Diabetes Volume 68, February 2019 337 Examining How the MAFB Transcription Factor Affects Islet b-Cell Function Postnatally Holly A. Cyphert,1 Emily M. Walker,1 Yan Hang,1 Sangeeta Dhawan,2 Rachana Haliyur,1,3 Lauren Bonatakis,1 Dana Avrahami,4 Marcela Brissova,1,3 Klaus H. Kaestner,5 Anil Bhushan,6 Alvin C. Powers,1,3,7 and Roland Stein1 Diabetes 2019;68:337–348 | https://doi.org/10.2337/db18-0903 The sustained expression of the MAFB transcription (1). Although many distinct genetic lesions appear to factor in human islet b-cells represents a distinct difference contribute to disease susceptibility, islet-enriched tran- in mice. Moreover, mRNA expression of closely related scription factor mutations commonly are associated with and islet b-cell–enriched MAFA does not peak in humans a monogenic form of diabetes termed maturity-onset di- until after 9 years of age. We show that the MAFA protein abetes of the young (e.g., HNF1a [2], HNF1b [3], PDX1 also is weakly produced within the juvenile human islet [4], MAFA [5]). As a consequence of extensive mutational b-cell population and that MafB expression is postnatally analysis of these and other islet-enriched transcription ISLET STUDIES b restricted in mouse -cells by de novo DNA methylation. factors in mice, many were shown to play essential roles in b To gain insight into how MAFB affects human -cells, we islet cell development and/or function (6). However, strik- developed a mouse model to ectopically express MafB ing differences exist in the expression pattern of a few of in adult mouse b-cells using MafA transcriptional control these key regulators between humans and mice. For ex- sequences. Coexpression of MafB with MafA had no overt MAFA b ample, mRNA is produced at lower levels in juvenile impact on mouse -cells, suggesting that the human adult , . b ( 9 years of age) than in adult ( 29 years of age) human -cell MAFA/MAFB heterodimer is functionally equivalent b to the mouse MafA homodimer. However, MafB alone was islet -cells (7), whereas expression peaks soon after birth MAFB unable to rescue the islet b-cell defects in a mouse mutant in mice (8,9). In addition, the closely related gene b lacking MafA in b-cells. Of note, transgenic production of is expressed in primate islet -cells postnatally (10) but MafB in b-cells elevated tryptophan hydroxylase 1 mRNA not in rodents (8,9). production during pregnancy, which drives the seroto- Both MafA and MafB are made relatively late during nin biosynthesis critical for adaptive maternal b-cell mouse islet cell development compared with other islet- responses. Together, these studies provide novel in- enriched transcription factors (11). Thus, MafB expression sight into the role of MAFB in human islet b-cells. begins around embryonic day 10.5 (e10.5) in both insulin- positive (i.e., b-cell) and glucagon-positive (i.e., a-cell) progenitors, whereas MafA is first detected at e13.5 and Type 2 diabetes is characterized by peripheral insulin only in insulin-positive cells (8,9). In contrast, most other resistance and impaired pancreatic a- and b-cell activity islet-enriched transcription factors are produced much 1Department of Molecular Physiology and Biophysics, Vanderbilt University, Received 21 August 2018 and accepted 31 October 2018 Nashville, TN This article contains Supplementary Data online at http://diabetes 2 Department of Translational Research and Cellular Therapeutics, Beckman .diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0903/-/DC1. Research Institute, City of Hope, Duarte, CA H.A.C. and E.M.W. are co-first authors. 3Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN H.A.C. is currently affiliated with the Department of Biological Sciences, Marshall 4Endocrinology and Metabolism Service, Hadassah-Hebrew University Medical University, Huntington, WV. Center, Jerusalem, Israel Y.H. is currently affiliated with the Department of Developmental Biology, Stanford 5Department of Genetics and Institute for Diabetes, Obesity, and Metabolism, University School of Medicine, Stanford, CA. University of Pennsylvania Perelman School of Medicine, Philadelphia, PA © 2018 by the American Diabetes Association. Readers may use this article as 6Diabetes Center, University of California, San Francisco, San Francisco, CA long as the work is properly cited, the use is educational and not for profit, and the 7Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, work is not altered. More information is available at http://www.diabetesjournals TN .org/content/license. Corresponding author: Roland Stein, [email protected] 338 MAFB Effects on Postnatal Islet b-Cell Function Diabetes Volume 68, February 2019 earlier during development (e.g., Pdx1 [e8.5 (12)]) and Islet Isolation, Cell Sorting, and DNA Methylation within a larger fraction of the adult islet cell population Mouse islets were isolated using standard islet isolation (e.g., a, b, d, PP, Pax6 [13], and FoxA1/2 [14]). MafA conditions (18). Islet a- and b-cells were isolated as pre- expression persists in the mouse islet b-cell population viously described (19). a-Cells were sorted by FACS to an postnatally, whereas MafB is restricted to a-cells (8,9). average purity of 80–85%. For isolation of b-cells from However, MafB is re-expressed in a small fraction of islet neonatal (postnatal day 5) and adult (age 2.5 months) fl b-cells during pregnancy (15). Analysis of mice that lack mice, islets from mouse INS-1 gene promoter-green uo- MafA or MafB during pancreas development has demon- rescent protein transgenic mice were sorted for green fl – strated that the MafA mutant has the most significant uorescent protein by FACS to an average purity of 85 Dpancreas phenotype (MafA [16]), which is manifested as 95%. Cadaveric human islets and mouse islets were pro- fi defects in b-cell activity and islet cell architecture after cessed for DNA extraction and bisul te treatment as de- Dpancreas birth. In contrast, there is no overt effect in MafB tailed previously (19). Primers and additional details are islet b-cells as a result of postnatal compensation by MafA, available upon request. although plasma glucagon secretion levels from a-cells are siRNA-Based Knockdown, RNA Isolation, cDNA reduced (10). Synthesis, and Real-time PCR Of note, human MAFA mRNA is made at low levels in Knockdown of Dnmt3a in Min6 cells was performed by juvenile b-cells in relation to adult islets (7), and MAFB is transfection with a pool of specific targeting siRNAs expressed throughout the lifetime of these cells in non- or scrambled controls (Dharmacon) using Lipofectamine human primates (NHPs) and humans (7,10,17). Here, we 2000 (Invitrogen) every 2 days (average transfection effi- fi rst show that the MAFA protein is found in relatively few ciency ;80%). RNA was isolated 4 days posttransfection b juvenile and adolescent human islet -cells in relation to using the RNeasy Micro Kit (QIAGEN). cDNA was gen- 9 fl MAFB and that DNA methylation within the 5 anking erated using Superscript III Reverse Transcriptase (Invi- MafB region of mouse correlates with gene silencing in trogen) by the oligo(dT) priming method. Real-time PCR b b -cells. The impact of MafB on adult islet -cells was next assays were performed using the LightCycler FastStart examined in bMafBTg transgenic mice that sustain pro- DNA Master PLUS SYBR Green kit (Roche) and a Light- duction of this transcription factor postnatally, thus mim- Cycler PCR instrument (Roche). The expression levels icking the expression pattern in humans. Although little were normalized to cyclophilin with previously published impact was observed on coexpression of MafB with en- primers (19). dogenous MafA in islet b-cells, production of MafB alone Mouse islet RNA was collected using the TRIzol reagent was unable to rescue any of the islet deficiencies of (Life Technologies) and a DNA-free RNA kit (Zymo Re- Dpancreas MafA mice. These results suggest that the juvenile search). cDNAs were produced using the iScript cDNA MAFB2 homodimer and adult MAFA/MAFB heterodimer Synthesis Kit (Bio-Rad), and real-time PCR was performed regulators could be involved in controlling age-dependent on a LightCycler 480 system (Roche) with primers listed in differences in human b-cell gene expression (7). Of note, Supplementary Table 2. Real-time PCR results were ana- maternal bMafBTg mice displayed increased b-cell pro- lyzed using the DDCt method; Gapdh was used to normal- liferation and function compared with wild-type mice. ize the mouse islet data. These data illustrate the many distinct ways MAFB may regulate human islet b-cells. Chromatin Immunoprecipitation Assays Chromatin immunoprecipitation (ChIP) experiments with purified a- and b-cells were carried out using the micro- RESEARCH DESIGN AND METHODS ChIP protocol (20). Islets from 2-month-old mice were fl Tissue Collection and Immuno uorescence used for MafB (Bethyl Laboratories) and Dnmt3a (Novus , Human pancreata from healthy juvenile ( 10 years of age) Biologicals) ChIP. Approximately 500 islets per data point donors were received in partnership with the International were cross-linked using 1% formaldehyde for 10 min at Institute for the Advancement of Medicine and National 37°C and quenched using glycine. Sonication was per- Disease Research Interchange for histological analysis formed for 3 3 5 min using a Diagenode Bioruptor (Supplementary Table 1). The Vanderbilt University in- (Diagenode Diagnostics). Approximately 4 mg antibody stitutional review board declared that studies on dei- was incubated at 4°C overnight. Protein A Dynabeads dentified human pancreatic specimens do not qualify as (Thermo Fisher Scientific) were treated overnight with human subject research. BSA, herring sperm, and protease inhibitors before being Human and mouse pancreata were fixed in 4% para- added to the chromatin-antibody mixture for 4 h at 4°C.