The SNAG Domain of Insm1 Regulates Pancreatic Endocrine Cell Differentiation and Represses B-To D-Cell Transdifferentiation

1084 Diabetes Volume 70, May 2021

The SNAG Domain of Insm1 Regulates Pancreatic Endocrine Cell Differentiation and Represses b-to d-Cell Transdifferentiation

Xuehua Liang,1 Hualin Duan,1 Yahui Mao,1 Ulrich Koestner,2 Yiqiu Wei,1 Feng Deng,1 Jingshen Zhuang,1 Huimin Li,1 Cunchuan Wang,1 Luis R. Hernandez-Miranda,3 Weihua Tao,1 and Shiqi Jia1,4

Diabetes 2021;70:1084–109 7 | https://doi.org/10.2337/db20-0883

1 The allocation and specification of pancreatic endocrine Pancreatic endocrine cells are differentiated from Ngn3 lineages are tightly regulated by transcription factors. progenitor cells and undergo terminal differentiation to Disturbances in differentiation of these lineages contrib- form mature a, b, d, pancreatic polypeptide (PP), and e ute to the development of various metabolic diseases, cells via complex regulatory networks (1,2). During differ- including diabetes. The insulinoma-associated protein entiation, glucagon-positive a-cells and PP-positive cells 1(Insm1), which encodes a protein containing one SNAG are first detected at embryonic day 10.5 (E10.5), and the domain and five zinc fingers, plays essential roles in coexpression of these two hormones is observed following pancreatic endocrine cell differentiation and in mature the early developmental stages (3). Insulin expression in b -cell function. In the current study, we compared the b-cells is initially detected at E11.5 (3), whereas somato- differentiation of pancreatic endocrine cells between Insm1 statin-positive d-cells appear at ;E13.5 (3). Ghrelin ex- null and Insm1 SNAG domain mutants (Insm1delSNAG) pression can be detected as early as on E10.5 (2). From to explore the specific function of the SNAG domain of d E15.5 to E18.5, endocrine cells undergo a rapid expansion;

ISLET STUDIES Insm1. We show that the -cell number is increased in however, b-cells expand much more rapidly than the other Insm1delSNAG but not in Insm1 null mutants as com- types of endocrine cells and become the major population pared with the control mice. We also show a less severe of pancreatic endocrine cells (3,4). By E18.5, islets of reduction of the b-cell number in Insm1delSNAG as that fi Langerhans start to form and the expression of hormone in Insm1 null mutants. In addition, similar de cits are – observed in a-, PP, and «-cells in Insm1delSNAG and acquires the characteristic one cell one hormone pattern Insm1 null mutants. We further identified that the in- (3). creased d-cell number is due to b-tod-cell transdiffer- The differential expression of distinct transcription fi entiation. Mechanistically, the SNAG domain of Insm1 factors determines the allocation and speci cation of the interacts with Lsd1, the demethylase of H3K4me1/2. Mu- pancreatic endocrine cell lineages (5). Among these, Ngn3 tation in the SNAG domain of Insm1 results in impaired is transiently expressed in endocrine progenitor cells and recruitment of Lsd1 and increased H3K4me1/2 levels at determines their differentiation fate (1,6,7). Furthermore, hematopoietically expressed homeobox (Hhex) loci that Pdx1 is initially expressed in the pancreatic anlagen, and are bound by Insm1, thereby promoting the transcrip- its expression is essential for the development of all tional activity of the d-cell–specific gene Hhex. Our study pancreatic lineages (8). During development, the expres- has identified a novel function of the SNAG domain of sion of Pdx1 is gradually enriched in pancreatic endocrine Insm1 in the regulation of pancreatic endocrine cell cells, particularly in b-cells (4). The combinatorial expres- differentiation, particularly in the repression of b-tod-cell sion of Pdx1, Nkx2.2, and Nkx6.1 has been shown to be transdifferentiation. essential for b-cell differentiation (4,9,10). Additional

1The First Affiliated Hospital of Jinan University, Guangzhou, China This article contains supplementary material online at https://doi.org/10.2337/ 2Max Delbrück Center for Molecular Medicine, Berlin-Buch, Berlin, Germany figshare.13667342. 3 Institute for Cell Biology and Neurobiology, Charité Universitätsmedizin Berlin, X.L., H.D., and Y.M. contributed equally to this work. Berlin, Germany © 2021 by the American Diabetes Association. Readers may use this article as 4The Institute of Clinical Medicine, Jinan University, Guangzhou, China long as the work is properly cited, the use is educational and not for profit, and the Corresponding author: Shiqi Jia, [email protected], or Weihua Tao, 24249724@ work is not altered. More information is available at https://www.diabetesjournals qq.com .org/content/license. Received 31 August 2020 and accepted 29 January 2021

Diabetes Publish Ahead of Print, published online May 14, 2021 diabetes.diabetesjournals.org Liang and Associates 1085 factors, such as Pax4, promote the specification of b- and In the current study, we used Insm1 SNAG mutant and d-cell fate and suppress a-cell fate (11–13), whereas Pax6 Insm1 null mutant mice to explore the SNAG domain– and Arx are required for a-cell differentiation (12,14). In specific function of Insm1 in the regulation of pancreatic addition, MafB is required for both a-andb-cell differen- endocrine cell differentiation. tiation (15,16). Lastly, hematopoietically expressed homeobox (Hhex) is the only identified d-cell–specific RESEARCH DESIGN AND METHODS factor that is required for d-cell differentiation (17). Animals Functionally, a- and b-cells inversely regulate blood Comparison analysis was performed in Insm1delSNAG mu- glucose levels and maintain euglycemia by tightly regulat- tant (Insm1delSNAG/lacZ), Insm1 null mutant (Insm1lacZ/lacZ), 1 1 1 ing glucagon or insulin secretion. In contrast, d-cells and heterozygous control (Insm1 /lacZ)mice.TheInsm1 / tonically inhibit glucagon and insulin secretion and con- and Insm1delSNAG/delSNAG mice were used to verify the phe- tribute to the therapeutic effects observed in patients with notypes observed in the comparison analysis. The b-cell diabetes (18,19). In mouse islets, b-cells are located in the conditional knockout (InsCre;Insm1f/delSNAG;CKO),Cre- 1 central region, whereas the other endocrine cells locate in heterozygous control (InsCre;Insm1f/ ), and Cre control 1 1 the periphery. Morphologically, d-cells display a complex (InsCre;Insm1 / ) mice were used to investigate the morphology with long neurite-like processes and can di- transdifferentiation of b-tod-cells. The mTmG transgene rectly come into contact with other endocrine cells in was introduced into the CKO and the Cre-heterozygous the islets. This suggests that d-cells are an important control mice for tracing analysis. 1 1 regulator and a novel pharmacological target in the islets The development of Insm1 /lacZ, Insm1 /delSNAG, and 1 fl (20). Furthermore, d-cells rapidly contribute to the in- Insm1 / ox mice has been described previously (25,26,31). crease in the number of b-cells in individuals with type InsCre mice were provided by Dr. Pedro Luis Herrera 1 diabetes (21). (Department Genetic Medicine and Development, Univer- The insulinoma-associated protein 1 (Insm1) gene encodes sity of Geneva, Geneva, Switzerland) (32). mTmG (007576; a DNA-binding protein that contains the SNAG domain and The Jackson Laboratory) mice were provided by Dr. Car- five zinc finger domains (22). Insm1 null mutation results in men Birchmeier (Developmental Biology/Signal Transduc- delayed pancreatic a-cell differentiation and interferes tion, Max Delbrück Center for Molecular Medicine). All with terminal differentiation of b-cells. In addition, de- animals were housed in a 12-h light/dark environment at creased numbers of d- and e-cells are found in Insm1 null a temperature of 21–23°C, with ad libitum access to food 1 1 mutants, whereas an increase in PP cell numbers is and water. Pregnant Insm1 /lacZ or Insm1 /delSNAG females 1 1 detected in these mutant animals (23,24). Insm1 also who had been mated with Insm1 /lacZ or Insm1 /delSNAG plays an essential role in b-cell function in adults. Muta- males were provided drinking water containing 0.1 mg/mL tion of Insm1 in adult b-cells, disturbing the function of phenylephrine hydrochloride, 0.1 mg/mL isoprenaline hy- the Insm1–NeuroD1–FoxA2 complex, leads to an imma- drochloride, and 0.1% ascorbic acid to prevent fetal death ture b-cell phenotype and hyperglycemia (25). Haploin- in the mutants (33). Mice numbers are indicated in each sufficiency of Insm1 delays cell cycle progression in b-cells figure legend. All animal experiments were approved by the during the early postnatal period, resulting in decreased Institutional Animal Care and Use Committee of Jinan b-cell numbers in adults (26). University (IACUC-20201231-03). The SNAG domain, first identified in Snail/Slug and Gfi1/Gfi1b zinc finger transcription factors, functions as Immunofluorescence Analysis a molecular hook to directly recruit the histone demethy- Immunofluorescence was performed as described previ- lase Lsd1 (Kdm1a). Lsd1 participates in multiple histone ously (26). Rabbit anti-insulin (20056,1:1,000; Immuno‐ modification complexes, including the Lsd1–Rcor1–Hdac1/ Star), guinea pig anti-insulin (4011-01F,1:100; LINCO), rat 2 complex (27–29). Lsd1 itself can specifically remove the anti-insulin (MAB1417, 1:500; R&D Systems), rabbit anti- mono- or dimethyl groups from the fourth amino acid glucagon (20076,1:2,000; ImmunoStar), guinea pig anti- lysine on the histone 3 tail (H3K4me1/2). The histone glucagon (1:500; LINCO), rabbit anti-somatostatin (A0566 H3K4me1 and H3K4me2 provides the active chromatin 1:500, DakoCytomation; and HPA019472, 1:2,500, Sigma- loci for transcriptional activity, removing these modifica- Aldrich), rabbit anti-PP (AB939,1:500; Chemicon Interna- tion results in transcriptional repression on associated tional), chicken anti–b-galactosidase (b-gal) (ab9361,1:1,000; genes (30). The function of the N-terminus SNAG domain Abcam), mouse and rabbit anti-ghrelin (ab57222, 1:100, of Insm1 has been studied in pituitary endocrine cells (31). Abcam; and H-031–31, 1:1,000, Phoenix Pharmaceuticals), This study showed that Insm1SNAG can directly recruit rabbit anti-MafA (34), rabbit anti-MafB (HPA005653,1:5,000; Lsd1 to regulate the specification of pituitary endocrine Sigma-Aldrich), goat anti-Pdx1 (ab47383,1:5,000; Abcam), cells; in addition, similar deficits in pituitary differentia- mouse anti-Isl1/2 (39.4D5, 1:50; DSHB), mouse anti-Ngn3 tion have been observed in Insm1 null and Insm1 SNAG (F25A1B3, 1:50; DSHB), mouse anti-Pax6 (PAX6-S, 1:20; domain–specific mutant mice (31). Whether this is a cell- DSHB), mouse anti-Nkx6.1 (F55A10-C, 1:200; DSHB), and type–specific function of the Insm1 SNAG domain or a rat anti-Ki67 (14–5698, 1:50; Invitrogen) were used as broad function in endocrine cells has yet to be determined. primary antibodies. Secondary antibodies coupled to Cy3, 1086 SNAG Domain of Insm1 and Endocrine Lineage Diabetes Volume 70, May 2021

Cy2, or Cy5 against rabbit, rat, mouse, chicken, or guinea P value of ,0.05 was considered statistically significant. pig (Jackson ImmunoResearch Laboratories) were used. All graphical and statistical analyses were performed using Fluorescence imaging was performed using a Leica confocal Prism 8 software (GraphPad Software, San Diego, CA) and microscope, and image processing was performed using Microsoft Excel. For the apoptosis analysis, we presented Adobe Photoshop software. the counting results without a further statistical analysis, due to the very low rates of apoptosis in pancreatic RNA Sequencing and Real-time RT-PCR endocrine cells. Total RNA was isolated from E18.5 pancreata using the TRIzol reagent (Invitrogen). For RNA-sequencing (RNA-seq) Data and Resource Availability 1 analysis, four animals per genotype, Insm1 /lacZ (control), The data sets generated during the current study are avail- Insm1lacZ/lacZ (Insm1 null), and Insm1delSNAG/lacZ (Insm1del- able from the corresponding authors on reasonable request. SNAG), were collected. Sequencing libraries were generated using the NEBNext Ultra Directional RNA Library Prep Kit RESULTS for Illumina (New England Biolabs). Sequencing was per- Pancreatic Endocrine Lineage Differentiation Is Altered formed on an Illumina NovaSeq 6000, and 150-nucleotide in Insm1 SNAG Domain Mutant Mice paired-end reads were generated. At least 6 GB of clean data The Insm1 null mutation results in severe developmental with .94% of them above Q30 were produced for each phenotypes in pancreatic endocrine cells (23,24). However, sample. HISAT2 (35) and StringTie (36) were used to align whether the SNAG domain of Insm1 accounts for the the reads and to analyze the transcripts. The DEGseq R dysfunctions observed in Insm1 mutant mice is presently package was used to identify differentially expressed genes unknown. In this study, we aimed to determine the specific (37). The whole analysis was performed on BMKCloud function of the SNAG domain of Insm1 in the develop- (www.biocloud.net). ment of pancreatic endocrine cells by comparing the de- For real-time RT-PCR analysis, cDNA was synthesized velopmental consequences of Insm1delSNAG and Insm1 using the PrimeScript RT Reagent Kit with gDNA Eraser null mutations. In particular, we analyzed Insm1 delSNAG/lacZ (Takara Bio, Beijing, China), and real-time RT-PCR was (Insm1delSNAG mutation), Insm1lacZ/lacZ (Insm1 null mu- 1 performed using the SYBR Fast qPCR Mix (Takara Bio) on tation), and Insm1 /lacZ (control) littermate embryos by a CFX96 RT-PCR system (Bio-Rad Laboratories). Expres- immunofluorescence. 2DDCt sion levels were determined using the 2 method. We stained b-gal (expressed by lacZ allele) to indicate Actb was used as the internal control, and the values are Insm1 expression in these control and mutant embryos. presented relative to the heterozygous control. The primer We observed a decreased number of insulin-positive b-cell sequences are listed in Supplementary Table 1. as assessed by insulin and b-gal antibody costaining of the pancreas of Insm1delSNAG mutants at E18.5, albeit this Immunoprecipitation and Western Blotting deficit was less severe when compared with that observed Immunoprecipitation and Western blotting were performed in Insm1 null mutant mice (Fig. 1A–A ’’’). No change of b as described previously using SJb -cells (25,31). Anti-Insm1 a-cell numbers was observed in Insm1delSNAG or Insm1 (25), anti-Lsd1 (ab17721; Abcam), anti-NeuroD1 (cs-4373; null mutants as compared with the control (Fig. 1B). Cell Signaling Technology), anti-FoxA2 (sc-6554X; Santa Comparable increased numbers of PP and decreased num- Cruz Biotechnology), and anti-Flag (F1804; Sigma-Aldrich) bers of e-cells, indicated by PP and ghrelin staining, respec- antibodies were used. ImageQuant LAS 500 (GE Healthcare) tively, were observed in Insm1delSNAG and Insm1 null was used for chemiluminescent Western blot imaging. mutants when compared with the control and to each other Chromatin Immunoprecipitation and PCR (Fig. 1D and E). However, the number of somatostatin- d fi Chromatin immunoprecipitation (ChIP) was performed as positive -cells was signi cantly increased in the previously described (25). SJb cells and freshly isolated Insm1delSNAG mutants and decreased in Insm1 null – ’’’ islets were used for the ChIP assays. Insm1 mutation in SJb mutants as compared with the control mice (Fig. 1C C ). fi cells was introduced by transfecting a Cre expression plas- To investigate whether these de cits occur in an earlier fl fl fi mid into the SJb cell line containing the Insm1 ox/ ox alleles. developmental stage (i.e., E15.5), the end of the rst wave A wild-type control and InsmdelSNAG mutant islets were of endocrine cell development and the start of the rapid used for ChIP. The Amy1 gene promoter region was used as expansion of the endocrine cells, we examined the pan- the negative control for ChIP-PCR. Anti-Lsd1 (ab17721; creatic endocrine lineages at E15.5. We observed a similar Abcam), anti-H3K4me1 (ab8895; Abcam), anti-H3K4me2 decrease in the number of insulin-, glucagon-, PP-, and (ab32356; Abcam), and anti-Insm1 (25) antibodies were ghrelin-positive cells in Insm1delSNAG and Insm1 null used. The primers used for ChIP-PCR are listed in Supple- mutants as compared with the control and to each other mentary Table 1. (Fig. 2). However, the number of somatostatin-positive cells was not altered in either Insm1delSNAG or Insm1 null Statistical Analysis mutants compared with the control mice at E15.5 (Fig. 2C– Data are expressed as mean 6 SD. Pairwise comparisons C’’’). The expression of hormone genes was further verified were made using the two-tailed unpaired Student t test. A by real-time RT-PCR (Supplementary Fig. 1). Therefore, diabetes.diabetesjournals.org Liang and Associates 1087

Figure 1—Impaired differentiation of pancreatic endocrine cells in Insm1delSNAG mutant mice at E18.5. Immunoﬂuorescence analysis of insulin (Ins) (A–A’’’), glucagon (Gcg) (B–B’’’), Sst (C–C’’’), PP (D–D’’’), and ghrelin (Ghrl) (E–E’’’)inInsm11/lacZ (A–E), Insm1lacZ/lacZ (A’, B’, C’, D’,andE’), and Insm1 delSNAG/lacZ (A’’, B’’, C’’, D’’,andE’’) mice. The proportions of the indicated endocrine cell types are presented (A’’’, B’’’, C’’’, D’’’,and E’’’). b-Gal staining of Insm1-expressing cells is shown. Hormones were stained green. Eight to 12 sections and 600–1,300 endocrine cells were counted per hormone per mouse (animal numbers: n 5 7forInsm11/lacZ and Insm1 delSNAG/lacZ and n 5 6forInsm1lacZ/lacZ in Ins1 and Sst1 cell counting; n 5 6inGcg1 and PP1 cell counting; and n 5 5inGhrl1 cell counting). Data are expressed as mean 6 SD; the two-tailed unpaired Student t test was used to examine the signiﬁcance between two groups: control vs. null mutants, control vs. delSNAG mutants, and null vs. delSNAG mutants. ns, P . 0.05; *P , 0.05; **P , 0.01; ***P , 0.001. 1088 SNAG Domain of Insm1 and Endocrine Lineage Diabetes Volume 70, May 2021

Figure 2—Impaired differentiation of pancreatic endocrine cells in Insm1delSNAG mutant mice at E15.5. Immunoﬂuorescence analysis of insulin (Ins) (A–A’’’), glucagon (Gcg) (B–B’’’), Sst (C–C’’’), PP (D–D’’’), and ghrelin (Ghrl) (E–E’’’)inInsm11/lacZ (A–E), Insm1lacZ/lacZ (A’, B’, C’, D’, and E’), and Insm1 delSNAG/lacZ (A’’, B’’, C’’, D’’, and E’’) mice. The proportion of the indicated endocrine cell types are presented (A’’’, B’’’,C’’’, D’’’, and E’’’). b-Gal staining of Insm1-expressing cells is shown. Hormones were stained green. Eight to 10 sections and 400–1,200 endocrine cells were counted per hormone per mouse (animal numbers: n 5 5 in Ins1 cell counting, n 5 5 for Insm11/lacZ and Insm1 delSNAG/ lacZ, and n 5 4 for Insm1lacZ/lacZ in Gcg1 cells counting, n 5 4 in Sst1 and PP1 cell counting, n 5 7 for Insm11/lacZ, and n 5 6 for Insm1lacZ/lacZ and Insm1 delSNAG/lacZ in Ghrl1 cell counting). Data are expressed as mean 6 SD; the two-tailed unpaired Student t test was used to examine the signiﬁcance between two groups: control vs. null mutants, control vs. delSNAG mutants, and null vs. delSNAG mutants. ns, P . 0.05; **P , 0.01; ***P , 0.001. diabetes.diabetesjournals.org Liang and Associates 1089

the increased d-cell number in Insm1delSNAG mutants protein 4 (Rbp4) were upregulated in the pancreas of occurred at E18.5 but not at E15.5. Insm1delSNAG mutant mice compared with control or To exclude the contribution of lacZ allele or heterozy- Insm1 null mutant mice (Fig. 5C). This is consistent with gous Insm1 to the deficits, we examined the phenotypes theincreaseinthenumberofd-cells observed in immu- using the Insm1delSNAG/delSNAG mutants and wild-type con- nostaining analysis (Fig. 1C). Most of the dysregulated 1 1 trol (Insm1 / ) mice at both E15.5 and E18.5. We ob- genes can be verified by real-time RT-PCR using the RNA served similar alteration of the endocrine cell lineages as isolated from an independent set of animals (Fig. 5D). that observed in Insm1delSNAG mutants versus the heterozygous control at both development stages (Supple- Transdifferentiation of b- to d-Cells mentary Figs. 2 and 3 vs. Figs. 1 and 2). Thus, the SNAG Because the increased d-cell numbers occurred after E15.5 domain mutation in Insm1 results in the deficits observed in the Insm1delSNAG mutants (Figs. 1C and 2C), we in the endocrine cell lineage differentiation. investigated whether it is the d-cell proliferation that We next investigated the expression of islet-specific contributed to this increase. We performed Ki67 and transcription factors in Insm1delSNAG mutants. To this somatostatin costaining in Insm1delSNAG mutant mice at end, we introduced both the wild-type and heterozygous E15.5 and E18.5. We observed comparable proliferation 1 1 1 as controls and costained Insm1 with the transcription rates in d-cells, as indicated by the ratio of Ki67 Sst /Sst factors MafA, MafB, Pdx1, Pax6, Ngn3, and Isl1/2 (Figs. 3 in control and Insm1 null or Insm1delSNAG mutants and 4). We detected dramatically decreased MafA and (Supplementary Fig. 4A and B). MafB expression and mild decreased Pdx1 expression in We also examined the possibility that a lower apopto- islets of Insm1delSNAG mutants compared with wild- tic rate in d-cells could account for their increased num- type or heterozygous controls at both E18.5 (Fig. 3A–C) bers in Insm1delSNAG mutants. However, the apoptotic and E15.5 (Fig. 4A–C). The expression of Pax6 was de- rate of somatostatin-positive cells was very low, and no creased at E18.5 but had little change at E15.5 (Figs. 3D difference was detected among Insm1delSNAG, Insm1 and 4D). The expression of Isl1/2 and the endocrine cell null mutants, and the control mice (Supplementary Fig. progenitor marker Ngn3 had no alteration at both stages 5). (Figs. 3E and F and 4E and F). Therefore, Insm1delSNAG The costaining of somatostatin with insulin or glucagon mutation results in decreased expression of the a-and was seldom observed, and no difference was detected b-lineage–specific transcription factors but not the pro- between Insm1delSNAG mutants and controls (Supple- genitororpan-isletfactors. mentary Fig. 6A–D). However, increased costaining of Sst- and b-cell–specific transcriptional factor Nkx6.1 was Gene Expression in the Pancreas of Insm1delSNAG detected in Insm1delSNAG mutants at both E15.5 and Mutants E18.5 (Fig. 6A and B and Supplementary Fig. 6E). The To investigate the specific gene expression in the pancreas coexpression of Sst and Nkx6.1 indicates the possibility of Insm1delSNAG mutants, we performed RNA-seq anal- that the increased d-cells may convert from the b-cells in ysis using the E18.5 pancreas of Insm1delSNAG, Insm1 Insm1delSNAG. fl null, and control mice. We observed that the expression of We therefore used InsCre;Insm1delSNAG/ ox (CKO) mice 231 genes was significantly dysregulated in Insm1delSNAG to investigate whether the d-cell number was increased in mutant pancreas as compared with the control using animals harboring a b-cell–specific SNAG mutation. We a cutoff of 1.5-fold and false discovery rate of ,0.05 (Sup- observed a significant increase in d-cell numbers in pan- plementary Table 2). Among these genes, those participat- creas of the neonatal CKO mice (Fig. 7A). Therefore, b-cell– ing in development and hormone secretion were identified specific Insm1delSNAG mutation results in increased d-cell (Supplementary Table 3). We observed a significant over- numbers. We further used a b-cell–specifictracingmouse fl lap in the altered gene expression of Insm1delSNAG and model mTmG;InsCre;Insm1delSNAG/ ox (tracing-CKO) to inves- null mutations, particularly in the genes that code for tigate the transdifferentiation of b-tod-cells. This allowed us hormones and those involved in glucose metabolism and to trace the Insm1delSNAG mutant b-cells using GFP. We secretion regulation (Fig. 5A and B and Supplementary Tables observed significant costaining of GFP with Sst in the pancreas 3 and 4). Approximately 72.7% of the differentially expressed of b-cell–specific Insm1delSNAG mutants but significantly less 1 fl genes in the Insm1delSNAG mutants were also identified costaining in the control mTmG;InsCre;Insm1 / ox mice (Fig. dysregulated in the Insm1 null mutants. However, the Insm1 7B). Therefore, b-cell to d-cell transdifferentiation null mutation resulted in more pronounced changes in occurs in Insm1delSNAG mutant pancreatic b-cells. both gene expression and numbers compared with Insm1delSNAG mutation (Supplementary Table 2 vs. Sup- Insm1delSNAG Mutation Alters the Interaction plementary Table 5). These results are consistent with the Between Insm1 and Lsd1 less severe defects observed in b-cells in Insm1delSNAG To further elucidate the molecular function of the SNAG mutants compared with those in Insm1 null mutants. domain of Insm1, we investigated the proteins that in- Among the cell lineage–specific genes, the d-cell–specific teract with Insm1delSNAG via coimmunoprecipitation. genes somatostatin (Sst), Hhex, and retinol binding We detected that Lsd1 could not be pulled down by the 1090 SNAG Domain of Insm1 and Endocrine Lineage Diabetes Volume 70, May 2021

Figure 3—The expression of pancreatic islet-speciﬁc transcription factors at E18.5. Immunoﬂuorescence analysis of Insm1 and MafA (green) (A), Insm1 and MafB (green) (B), Insm1 and Pdx1 (green) (C), Insm1 and Pax6 (green) (D), Insm1 and Ngn3 (green) (E), and Insm1 and Isl1/2 (green) (F). Double staining of Insm1 and transcription factors is shown in the top panel; single staining of each transcription factor is shown in the bottom panel in A–F. Wild-type (Wt): Insm11/1; heterozygous (Het): Insm11/lacZ; delSNAG: Insm1delSNAG/lacZ. Scale bar 5 25 mm.

Flag antibody in SJb b-cells transfected with Insm1del- FoxA2 (Fig. 8A). Therefore, Insm1delSNAG mutation dis- SNAG-Flag (Fig. 8A). This result is consistent with a previous rupts the interaction between Insm1 and Lsd1 but not observation in pituitary cells (31). Insm1 can interact with between Insm1 and NeuroD1 or FoxA2. NeuroD1 and FoxA2 in pancreatic b-cells (25). Therefore, Analysis of our ChIP-sequencing (ChIP-seq) data pre- we performed immunoprecipitation using NeuroD1 and viously performed in a pancreatic b-cell line (25) revealed FoxA2 antibodies in the same cell lysate containing the that Insm1 can bind to the Hhex locus on an intron and Insm1delSNAG-Flag or Insm1-Flag protein. We observed 18-kb downstream of the gene (Fig. 8B). An H3K4me1- that mutation in the SNAG domain of Insm1 did not enriched region surrounds these binding sites (Fig. 8B, top prevent the interaction between Insm1 and NeuroD1 or panel). These features are not observed on the locus of diabetes.diabetesjournals.org Liang and Associates 1091

Figure 4—The expression of pancreatic islet-speciﬁc transcription factors at E15.5. Immunoﬂuorescence analysis of Insm1 and MafA (green) (A), Insm1 and MafB (green) (B), Insm1 and Pdx1 (green) (C), Insm1 and Pax6 (green) (D), Insm1 and Ngn3 (green) (E), and Insm1 and Isl1/2 (green) (F). Double staining of Insm1 and transcription factors is shown in the top panel; single staining of transcription factors is shown in the bottom panel in A–F. Wild-type (Wt): Insm11/1; heterozygous (Het): Insm11/lacZ; and delSNAG: Insm1delSNAG/lacZ. Scale bar 5 25 mm.

Amy1, a pancreatic exocrine cell–specific gene (Fig. 8B, demethylase activity in histones H3K4me2 and H3K4me1 bottom panel). To investigate the site-specific DNA bind- (38). H3K4me1 indicates the enhancer sequences while ing of Lsd1 recruited by Insm1, we performed ChIP-PCR H3K4me2 is enriched around the promoter and gene body using antibodies against Insm1 and Lsd1. We observed regions (39,40). To investigate the histone modification of enrichment of the Lsd1 binding at the Insm1 biding sites, H3K4me1 and H3K4me2 mediated by Insm1SNAG-Lsd1, the intron, and the 18-kb downstream region of Hhex1,as we performed ChIP-PCR using antibodies against H3K4me1 compared with Amy1 locus, while decreased Lsd1 binding and H3K4me2 in control and Insm1delSNAG mutant islets. at these two sites were observed in Insm1 mutant SJb We detected two- to threefold enrichment of H3K4me2 at cells (Fig. 8C and D). Lsd1 specifically exhibits histone the intron and 18-kb downstream sites of Hhex gene and 1092 SNAG Domain of Insm1 and Endocrine Lineage Diabetes Volume 70, May 2021

Figure 5—Gene expression in the pancreas of InsmdelSNAG mutant mice. A: Comparison of the differentially expressed genes identified by RNA-seq in the isolated pancreas of Insm1 null and Insm1delSNAG mutant mice at E18.5. Heterozygous (Het) was used as the control (n 5 4). B: Genes with altered expression levels detected by RNA-seq in Insm1 null and Insm1delSNAG mutations. C: Heat map showing the b-cell (blue), a-cell, and d-cell (black) lineage-specific genes. D: Real-time RT-PCR confirmation of the differentially expressed genes identified by RNA-seq (n 5 4 for Sst and n 5 3 for other genes). Data are expressed as mean 6 SD; the two-tailed unpaired Student t test was used to examine the significance between two groups: control vs. null mutants and control vs. delSNAG mutants. ns, P . 0.05; *P , 0.05; **P , 0.01; ***P , 0.001. FC, fold change.

a twofold enrichment of H3K4me1 at the 18-kb down- and analyzed the gene expression. We observed that the stream site of Hhex in Insm1delSNAG mutant islets than expression of hormone gene Ins1 was downregulated and in wild-type islets (Fig. 8E and F). Therefore, the Insm1del- Ppy was upregulated. However, the expression of Ins2, Gcg, SNAG mutation disrupts the recruitment of Lsd1 to Insm1- and Sst had no changes. The b-cell–specific transcription binding loci and results in the increased H3K4me1/ factors Pdx1 and Pax6 were downregulated, as observed in H3K4me2 levels, particularly on the d-cell–specificgene Insm1delSNAG mutation, whereas the expression of Mafa Hhex. and Nkx6.1 was upregulated. The expression of d-cell– To investigate whether the pan-Lsd1 inhibition resem- specific factor Rbp4 was increased but Hhex only increased bles the Insm1delSNAG-mediated Lsd1 ablation, we trea- slightly and did not reach a statistical significance (Sup- ted isolated neonatal pancreatic islets with Lsd1 inhibitor plementary Fig. 7). These data indicated that pan-Lsd1 diabetes.diabetesjournals.org Liang and Associates 1093

Figure 6—Increased Nkx6.11Sst1 cells in Nkx6.1 expression b-cells. Nkx6.1, Sst (green), and DAPI staining in pancreas of E15.5 (A) and E18.5 (B) embryos. Top panels in A and B showed the triple channels, while the bottom panels showed the double channels of Nkx6.1 and Sst. Arrows indicate the costaining of Nkx6.1 and Sst. Wild-type: Insm11/1; heterozygous: Insm11/lacZ; and delSNAG: Insm1delSNAG/lacZ. Scale bar 5 10 mm. The cell counting was shown at right. Embryo number, n 5 6, 1,000–3,000 Nkx6.11 cells were counted per animal. Data are expressed as mean 6 SD; the two-tailed unpaired Student t test was used to examine the signiﬁcance between two groups: wild-type vs. delSNAG and heterozygous vs. delSNAG. *P , 0.05.

inhibition results in only partial similarity of the gene roles in the development of pancreatic b-cells; the main- expression changes as those observed in Insm1delSNAG tenance of the interaction between Insm1delSNAG and mutants (Fig. 5D and Supplementary Fig. 7). these factors partially explains the less severe developmental deficits in b-cells of Insm1delSNAG mutant mice DISCUSSION compared with that in the Insm1 null mutants. In the current study, we showed that mutation in the We showed that mutation in the SNAG domain of SNAG domain of Insm1 results in similar developmental Insm1 results in b-tod-cell transdifferentiation. We deficits in a, PP, and e cells but less severe deficits in b-cells identified that the SNAG domain of Insm1 interacts compared with that in Insm1 null mutation. The SNAG with and recruits Lsd1 onto DNA. Mutation of the mutation in Insm1 impairs the interaction between Insm1 SNAG domain interrupts the recruitment of Lsd1 and and Lsd1. However, the interaction of Insm1-NeuroD1 or results in increased H3K4me1 and H3K4me2 levels at the Insm1-FoxA2 is preserved. NeuroD1 and FoxA2 play essential locus of the d-cell–specific gene Hhex. Therefore, we 1094 SNAG Domain of Insm1 and Endocrine Lineage Diabetes Volume 70, May 2021

Figure 7—b-Cell to d-cell transdifferentiation in Insm1delSNAG mutant mice. A: Immunostaining of Insm1, Sst (green), and DAPI (blue) in InsCre;Insm11/1, InsCre;Insm11/flox, and InsCre;Insm1delSNAG/flox mice at E18.5. B: Immunostaining of Sst in mTmG;InsCre;Insm11/flox and mTmG;InsCre;Insm1delSNAG/flox mice at E18.5. GFP indicates the presence of insulin-expressing b-cells. Tomato channel was removed, and Sst showed red. White arrows indicate the Sst and GFP double-positive cells. Statistical analysis is shown in the right panels of A and B. Data are expressed as mean 6 SD; the two-tailed unpaired Student t test was used to examine the significance between two groups: wild-type vs. delSNAG or heterozygous vs. delSNAG mutants. *P , 0.05. Animal numbers: n 5 4inA,8–12 sections, and 600–1,300 Insm11 endocrine cells were counted per mouse; n 5 5 in B, 800–1,000 eGFP1 cells were counted per mouse. diabetes.diabetesjournals.org Liang and Associates 1095

Figure 8—The SNAG domain of Insm1 recruits Lsd1. A: Coimmunoprecipitation (Co-IP) analysis of the lysates of SJb cells transfected with Insm1-Flag or Insm1delSNAG-Flag plasmid. Anti-Flag, anti-NeuroD1, anti-FoxA2, and anti-IgG antibodies were used for Co-IP analysis. Antibodies used for Western blotting (Wb) are shown beside the blot (representative of three independent experiments). The 1% of the lysates used for IP was used as input in Western blot analysis. B: ChIP-seq reads on Hhex (top) and Amy1 (bottom) loci. The reads of ChIPs using antibodies against H3k4me1, Insm1, NeuroD1, and FoxA2 were shown. The reads range was 0–150 for each ChIP and showed on x-axis. Data from independent Insm1 ChIP-seq experiments were shown as Insm1-rep1 and Insm1-rep2. ChIP-PCR analysis using antibodies against Insm1 (C), Lsd1 (D), H3K4me1 (E), and H3K4me2 (F). Insm1 and Lsd1 ChIP assays were performed using SJb cells. H3K4me1 and H3K4me2 ChIP assays were performed using islets isolated from neonatal animals. The Amy1 locus was used as the negative control. The 5% of the chromatin that was used for ChIP was used as an input. Cycle threshold value of quantitative PCR was used for calculation; that is, ChIP divided by input. Data are shown as percentages. Three biologically independent experiments were performed for each ChIP assay. Data are expressed as mean 6 SD; the two-tailed unpaired Student t test was used. ns, P . 0.05; *P , 0.05; **P , 0.01; ***P , 0.001. mut, mutant; wt, wild-type. 1096 SNAG Domain of Insm1 and Endocrine Lineage Diabetes Volume 70, May 2021 conclude that during pancreatic b-cell development Insm1 Foundation of Guangdong Province (grant 2017A030313527 to S.J.), and the represses d-cell fate partially through the recruitment of Guangzhou Science and Technology Program (grant 201704020209 to S.J.). Lsd1 via its SNAG domain. However, considering the Duality of Interest. No potential conflicts of interest relevant to this article various histone modification complexes that can be further were reported. Author Contributions. X.L. and H.D. performed the experiments and recruited by Lsd1 in a temporal- and cellular-dependent fi analyzed the data. Y.M. performed the animal mating, immunostaining, cell manner, the molecular mechanism we identi ed may re- counting, and most of the experiments during the revision. U.K. contributed to veal only partial regulatory scenarios. the preliminary data production and analysis. Y.W., F.D., and H.L. contributed fi Vinckier et al. (41) identi ed a regulatory role of Lsd1 in to the animal mating, immunostaining, and cell counting. J.Z. performed the pancreatic endocrine cell development via repressor activ- immunoprecipitation experiments. C.W., L.R.H.-M., and W.T. contributed to ity in a human embryonic stem cell differentiation system the study and experiments design. S.J. supervised the project and wrote the in vitro. During the differentiation of embryonic stem cells manuscript. S.J. is the guarantor of this work and, as such, had full access to all of to pancreatic endocrine cells, they observed that Lsd1 the data in the study and takes responsibility for the integrity of the data and the plays a strong role in the early differentiation stages and accuracy of the data analysis. appears to have little effect at later stages (41). However, the decreased expression, though not significant, of the References b-cell–specific genes Ins and Pdx1 was observed upon Lsd1 1. Gradwohl G, Dierich A, LeMeur M, Guillemot F. neurogenin3 is required for inhibition in the later stage of development (41). In the the development of the four endocrine cell lineages of the pancreas. Proc Natl Acad Sci U S A 2000;97:1607–1611 current study, we observed that inhibition of Lsd1 by its 2. Heller RS, Jenny M, Collombat P, et al. 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