Pancreas agenesis mutations disrupt a lead enhancer controlling a developmental enhancer cluster

Miguel-Escalada, Irene; Maestro, Miguel A.; Balboa, Diego; Elek, Anamaria; Bernal, Aina; Bernardo, Edgar; Grau, Vanessa; García-Hurtado, Javier; Sebé-Pedrós, Arnau; Ferrer, Jorge

doi:10.1016/j.devcel.2022.07.014

“…This may appear surprising because ePP cells contained a much higher percentage of PDX1 + /SOX9 + /NKX6.1 + cells. It should be noted, however, that the liver marker AFP and, particularly, the gut marker CDX2 were upregulated, whereas expression of PTF1A , recently shown to promote endocrine differentiation of hPS cells (Miguel-Escalada et al, 2022), was essentially lost. The same pattern, and even higher upregulation of gut and liver markers, was also seen in the other expansion procedures (Ma et al, 2022; Nakamura et al, 2022).…”

Section: Discussionmentioning

confidence: 92%

Regulation of multiple signaling pathways promotes the consistent expansion of human pancreatic progenitors in defined conditions

Jarc,

Bandral,

Zanfrini

et al. 2023

Preprint

0

View full text Add to dashboard Cite

The unlimited expansion of human progenitor cells in vitro could unlock many prospects for regenerative medicine but it remains an important challenge as it requires the decoupling of the mechanisms supporting progenitor self-renewal and expansion from feed-forward mechanisms promoting their differentiation. The expansion of human pluripotent stem (hPS) cell derived pancreatic progenitors (PP) will accelerate the development of novel therapies for diabetes. We obtained mechanistic insights into the expansion requirements of PP cells and leveraged them to conduct a hypothesis-driven iterative search to identify conditions for the robust and unlimited expansion of hPS cell derived PP cells under GMP-compliant conditions. We show that the combined stimulation of specific mitogenic pathways, suppression of retinoic acid signaling and inhibition of selected branches of the TGFbeta and Wnt signaling pathways are necessary for the effective decoupling of PP proliferation from differentiation. This enabled the selection of PDX1+/SOX9+/NKX6.1+ PP cells and their consistent, 2000-fold, expansion over ten passages and 40-45 days. Transcriptome analyses confirmed the stabilisation of PP identity and the effective suppression of differentiation. Using these conditions, PDX1+/SOX9+/NKX6.1+ PP cells, derived from different, both XY and XX, hPS cells lines, were enriched to nearly 90% homogeneity and expanded with very similar kinetics and efficiency. Furthermore, non-expanded and expanded PP cells, from different hPS cell lines, were differentiated in micropatterned wells into homogeneous islet-like clusters (SC-islets) with very similar efficiency. These clusters contained abundant beta-cells of comparable functionality as assessed by glucose-stimulated insulin secretion assays. These findings established the signaling requirements to decouple PP proliferation from differentiation and allowed the consistent expansion of hPS cell derived PP cells. They will enable the establishment of large banks of PP cells derived under GMP conditions from diverse hPS cell lines. This will also streamline the generation of SC-islet clusters for further development of the differentiation process, diabetes research, personalized medicine and cell therapies.

show abstract

“…Literature both reports potential for significant impact of the deletions on phenotype in vitro or in vivo. 48,49 and, no less importantly, no differences in knockout strains. 50 To our knowledge, this is the first report on the functionality of an enhancer of a known renal disease marker.…”

Section: Discussionmentioning

confidence: 93%

Sexually dimorphic renal expression of Klotho is directed by a kidney-specific distal enhancer responsive to HNF1b

Jankowski,

Lee,

Liu

et al. 2024

Preprint

0

View full text Add to dashboard Cite

Transcription enhancers are genomic sequences regulating common and tissue-specific genes and their disruption can contribute to human disease development and progression. Klotho, a sexually dimorphic gene specifically expressed in kidney, is well-linked to kidney dysfunction and its deletion from the mouse genome leads to premature aging and death. However, the sexually dimorphic regulation of Klotho is not understood. Here, we characterize two candidate Klotho enhancers using H3K27ac epigenetic marks and transcription factor binding and investigate their functions, individually and combined, through CRISPR-Cas9 genome engineering. We discovered that only the distal (E1), but not the proximal (E2) candidate region constitutes a functional enhancer, with the double deletion not causing Klotho expression to further decrease. E1 activity is dependent on HNF1b transcription factor binding site within the enhancer. Further, E1 controls the sexual dimorphism of Klotho as evidenced by qPCR and RNA-seq. Despite the sharp reduction of Klotho mRNA, unlike germline Klotho knockouts, mutant mice presented normal phenotype, including weight, lifespan, and serum biochemistry. Lastly, only males lacking E1 display more prominent acute, but not chronic kidney injury responses, indicating a remarkable range of potential adaptation to isolated Klotho loss, especially in female E1 knockouts, retaining renoprotection despite over 80% Klotho reduction.

show abstract

“…Although validating the activity of potential enhancers through animal models is essential, it remains infrequent due to its time-consuming nature, expenses and uncertain outcomes. Literature both reports potential for significant impact of the deletions on phenotype in vitro or in vivo (48, 49), and, no less importantly, no differences in knockout strains (50). To our knowledge, this is the first report on the functionality of an enhancer of a known renal disease marker.…”

Section: Discussionmentioning

confidence: 99%

Sexually dimorphic renal expression ofKlothois directed by a kidney-specific distal enhancer responsive to HNF1b

Jankowski,

Lee,

Liu

et al. 2024

Preprint

1

0

View full text Add to dashboard Cite

Transcription enhancers are genomic sequences regulating common and tissue-specific genes and their disruption can contribute to human disease development and progression. Klotho, a sexually dimorphic gene specifically expressed in kidney, is well-linked to kidney dysfunction and its deletion from the mouse genome leads to premature aging and death. However, the sexually dimorphic regulation of Klotho is not understood. Here, we characterize two candidate Klotho enhancers using H3K27ac epigenetic marks and transcription factor binding and investigate their functions, individually and combined, through CRISPR-Cas9 genome engineering. We discovered that only the distal (E1), but not the proximal (E2) candidate region constitutes a functional enhancer, with the double deletion not causing Klotho expression to further decrease. E1 activity is dependent on HNF1b transcription factor binding site within the enhancer. Further, E1 controls the sexual dimorphism of Klotho as evidenced by qPCR and RNA-seq. Despite the sharp reduction of Klotho mRNA, unlike germline Klotho knockouts, mutant mice presented normal phenotype, including weight, lifespan, and serum biochemistry. Lastly, only males lacking E1 display more prominent acute, but not chronic kidney injury responses, indicating a remarkable range of potential adaptation to isolated Klotho loss, especially in female E1 knockouts, retaining renoprotection despite over 80% Klotho reduction.

show abstract

Pancreas agenesis mutations disrupt a lead enhancer controlling a developmental enhancer cluster

Cited by 16 publications

References 88 publications

Regulation of multiple signaling pathways promotes the consistent expansion of human pancreatic progenitors in defined conditions

Regulation of multiple signaling pathways promotes the consistent expansion of human pancreatic progenitors in defined conditions

Sexually dimorphic renal expression of Klotho is directed by a kidney-specific distal enhancer responsive to HNF1b

Sexually dimorphic renal expression ofKlothois directed by a kidney-specific distal enhancer responsive to HNF1b

Contact Info

Product

Resources

About