Paternal allelic mutation at the Kcnq1 locus reduces pancreatic β-cell mass by epigenetic modification of Cdkn1c Shun-ichiro Asaharaa, Hiroaki Etohb, Hiroyuki Inoueb, Kyoko Teruyamab, Yuki Shibutania, Yuka Iharab, Yukina Kawadab, Alberto Bartolomeb, Naoko Hashimotoa, Tomokazu Matsudaa, Maki Koyanagi-Kimuraa, Ayumi Kannoa, Yushi Hirotaa, Tetsuya Hosookaa, Kazuaki Nagashimac, Wataru Nishimurad, Hiroshi Inouee, Michihiro Matsumotof, Michael J. Higginsg, Kazuki Yasudah, Nobuya Inagakic, Susumu Seinoa,i, Masato Kasugaj, and Yoshiaki Kidoa,b,1 aDivision of Diabetes and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, Hyogo 650-0017, Japan; bDivision of Medical Chemistry, Department of Biophysics, Kobe University Graduate School of Health Sciences, Hyogo 654-0142, Japan; cDepartment of Diabetes and Clinical Nutrition, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan; dDepartment of Anatomy, Jichi Medical University, Tochigi 329-0498, Japan; eFrontier Science Organization, Kanazawa University, Ishikawa 320-8641, Japan; fDepartment of Molecular Metabolic Regulation, Diabetes Research Center, Research Institute, National Center for Global Health and Medicine, Tokyo 162-8655, Japan; gDepartment of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263; hDepartment of Metabolic Disorder, Diabetes Research Center, Research Institute, National Center for Global Health and Medicine, Tokyo 162-8655, Japan; iDivision of Molecular and Metabolic Medicine, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Hyogo 650-0017, Japan; and jNational Center for Global Health and Medicine, Tokyo 162-8655, Japan Edited by C. Ronald Kahn, Joslin Diabetes Center, Harvard Medical School, Boston, MA, and approved May 26, 2015 (received for review December 1, 2014) Genetic factors are important determinants of the onset and on parental origin (11). Although the underlying mechanism progression of diabetes mellitus. Numerous susceptibility genes for remains unknown, this finding indicates that SNPs of KCNQ1 type 2 diabetes, including potassium voltage-gated channel, KQT-like influence imprinting control of this genomic region. subfamily Q, member1 (KCNQ1), have been identified in humans by With the use of genetically engineered mutant mice, we have genome-wide analyses and other studies. Experiments with geneti- now found that a paternal allelic mutation at the Kcnq1 locus cally modified mice have also implicated various genes in the patho- resulted in an abnormality of imprinting control at this locus and an genesis of diabetes. However, the possible effects of the parent associated decrease in pancreatic β-cell mass. Our results suggest of origin for diabetes susceptibility alleles on disease onset have that defective imprinting control at the KCNQ1 locus might con- Kcnq1 remained unclear. Here, we show that a mutation at the locus tribute to the pathogenesis of pancreatic β-cell failure and type reduces pancreatic β-cell mass in mice by epigenetic modulation only 2 diabetes by affecting the expression of neighboring genes. when it is inherited from the father. The noncoding RNA KCNQ1 overlapping transcript1 (Kcnq1ot1) is expressed from the Kcnq1 locus Results and regulates the expression of neighboring genes on the paternal Insulin Secretion by Pancreatic β-Cells Is Not Impaired in Kcnq1 allele. We found that disruption of Kcnq1 results in reduced Kcnq1ot1 Homozygous KO Mice. To investigate whether loss of function of expression as well as the increased expression of cyclin-dependent Cdkn1c KCNQ1 affects insulin secretion, we evaluated this process in mice kinase inhibitor 1C ( ), an imprinted gene that encodes a cell Kcnq1 cycle inhibitor, only when the mutation is on the paternal allele. in which exon 2 of on chromosome 7 has been replaced by a Furthermore, histone modification at the Cdkn1c promoter region in pancreatic islets was found to contribute to this phenomenon. Significance Our observations suggest that the Kcnq1 genomic region directly regulates pancreatic β-cell mass and that genomic imprinting may Recently, the potassium voltage-gated channel, KQT-like sub- be a determinant of the onset of diabetes mellitus. family Q, member1 (KCNQ1) gene has received much attention as a candidate susceptibility gene for type 2 diabetes in Asian, Eu- pancreatic β-cells | imprinting | Kcnq1 ropean, and other populations. The molecular mechanism un- derlying the association of KCNQ1 with the onset of type 2 enetic and environmental factors are important determinants diabetes has remained unclear; however, we have now found Gof the development of type 2 diabetes. Recent large-scale that a paternal allelic mutation of Kcnq1 results in the up-regu- studies, including genome-wide association studies, have identified lation of the neighboring imprinted gene cyclin-dependent kinase Cdkn1c β many susceptibility genes for this disease (1–3). However, the inhibitor 1C ( ), a cell cycle inhibitor, in pancreatic -cells of mechanisms by which these genes contribute to the pathogen- mice, with this effect being mediated by epigenetic modification of the Cdkn1c promoter. These changes seem to be responsible esis of type 2 diabetes remain unclear. for the reduced pancreatic β-cell mass and impaired glucose tol- Potassium voltage-gated channel, KQT-like subfamily Q, erance characteristics of Kcnq1 mutant mice. member1 (KCNQ1) was identified as a susceptibility gene for type 2 diabetes in 2008 (3, 4). Exonic mutations in KCNQ1, + Author contributions: S.A., M.K., and Y. Kido designed research; S.A., H.E., Hiroyuki Inoue, which encodes a voltage-gated K channel, have been implicated K.T., Y.S., Y.I., Y. Kawada, A.B., T.M., M.K.-K., A.K., K.N., and W.N. performed research; in familial atrial fibrillation and long QT syndrome (5, 6). H.E., Hiroyuki Inoue, K.T., Y.S., Y.I., and Y. Kawada contributed new reagents/analytic tools; S.A., H.E., Hiroyuki Inoue, K.T., Y.S., Y.I., Y. Kawada, A.B., N.H., T.M., M.K.-K., A.K., Y.H., T.H., KCNQ1 contributes to cell repolarization and plays a role in both K.N., Hiroshi Inoue, M.M., M.J.H., K.Y., N.I., S.S., M.K., and Y. Kido analyzed data; and S.A., K.N., gastric acid secretion and secretion in the respiratory tract (7, 8). and Y. Kido wrote the paper. Intronic single nucleotide polymorphism (SNPs) in the KCNQ1 The authors declare no conflict of interest. genomic region have also been associated with reduced insulin This article is a PNAS Direct Submission. β secretion by pancreatic -cells in individuals with diabetes mellitus Freely available online through the PNAS open access option. (9, 10), although the mechanism underlying this association has 1To whom correspondence should be addressed. Email: [email protected]. KCNQ1 remained unclear. SNPs of have been associated with This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. diabetes mellitus in the Icelandic population in a manner dependent 1073/pnas.1422104112/-/DCSupplemental. 8332–8337 | PNAS | July 7, 2015 | vol. 112 | no. 27 www.pnas.org/cgi/doi/10.1073/pnas.1422104112 Downloaded by guest on October 1, 2021 neomycin resistance gene (12). Static incubation of pancreatic the insulin-secreting capacity of pancreatic β-cells in the mice − − islets isolated from Kcnq1 homozygous KO (Kcnq1 / )mice used in this study. revealed no difference in the extent of basal or stimulated insulin secretion compared with islets from WT mice (Fig. 1A). In addi- Effect of Kcnq1 Mutation on Pancreatic β-Cell Mass Depends on the tion, perfusion of the pancreas revealed no difference in glucose- Parent from Which the Mutant Allele Was Inherited. Kcnq1 is an − − induced insulin secretion between Kcnq1 / and WT mice (Fig. S1). imprinted gene that is expressed exclusively from the maternal Together, these results showed that loss of KCNQ1 did not affect allele during fetal development (13). However, although im- printing of Kcnq1 is lost after birth (14), neighboring genes are also imprinted and expressed exclusively from the maternal allele even after birth (15). The noncoding RNA KCNQ1 overlapping transcript1 (Kcnq1ot1) is expressed from the paternally derived Kcnq1 genomic region and regulates the imprinted expression of neighboring target genes by silencing them on the paternal allele (16). The Kvdmr1 locus, which is located in intron 10 of Kcnq1 and has been referred to as an imprinting control region, includes the Kcnq1ot1 promoter. Methylation of DNA in the Kvdmr1 region of the maternal allele inhibits Kcnq1ot1 expression, thereby allowing expression of the gene cluster at the Kcnq1 locus on this allele. Mice with a deletion of the Kvdmr1 region on the paternal allele show biallelic expression of the imprinted gene cluster at the Kcnq1 locus, resulting in systemic growth deficiency during fetal development. This growth defect is attributable in large part to the increased expression of the cyclin-dependent kinase inhibitor 1C (Cdkn1c) gene (17), which encodes a cell cycle regulator and is located on the telomeric side of Kcnq1. We hypothesized that mutations in the Kcnq1 region might affect pancreatic islets by altering the expression of imprinted +/− genes. Therefore, we categorized Kcnq1 heterozygous KO (Kcnq1 ) CELL BIOLOGY mice as either paternal heterozygous (PH) or maternal hetero- zygous (MH) depending on whether they inherited the mutant allele from their father or mother, respectively. The birth weight of these two types of KO