Chad S. Hunter

LIM-homeodomain genes in mammalian development and human disease.

Abstract.The human and mouse genomes each contain at least 12 genes encoding LIM homeodomain (LIM-HD) transcription factors. These gene regulatory proteins feature two LIM domains in their amino termini and a characteristic DNA binding homeodomain. Studies of mouse models and human patients have established that the LIM-HD factors are critical for the development of specialized cells in multiple tissue types, including the nervous system, skeletal muscle, the heart, the kidneys, and endocrine organs such as the pituitary gland and the pancreas. In this article, we review the roles of the LIM-HD proteins in mammalian development and their involvement in human diseases.

Islet-1 is required for the maturation, proliferation and survival of the endocrine pancreas

OBJECTIVE The generation of mature cell types during pancreatic development depends on the expression of many regulatory and signaling proteins. In this study, we tested the hypothesis that the transcriptional regulator Islet-1 (Isl-1), whose expression is first detected in the mesenchyme and epithelium of the developing pancreas and is later restricted to mature islet cells, is involved in the terminal differentiation of islet cells and maintenance of islet mass. RESEARCH DESIGN AND METHODS To investigate the role of Isl-1 in the pancreatic epithelium during the secondary transition, Isl-1 was conditionally and specifically deleted from embryonic day 13.5 onward using Cre/LoxP technology. RESULTS Isl-1–deficient endocrine precursors failed to mature into functional islet cells. The postnatal expansion of endocrine cell mass was impaired, and consequently Isl-1 deficient mice were diabetic. In addition, MafA, a potent regulator of the Insulin gene and β-cell function, was identified as a direct transcriptional target of Isl-1. CONCLUSIONS These results demonstrate the requirement for Isl-1 in the maturation, proliferation, and survival of the second wave of hormone-producing islet cells.

Islet β-Cell-Specific MafA Transcription Requires the 5′-Flanking Conserved Region 3 Control Domain

ABSTRACT MafA is a key transcriptional activator of islet β cells, and its exclusive expression within β cells of the developing and adult pancreas is distinct among pancreatic regulators. Region 3 (base pairs −8118 to −7750 relative to the transcription start site), one of six conserved 5′ cis domains of the MafA promoter, is capable of directing β-cell-line-selective expression. Transgenic reporters of region 3 alone (R3), sequences spanning regions 1 to 6 (R1-6; base pairs −10428 to +230), and R1-6 lacking R3 (R1-6ΔR3) were generated. Only the R1-6 transgene was active in MafA+ insulin+ cells during development and in adult cells. R1-6 also mediated glucose-induced MafA expression. Conversely, pancreatic expression was not observed with the R3 or R1-6ΔR3 line, although much of the nonpancreatic expression pattern was shared between the R1-6 and R1-6ΔR3 lines. Further support for the importance of R3 was also shown, as the islet regulators Nkx6.1 and Pax6, but not NeuroD1, activated MafA in gel shift, chromatin immunoprecipitation (ChIP), and transfection assays and in vivo mouse knockout models. Lastly, ChIP demonstrated that Pax6 and Pdx-1 also bound to R1 and R6, potentially functioning in pancreatic and nonpancreatic expression. These data highlight the nature of the cis- and trans-acting factors controlling the β-cell-specific expression of MafA.

Islet-1 regulates Arx transcription during pancreatic islet α-cell development

Aristaless related homeodomain protein (Arx) specifies the formation of the pancreatic islet α-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 α-cells, a process mediated by Isl-1 in overexpression, knockdown, and site-directed mutation experiments. Moreover, Arx mRNA levels were up-regulated in islet α-cells upon Isl-1 overexpression in vivo. Isl-1 represents the first known activator of Arx transcription in α-cells, here established to be acting through the conserved Re1 and Re2 control domains.

LIM domain–binding 1 maintains the terminally differentiated state of pancreatic β cells

The recognition of &bgr; cell dedifferentiation in type 2 diabetes raises the translational relevance of mechanisms that direct and maintain &bgr; 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 &bgr; cells. Inducible ablation of LDB1 in mature &bgr; cells impaired insulin secretion and glucose homeostasis. Transcriptomic analysis of LDB1-depleted &bgr; cells revealed the collapse of the terminally differentiated gene program, indicated by a loss of &bgr; cell identity genes and induction of the endocrine progenitor factor neurogenin 3 (NEUROG3). Lineage tracing confirmed that LDB1-depleted, insulin-negative &bgr; 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 &bgr; cells and is a component of active enhancers in both murine and human islets.

Transcriptional Activity of the Islet β Cell Factor Pdx1 Is Augmented by Lysine Methylation Catalyzed by the Methyltransferase Set7/9

Background: Pdx1 interacts with the methyltransferase Set7/9 to transactivate β cell genes. Results: Methylation of Pdx1 residue Lys-131 by Set7/9 augments Pdx1 activity. Conclusion: The ability of Pdx1 to regulate genes in β cells is partially dependent upon its methylation by Set7/9. Significance: This study reveals a previously unappreciated role for Lys methylation in the maintenance of Pdx1 activity and β cell function. The transcription factor Pdx1 is crucial to islet β cell function and regulates target genes in part through interaction with coregulatory factors. Set7/9 is a Lys methyltransferase that interacts with Pdx1. Here we tested the hypothesis that Lys methylation of Pdx1 by Set7/9 augments Pdx1 transcriptional activity. Using mass spectrometry and mutational analysis of purified proteins, we found that Set7/9 methylates the N-terminal residues Lys-123 and Lys-131 of Pdx1. Methylation of these residues occurred only in the context of intact, full-length Pdx1, suggesting a specific requirement of secondary and/or tertiary structural elements for catalysis by Set7/9. Immunoprecipitation assays and mass spectrometric analysis using β cells verified Lys methylation of endogenous Pdx1. Cell-based luciferase reporter assays using wild-type and mutant transgenes revealed a requirement of Pdx1 residue Lys-131, but not Lys-123, for transcriptional augmentation by Set7/9. Lys-131 was not required for high-affinity interactions with DNA in vitro, suggesting that its methylation likely enhances post-DNA binding events. To define the role of Set7/9 in β cell function, we generated mutant mice in which the gene encoding Set7/9 was conditionally deleted in β cells (SetΔβ). SetΔβ mice exhibited glucose intolerance similar to Pdx1-deficient mice, and their isolated islets showed impaired glucose-stimulated insulin secretion with reductions in expression of Pdx1 target genes. Our results suggest a previously unappreciated role for Set7/9-mediated methylation in the maintenance of Pdx1 activity and β cell function.

DNA recognition properties of the LHX3b LIM homeodomain transcription factor

LHX3 is a LIM homeodomain transcription factor with established roles in pituitary and nervous system development. Mutations in the human LHX3 gene are associated with severe hormone deficiency diseases. Previous studies have shown that the human LHX3 gene produces at least three protein isoforms: LHX3a, LHX3b, and M2-LHX3. In gene activation assays, LHX3a and M2-LHX3 are significantly more active than LHX3b because the actions of LHX3b are repressed by an inhibitory domain in its amino terminus. In this report, we investigate the molecular characteristics that result in reduced transcriptional capacity of LHX3b by determining the optimal DNA binding preference of LHX3b. Site selection experiments using purified human LHX3b reveal that it selects AT-rich sequences that contain ATTA/TAAT motifs. The pool of sequences selected by LHX3b is similar to that selected by LHX3a but does not conform to as strict a consensus. Further, the LHX3b-selected sites are bound more avidly by LHX3a and M2-LHX3 suggesting that LHX3b does not act by recognizing LHX3b-specific binding sites in target genes. We conclude that the amino terminal repression domain of LHX3b mostly acts to reduce the transcriptional potency of LHX3 by inhibiting the DNA binding affinity of the homeodomain, with some reduction in DNA binding specificity.

LHX3 interacts with inhibitor of histone acetyltransferase complex subunits LANP and TAF-1β to modulate pituitary gene regulation.

LIM-homeodomain 3 (LHX3) is a transcription factor required for mammalian pituitary gland and nervous system development. Human patients and animal models with LHX3 gene mutations present with severe pediatric syndromes that feature hormone deficiencies and symptoms associated with nervous system dysfunction. The carboxyl terminus of the LHX3 protein is required for pituitary gene regulation, but the mechanism by which this domain operates is unknown. In order to better understand LHX3-dependent pituitary hormone gene transcription, we used biochemical and mass spectrometry approaches to identify and characterize proteins that interact with the LHX3 carboxyl terminus. This approach identified the LANP/pp32 and TAF-1β/SET proteins, which are components of the inhibitor of histone acetyltransferase (INHAT) multi-subunit complex that serves as a multifunctional repressor to inhibit histone acetylation and modulate chromatin structure. The protein domains of LANP and TAF-1β that interact with LHX3 were mapped using biochemical techniques. Chromatin immunoprecipitation experiments demonstrated that LANP and TAF-1β are associated with LHX3 target genes in pituitary cells, and experimental alterations of LANP and TAF-1β levels affected LHX3-mediated pituitary gene regulation. Together, these data suggest that transcriptional regulation of pituitary genes by LHX3 involves regulated interactions with the INHAT complex.

Islet encapsulation with polyphenol coatings decreases pro-inflammatory chemokine synthesis and T cell trafficking ☆

Type 1 Diabetes (T1D) is a chronic pro-inflammatory autoimmune disease consisting of islet-infiltrating leukocytes involved in pancreatic β-cell lysis. One promising treatment for T1D is islet transplantation; however, clinical application is constrained due to limited islet availability, adverse effects of immunosuppressants, and declining graft survival. Islet encapsulation may provide an immunoprotective barrier to preserve islet function and prevent immune-mediated rejection after transplantation. We previously demonstrated that a novel cytoprotective nanothin multilayer coating for islet encapsulation consisting of tannic acid (TA), an immunomodulatory antioxidant, and poly(N-vinylpyrrolidone) (PVPON), was efficacious in dampening inxa0vitro immune responses involved in transplant rejection and preserving inxa0vitro islet function. However, the ability of (PVPON/TA) to maintain islet function inxa0vivo and reverse diabetes has not been tested. Recent evidence has demonstrated that modulation of redox status can affect pro-inflammatory immune responses. Therefore, we hypothesized that transplanted (PVPON/TA)-encapsulated islets can restore euglycemia to diabetic mice and provide an immunoprotective barrier. Our results demonstrate that (PVPON/TA) nanothin coatings can significantly decrease inxa0vitro chemokine synthesis and diabetogenic T cell migration. Importantly, (PVPON/TA)-encapsulated islets restored euglycemia after transplantation into diabetic mice. Our results demonstrate that (PVPON/TA)-encapsulated islets may suppress immune responses and enhance isletxa0allograft acceptance in patients with T1D.

Minireview: Directed Differentiation and Encapsulation of Islet β-Cells—Recent Advances and Future Considerations

Diabetes mellitus has rapidly become a 21st century epidemic with the promise to create vast economic and health burdens, if left unchecked. The 2 major forms of diabetes arise from unique causes, with outcomes being an absolute (type 1) or relative (type 2) loss of functional pancreatic islet β-cell mass. Currently, patients rely on exogenous insulin and/or other pharmacologies that restore glucose homeostasis. Although these therapies have prolonged countless lives over the decades, the striking increases in both type 1 and type 2 diabetic diagnoses worldwide suggest a need for improved treatments. To this end, islet biologists are developing cell-based therapies by which a patients lost insulin-producing β-cell mass is replenished. Pancreatic or islet transplantation from cadaveric donors into diabetic patients has been successful, yet the functional islet demand far surpasses supply. Thus, the field has been striving toward transplantation of renewable in vitro-derived β-cells that can restore euglycemia. Challenges have been numerous, but progress over the past decade has generated much excitement. In this review we will summarize recent findings that have placed us closer than ever to β-cell replacement therapies. With the promise of cell-based diabetes therapies on the horizon, we will also provide an overview of cellular encapsulation technologies that will deliver critical protection of newly implanted cells.