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The Krüppel-like zinc finger transcription factor Gli-similar 3 (GLIS3) plays a critical role in the regulation of pancreatic beta cells, with global Glis3-knockout mice suffering from severe hyperglycemia and dying by post-natal day 11. In addition, GLIS3 has been shown to directly regulate the early endocrine marker Ngn3, as well as Ins2 gene expression in mature beta cells. We hypothesize that GLIS3 regulates several other genes critical to beta cell function, in addition to Ins2, by directly binding to regulatory regions. We therefore generated a pancreas-specific Glis3 deletion mouse model (Glis3Δ panc ) using a Pdx1-driven Cre mouse line. Roughly 20% of these mice develop hyperglycemia by 8 weeks and lose most of their insulin expression. However, this did not appear to be due to loss of the beta cells themselves, as no change in cell death was observed. Indeed, presumptive beta cells appeared to persist as PDX1+/INS−/MAFA−/GLUT2− cells. Islet RNA-seq analysis combined with GLIS3 ChIP-seq analysis revealed apparent direct regulation of a variety of diabetes-related genes, including Slc2a2 and Mafa. GLIS3 binding near these genes coincided with binding for other islet-enriched transcription factors, indicating these are distinct regulatory hubs. Our data indicate that GLIS3 regulates not only insulin expression, but also several additional genes critical for beta cell function.

Recent advances in high throughput RNA sequencing have revealed that, in addition to messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs) play an important role in the regulation of many cell functions and of organ development. While a number of lncRNAs have been identified in pancreatic islets, their function remains largely undetermined. Here, we identify a novel long ncRNA regulated by the transcription factor GLIS3, which we refer to as GLIS3 regulated 1 (G3R1). This lncRNA was identified for its significant loss of expression in GLIS3 knockout mouse pancreatic islets. G3R1 appears to be specifically expressed in mouse pancreatic β-cells and in a β-cell line (βTC-6). ChIP-seq analysis indicated that GLIS3 and other islet-enriched transcription factors bind near the G3R1 gene, suggesting they directly regulate G3R1 transcription. Similarly, an apparent human homolog of G3R1 displays a similar expression pattern, with additional expression seen in human brain. In order to determine the function of G3R1 in mouse pancreatic β-cells, we utilized CRISPR to develop a knockout mouse where ~80% of G3R1 sequence is deleted. Phenotypic analysis of these mice did not reveal any impairment in β-cell function or glucose regulation, indicating the complexity underlying the study of lncRNA function.

GLI-Similar 3 (GLIS3) is a member of the GLIS subfamily of Krüppel-like zinc finger transcription factors that functions as an activator or repressor of gene expression. Study of GLIS3-deficiency in mice and humans revealed that GLIS3 plays a critical role in the regulation of several biological processes and is implicated in the development of various diseases, including hypothyroidism and diabetes. This was supported by genome-wide association studies that identified significant associations of common variants in GLIS3 with increased risk of these pathologies. To obtain insights into the causal mechanisms underlying these diseases, it is imperative to understand the mechanisms by which this protein regulates the development of these pathologies. Recent studies of genes regulated by GLIS3 led to the identification of a number of target genes and have provided important molecular insights by which GLIS3 controls cellular processes. These studies revealed that GLIS3 is essential for thyroid hormone biosynthesis and identified a critical function for GLIS3 in the generation of pancreatic β cells and insulin gene transcription. These observations raised the possibility that the GLIS3 signaling pathway might provide a potential therapeutic target in the management of diabetes, hypothyroidism, and other diseases. To develop such strategies, it will be critical to understand the upstream signaling pathways that regulate the activity, expression and function of GLIS3. Here, we review the recent progress on the molecular mechanisms by which GLIS3 controls key functions in thyroid follicular and pancreatic β cells and how this causally relates to the development of hypothyroidism and diabetes.

Insulin produced by islet β-cells plays a critical role in glucose homeostasis, with type 1 and type 2 diabetes both resulting from inactivation and/or loss of this cell population. Islet-enriched transcription factors regulate β-cell formation and function, yet little is known about the molecules recruited to mediate control. An unbiased in-cell biochemical and mass spectrometry strategy was used to isolate MafA transcription factor–binding proteins. Among the many coregulators identified were all of the subunits of the mixed-lineage leukemia 3 (Mll3) and 4 (Mll4) complexes, with histone 3 lysine 4 methyltransferases strongly associated with gene activation. MafA was bound to the ∼1.5 MDa Mll3 and Mll4 complexes in size-fractionated β-cell extracts. Likewise, closely related human MAFB, which is important to β-cell formation and coproduced with MAFA in adult human islet β-cells, bound MLL3 and MLL4 complexes. Knockdown of NCOA6, a core subunit of these methyltransferases, reduced expression of a subset of MAFA and MAFB target genes in mouse and human β-cell lines. In contrast, a broader effect on MafA/MafB gene activation was observed in mice lacking NCoA6 in islet β-cells. We propose that MLL3 and MLL4 are broadly required for controlling MAFA and MAFB transactivation during development and postnatally.

Analysis of MafB−/−mice has suggested that the MAFB transcription factor was essential to islet α- and β-cell formation during development, although the postnatal physiological impact could not be studied here because these mutants died due to problems in neural development. Pancreas-wide mutant mice were generated to compare the postnatal significance of MafB ( MafBΔpanc) and MafA/B ( MafABΔpanc) with deficiencies associated with the related β-cell-enriched MafA mutant (MafAΔpanc). Insulin+cell production and β-cell activity were merely delayed in MafBΔpancislets until MafA was comprehensively expressed in this cell population. We propose that MafA compensates for the absence of MafB in MafBΔpancmice, which is supported by the death of MafABΔpancmice soon after birth from hyperglycemia. However, glucose-induced glucagon secretion was compromised in adult MafBΔpancislet α-cells. Based upon these results, we conclude that MafB is only essential to islet α-cell activity and not β-cell. Interestingly, a notable difference between mice and humans is that MAFB is coexpressed with MAFA in adult human islet β-cells. Here, we show that nonhuman primate (NHP) islet α- and β-cells also produce MAFB, implying that MAFB represents a unique signature and likely important regulator of the primate islet β-cell.

Recent studies have provided evidence for a regulatory role of GLI-similar (GLIS) transcription factors in reprogramming, maintenance and differentiation of several stem and progenitor cell populations. GLIS1, in conjunction with several other reprogramming factors, was shown to markedly increase the efficiency of generating induced pluripotent stem cells (iPSC) from somatic cells. GLIS2 has been reported to contribute to the maintenance of the pluripotent state in hPSCs. In addition, GLIS2 has a function in regulating self-renewal of hematopoietic progenitors and megakaryocytic differentiation. GLIS3 plays a critical role during the development of several tissues. GLIS3 is able to promote reprogramming of human fibroblasts into retinal pigmented epithelial (RPE) cells. Moreover, GLIS3 is essential for spermatogonial stem cell renewal and spermatogonial progenitor cell differentiation. During pancreas development, GLIS3 protein is first detectable in bipotent pancreatic progenitors and pro-endocrine progenitors and plays a critical role in the generation of pancreatic beta cells. Here, we review the current status of the roles of GLIS proteins in the maintenance and differentiation of these different stem and progenitor cells.

For over two decades now, studies into pancreatic development have relied largely on the mouse as a model organism. These studies have generated a wealth of information into the genetic regulatory networks involved in pancreas formation, identifying key transcription factors involved in early pancreatic differentiation, endocrine cell specification, and mature hormone-producing cell production [reviewed recently in (1)]. Many of these genes have been confirmed to play a role in β-cell development in humans as well, with some factors linked to monogenic forms of diabetes, while others through Genome-Wide Association Studies (GWAS) have been linked to increased risk of developing either type 1 or type 2 diabetes mellitus.