Dataset Information

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

ABSTRACT: The loss of insulin-secreting ? cells is characteristic among type I and type II diabetes. Stimulating proliferation to expand sources of ? cells for transplantation remains a challenge because adult ? cells do not proliferate readily. The cell cycle inhibitor p57 has been shown to control cell division in human ? 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 ? 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 ? cell replication compared with control islets. These findings demonstrate that epigenetic editing is a promising tool for inducing ? cell proliferation, which may one day alleviate the scarcity of transplantable ? cells for the treatment of diabetes.

SUBMITTER: Ou K

PROVIDER: S-EPMC6307972 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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

Ou Kristy K Yu Ming M Moss Nicholas G NG Wang Yue J YJ Wang Amber W AW Nguyen Son C SC Nguyen Son C SC Jiang Connie C Feleke Eseye E Kameswaran Vasumathi V Joyce Eric F EF Naji Ali A Glaser Benjamin B Avrahami Dana D Kaestner Klaus H KH

The Journal of clinical investigation 20181126 1

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 ...[more]

PMID: 30352048

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Project description:To evaluate the role of cyclin-dependent kinase inhibitors p57 and p15 in transforming growth factor (TGF)-beta1 or TGF-beta2 inhibited proliferation of primary cultured human limbal epithelial cells using short interfering RNA (siRNA).Primary cultured human limbal epithelial cells were treated with TGF-beta1 or TGF-beta2 for 6 and 24 h, and total RNA extracted for RT-PCR and real-time PCR using primers for p21, p27, and p57 (CipP/Kip family) and p15 and p19 (INK4 family). Proteins were extracted for western blot analysis of p57 and p15. For RNA interference, primary cultured human limbal epithelial cells were transfected with annealed double-stranded siRNA (67 nM) specific for p57, p15, or siRNA-Fluorescein (siRNA-F; as a negative control) followed by treatment with TGF-beta1 or TGF-beta2 at 1 ng/ml. P57 and p15 were quantitatively detected by real-time PCR and western blot; and immunolocalized by immunofluorescent staining. The effects of TGF-beta1 or TGF-beta2 on cell proliferation were evaluated by BrdU incorporation and MTT assay.TGF-beta1 or TGF-beta2 significantly inhibited primary cultured human limbal epithelial cell proliferation measured by BrdU incorporation and MTT assay. TGF-beta1 or TGF-beta2 upregulated the expression of p57 and p15 mRNA and protein, but did not effect the expression of p19, p21, or p27. The siRNA transfection efficiency of these cells was 75% and no cellular toxicity was observed by 24 h. The TGF-beta1 or TGF-beta2 stimulated expression of p57 and p15 mRNA were markedly blocked by siRNA-p57 or siRNA-p15, respectively, but not by siRNA-F. The TGF-beta1 or TGF-beta2 suppression of epithelial proliferation measured by BrdU incorporation and MTT generation was increased to near normal levels by siRNA-p57 or siRNA-p15. Western blot and immunofluorescent staining showed that levels of p57 and p15 proteins were equally reduced in the cytoplasm and nucleus.These findings demonstrate that TGF-beta1 and/or TGF-beta2 inhibit proliferation of primary cultured human limbal epithelial cells and that p57 and p15 play roles in this process.

Dataset Information

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

Publications

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

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