Missouri, United States
  • Ledru, Nicolas, Washington University School of Medicine, St. Louis, Missouri, United States
  • Miner, Jeffrey H., Washington University School of Medicine, St. Louis, Missouri, United States
  • Humphreys, Benjamin D., Washington University School of Medicine, St. Louis, Missouri, United States
Background

During development, changes in chromatin structure regulate gene expression dynamics. A detailed understanding of gene regulatory networks will improve our understanding of organogenesis and disease mechanisms. Kidney organoids derived from pluripotent stem cells provide unique opportunities to study kidney development in vitro. However, whether the epigenetic landscape during organoid differentiation is similar to that during nephrogenesis has not been elucidated.

Methods

We performed single nucleus RNA sequencing (snRNA-seq) and assay for transposase-accessible chromatin with sequencing (snATAC-seq) and cleavage under targets and release using nuclease (CUT&RUN) sequencing to map the epigenetic and gene expression signatures of human kidney organoids during the organoid differentiation time course.

Results

We identified cell type-specific chromatin accessibility and predicted cis-regulatory links between ATAC peaks and target genes. Gene body ATAC peaks correlated highly with gene expression levels. Putative enhancers were identified by examining peaks in intergenic and intronic regions through integration with genome-wide histone marks identified by CUT&RUN sequencing. Direct comparison with human adult kidney multiome datasets revealed that kidney organoids had fewer distinct cell-specific chromatin accessibility patterns in the gene regulatory elements of maturation-related genes, indicating organoid cell immaturity. Time-course analysis of organoid differentiation showed changes in transcription factor expression varied directly with enrichment of their corresponding DNA binding motifs in ATAC peaks and target gene expression. For validation, we applied CRISPR interference against the promoter and putative enhancer regions of the HNF1B gene, a critical transcription factor for proximal tubule differentiation. We observed decreased HNF1B expression in the proximal tubule cells by suppressing either promoter or distal enhancer activity, validating this gene regulatory network elucidated by multiome analysis.

Conclusion

We defined the cell-specific epigenetic landscape during organoid differentiation and validated select gene regulatory mechanisms in human kidney organoids. This study provides a new benchmark against which to judge the cell-specific maturation state of human kidney organoids.

Funding

  • Private Foundation Support