Islet-1 (Isl-1) is essential for the survival and ensuing differentiation of pancreatic endocrine progenitors. Isl-1 remains expressed in all adult pancreatic endocrine lineages; however, its specific function in the postnatal pancreas is unclear. Here we determine whether Isl-1 plays a distinct role in the postnatal β-cell by performing physiological and morphometric analyses of a tamoxifen-inducible, β-cell–specific Isl-1 loss-of-function mouse: Isl-1L/L; Pdx1-CreERTm. Ablating Isl-1 in postnatal β-cells reduced glucose tolerance without significantly reducing β-cell mass or increasing β-cell apoptosis. Rather, islets from Isl-1L/L; Pdx1-CreERTm mice showed impaired insulin secretion. To identify direct targets of Isl-1, we integrated high-throughput gene expression and Isl-1 chromatin occupancy using islets from Isl-1L/L; Pdx1-CreERTm mice and βTC3 insulinoma cells, respectively. Ablating Isl-1 significantly affected the β-cell transcriptome, including known targets Insulin and MafA as well as novel targets Pdx1 and Slc2a2. Using chromatin immunoprecipitation sequencing and luciferase reporter assays, we found that Isl-1 directly occupies functional regulatory elements of Pdx1 and Slc2a2. Thus Isl-1 is essential for postnatal β-cell function, directly regulates Pdx1 and Slc2a2, and has a mature β-cell cistrome distinct from that of pancreatic endocrine progenitors.

The recognition of β cell dedifferentiation in type 2 diabetes raises the translational relevance of mechanisms that direct and maintain β cell identity. LIM domain-binding protein 1 (LDB1) nucleates multimeric transcriptional complexes and establishes promoter-enhancer looping, thereby directing fate assignment and maturation of progenitor populations. Many terminally differentiated endocrine cell types, however, remain enriched for LDB1, but its role is unknown. Here, we have demonstrated a requirement for LDB1 in maintaining the terminally differentiated status of pancreatic β cells. Inducible ablation of LDB1 in mature β cells impaired insulin secretion and glucose homeostasis. Transcriptomic analysis of LDB1-depleted β cells revealed the collapse of the terminally differentiated gene program, indicated by a loss of β cell identity genes and induction of the endocrine progenitor factor neurogenin 3 (NEUROG3). Lineage tracing confirmed that LDB1-depleted, insulin-negative β cells express NEUROG3 but do not adopt alternate endocrine cell fates. In primary mouse islets, LDB1 and its LIM homeodomain-binding partner islet 1 (ISL1) were coenriched at chromatin sites occupied by pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1), forkhead box A2 (FOXA2), and NK2 homeobox 2 (NKX2.2) - factors that co-occupy active enhancers in 3D chromatin domains in human islets. Indeed, LDB1 was enriched at active enhancers in human islets. Thus, LDB1 maintains the terminally differentiated state of β cells and is a component of active enhancers in both murine and human islets.

Ldb1 and Ldb2 are coregulators that mediate Lin11-Isl1-Mec3 (LIM)–homeodomain (HD) and LIM-only transcription factor–driven gene regulation. Although both Ldb1 and Ldb2 mRNA were produced in the developing and adult pancreas, immunohistochemical analysis illustrated a broad Ldb1 protein expression pattern during early pancreatogenesis, which subsequently became enriched in islet and ductal cells perinatally. The islet-enriched pattern of Ldb1 was similar to pan-endocrine cell–expressed Islet-1 (Isl1), which was demonstrated in this study to be the primary LIM-HD transcription factor in developing and adult islet cells. Endocrine cell–specific removal of Ldb1 during mouse development resulted in a severe reduction of hormone+ cell numbers (i.e., α, β, and δ) and overt postnatal hyperglycemia, reminiscent of the phenotype described for the Isl1 conditional mutant. In contrast, neither endocrine cell development nor function was affected in the pancreas of Ldb2−/− mice. Gene expression and chromatin immunoprecipitation (ChIP) analyses demonstrated that many important Isl1-activated genes were coregulated by Ldb1, including MafA, Arx, insulin, and Glp1r. However, some genes (i.e., Hb9 and Glut2) only appeared to be impacted by Ldb1 during development. These findings establish Ldb1 as a critical transcriptional coregulator during islet α-, β-, and δ-cell development through Isl1-dependent and potentially Isl1-independent control.

Aristaless related homeodomain protein (Arx) specifies the formation of the pancreatic islet ␣-cell during development. This cell type produces glucagon, a major counteracting hormone to insulin in regulating glucose homeostasis in adults. However, little is known about the factors that regulate Arx transcription in the pancreas. In this study, we showed that the number of Arx ؉ cells was significantly reduced in the pancreata of embryos deficient for the Islet-1 (Isl-1) transcription factor, which was also supported by the reduction in Arx mRNA levels. Chromatin immunoprecipitation analysis localized Isl-1 activator binding sites within two highly conserved noncoding regulatory regions (Re) in the Arx locus, termed Re1 (؉5.6 to ؉6.1 kb) and Re2 (؉23.6 to ؉24 kb). Using cell line-based transfection assays, we demonstrated that a Re1-and Re2-driven reporter was selectively activated in islet ␣-cells, a process mediated by Isl-1 in overexpression, knockdown, and site-directed mutation experiments. Moreover, Arx mRNA levels were upregulated in islet ␣-cells upon Isl-1 overexpression in vivo. Isl-1 represents the first known activator of Arx transcription in ␣-cells, here established to be acting through the conserved Re1 and Re2 control domains.Pancreatic islets play an important role in regulating carbohydrate metabolism through the production and secretion of hormones. The five cell types found in the islet are ␣-, ␤-, ␦-, ⑀-, and pancreatic polypeptide cells that, respectively, produce the hormones glucagon, insulin, somatostatin, ghrelin, and pancreatic polypeptide (1). The hormonal products of the predominant ␣-and ␤-cells act in peripheral tissues in a counter-regulatory manner to control blood glucose homeostasis, with insulin promoting cellular glucose uptake and storage and glucagon promoting its release. Notably, although insulin resistance and ␤-cell dysfunction are the major causes of diabetes, the sustained, unregulated secretion of glucagon from ␣-cells also contributes to hyperglycemia and the associated complications (2-6). In fact, suppression of glucagon activity or levels has been shown to be a promising treatment for diabetics (3, 6 -9). Unfortunately, and in contrast to islet ␤-cells, our understanding of the factors involved in controlling ␣-cell differentiation and function is quite limited (1, 10).Glucagon ϩ cells first appear at embryonic day (E) 9.5 3 in mice, followed by insulin ϩ cells a day later (1). At around E13.5, a major expansion of a distinct population of hormone-producing cells begins to occur, with only these cells becoming mature islet ␣-and ␤-cells (11). A variety of distinct transcription factors are required during development for their production (1), including those that are necessary early in specifying the ␣-(e.g. Arx) and ␤-(e.g. Pax4 and Nkx6.1) lineages and others acting later in cell maintenance and maturation (e.g. MafB, Foxa2, Isl-1, and Pdx1) (12)(13)(14)(15)(16)(17)(18)(19)(20).Arx plays an essential role in islet ␣-cell formation. Expression of this transcrip...