Targeted demethylation at the CDKN1C/p57 locus induces human β cell replication
Kristy Ou, … , Dana Avrahami, Klaus H. Kaestner
Kristy Ou, … , Dana Avrahami, Klaus H. Kaestner
Published October 23, 2018
Citation Information: J Clin Invest. 2019;129(1):209-214. https://doi.org/10.1172/JCI99170.
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Concise Communication Endocrinology

Targeted demethylation at the CDKN1C/p57 locus induces human β cell replication

Abstract

The loss of insulin-secreting β cells is characteristic among type I and type II diabetes. Stimulating proliferation to expand sources of β cells for transplantation remains a challenge because adult β cells do not proliferate readily. The cell cycle inhibitor p57 has been shown to control cell division in human β cells. Expression of p57 is regulated by the DNA methylation status of the imprinting control region 2 (ICR2), which is commonly hypomethylated in Beckwith-Wiedemann syndrome patients who exhibit massive β cell proliferation. We hypothesized that targeted demethylation of the ICR2 using a transcription activator–like effector protein fused to the catalytic domain of TET1 (ICR2-TET1) would repress p57 expression and promote cell proliferation. We report here that overexpression of ICR2-TET1 in human fibroblasts reduces p57 expression levels and increases proliferation. Furthermore, human islets overexpressing ICR2-TET1 exhibit repression of p57 with concomitant upregulation of Ki-67 while maintaining glucose-sensing functionality. When transplanted into diabetic, immunodeficient mice, the epigenetically edited islets show increased β cell replication compared with control islets. These findings demonstrate that epigenetic editing is a promising tool for inducing β cell proliferation, which may one day alleviate the scarcity of transplantable β cells for the treatment of diabetes.

Authors

Kristy Ou, Ming Yu, Nicholas G. Moss, Yue J. Wang, Amber W. Wang, Son C. Nguyen, Connie Jiang, Eseye Feleke, Vasumathi Kameswaran, Eric F. Joyce, Ali Naji, Benjamin Glaser, Dana Avrahami, Klaus H. Kaestner

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Figure 1

Targeted demethylation of the ICR2 at the CDKN1C/p57 locus causes increased proliferation of human fibroblasts.

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Targeted demethylation of the ICR2 at the CDKN1C/p57 locus causes increa...
(A) Schematic of the imprinted chr11p15.5 locus. The ICR2 is methylated (depicted by black circles) at the promoter of the long noncoding RNA KCNQ1OT1 on the maternal allele, and correlates with maternal allele–specific expression of CDKN1C. A TALE-TET1 fusion protein was designed to target the ICR2 and remove the methylCpGs at the ICR2 in order to deactivate CDKN1C and increase cell proliferation. (B) Three regions within the ICR2 were amplified for methylation analysis by targeted bisulfite sequencing. Percentage CpG methylation at 3 regions of the ICR2 are shown (n = 3 for each condition). (C) p57 mRNA and protein levels in fibroblasts overexpressing ICR2-TET1dead or ICR2-TET1 (n = 3 for each condition). VCL, vinculin. (D) EdU incorporation in fibroblasts 72 hours after transduction with the ICR2-TET1dead or ICR2-TET1 lentivirus (n = 5 for each condition). Scale bar: 100 μm. *P < 0.05; **P < 0.01 by 1-way ANOVA (B), 1-tailed t test (C), or 2-tailed t test (D). NS, not significant.