GLIS3 binds pancreatic beta cell regulatory regions alongside other islet transcription factors.

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 (Glis3Δpanc) 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 indicates that GLIS3 not only regulates insulin expression, but several additional genes critical for beta cell function.

Loss of Glis3 causes dysregulation of retrotransposon silencing and germ cell demise in fetal mouse testis.

Fetal germ cell development is regulated by an elaborate combination of cell-extrinsic and cell-intrinsic signals. Here we identify a novel role for the Krüppel-like transcription factor Gli-Similar 3 (Glis3) in male germ cell development in the mouse embryos. Glis3 is expressed in male germ cells during the brief window of time prior to initiation of piRNA-dependent retrotransposon surveillance. Disruption of Glis3 function led to a widespread reduction in retrotransposon silencing factors, aberrant retrotransposon expression and pronounced germ cell loss. Experimental induction of precocious Glis3 expression in vivo before its normal expression resulted in premature expression of several piRNA pathway members, suggesting that GLIS3 is necessary for the activation of the retrotransposon silencing programs. Our findings reveal an unexpected role for GLIS3 in the development of male germ cells and point to a central role for GLIS3 in the control of retrotransposon silencing in the fetal germline.

GLIS3 is indispensable for TSH/TSHR-dependent thyroid hormone biosynthesis and follicular cell proliferation.

Deficiency in Krüppel-like zinc finger transcription factor GLI-similar 3 (GLIS3) in humans is associated with the development of congenital hypothyroidism. However, the functions of GLIS3 in the thyroid gland and the mechanism by which GLIS3 dysfunction causes hypothyroidism are unknown. In the current study, we demonstrate that GLIS3 acts downstream of thyroid-stimulating hormone (TSH) and TSH receptor (TSHR) and is indispensable for TSH/TSHR-mediated proliferation of thyroid follicular cells and biosynthesis of thyroid hormone. Using ChIP-Seq and promoter analysis, we demonstrate that GLIS3 is critical for the transcriptional activation of several genes required for thyroid hormone biosynthesis, including the iodide transporters Nis and Pds, both of which showed enhanced GLIS3 binding at their promoters. The repression of cell proliferation of GLIS3-deficient thyroid follicular cells was due to the inhibition of TSH-mediated activation of the mTOR complex 1/ribosomal protein S6 (mTORC1/RPS6) pathway as well as the reduced expression of several cell division-related genes regulated directly by GLIS3. Consequently, GLIS3 deficiency in a murine model prevented the development of goiter as well as the induction of inflammatory and fibrotic genes during chronic elevation of circulating TSH. Our study identifies GLIS3 as a key regulator of TSH/TSHR-mediated thyroid hormone biosynthesis and proliferation of thyroid follicular cells and uncovers a mechanism by which GLIS3 deficiency causes neonatal hypothyroidism and prevents goiter development.

Transcription Factor GLIS3: A New and Critical Regulator of Postnatal Stages of Mouse Spermatogenesis.

In this study, we identify a novel and essential role for the Krüppel-like zinc finger transcription factor GLI-similar 3 (GLIS3) in the regulation of postnatal spermatogenesis. We show that GLIS3 is expressed in gonocytes, spermatogonial stem cells (SSCs) and spermatogonial progenitors (SPCs), but not in differentiated spermatogonia and later stages of spermatogenesis or in somatic cells. Spermatogenesis is greatly impaired in GLIS3 knockout mice. Loss of GLIS3 function causes a moderate reduction in the number of gonocytes, but greatly affects the generation of SSCs/SPCs, and as a consequence the development of spermatocytes. Gene expression profiling demonstrated that the expression of genes associated with undifferentiated spermatogonia was dramatically decreased in GLIS3-deficient mice and that the cytoplasmic-to-nuclear translocation of FOXO1, which marks the gonocyte-to-SSC transition and is necessary for SSC self-renewal, is inhibited. These observations suggest that GLIS3 promotes the gonocyte-to-SSC transition and is a critical regulator of the dynamics of early postnatal spermatogenesis. Stem Cells 2016;34:2772-2783.

TRANSCRIPTION FACTOR GLI-SIMILAR 3 (GLIS3): IMPLICATIONS FOR THE DEVELOPMENT OF CONGENITAL HYPOTHYROIDISM.

Congenital hypothyroidism (CH) is the most frequent endocrine disorder in neonates. While several genetic mutations have been identified that result in developmental defects of the thyroid gland or thyroid hormone synthesis, genetic factors have yet to be identified in many CH patients along with the mechanisms underlying their pathophysiology. Mutations in the gene encoding the Krüppel-like transcription factor, GLI-similar 3 (GLIS3) have been associated with the development of a syndrome characterized by congenital hypothyroidism and neonatal diabetes and similar phenotypes were observed in mouse knockout models of Glis3. Patients with GLIS3-mediated CH exhibit diminished serum levels of thyroxine (T4) and triiodothyronine (T3) and elevated thyroid stimulating hormone (TSH) and thyroglobulin (TG). However, the inconsistent presentation of clinical features associated with this CH has made it difficult to ascertain a causative mechanism. Future elucidation of the biological functions of GLIS3 in the thyroid will be crucial to the discovery of new therapeutic opportunities for the treatment of CH.

Protection of primary neurons and mouse brain from Alzheimer's pathology by molecular tweezers.

Alzheimer's disease is a devastating cureless neurodegenerative disorder affecting >35 million people worldwide. The disease is caused by toxic oligomers and aggregates of amyloid ß protein and the microtubule-associated protein tau. Recently, the Lys-specific molecular tweezer CLR01 has been shown to inhibit aggregation and toxicity of multiple amyloidogenic proteins, including amyloid ß protein and tau, by disrupting key interactions involved in the assembly process. Following up on these encouraging findings, here, we asked whether CLR01 could protect primary neurons from Alzheimer's disease-associated synaptotoxicity and reduce Alzheimer's disease-like pathology in vivo. Using cell culture and brain slices, we found that CLR01 effectively inhibited synaptotoxicity induced by the 42-residue isoform of amyloid ß protein, including ∼80% inhibition of changes in dendritic spines density and long-term potentiation and complete inhibition of changes in basal synaptic activity. Using a radiolabelled version of the compound, we found that CLR01 crossed the mouse blood-brain barrier at ∼2% of blood levels. Treatment of 15-month-old triple-transgenic mice for 1 month with CLR01 resulted in a decrease in brain amyloid ß protein aggregates, hyperphosphorylated tau and microglia load as observed by immunohistochemistry. Importantly, no signs of toxicity were observed in the treated mice, and CLR01 treatment did not affect the amyloidogenic processing of amyloid ß protein precursor. Examining induction or inhibition of the cytochrome P450 metabolism system by CLR01 revealed minimal interaction. Together, these data suggest that CLR01 is safe for use at concentrations well above those showing efficacy in mice. The efficacy and toxicity results support a process-specific mechanism of action of molecular tweezers and suggest that these are promising compounds for developing disease-modifying therapy for Alzheimer's disease and related disorders.

Gli-similar proteins: their mechanisms of action, physiological functions, and roles in disease.

Gli-similar (Glis) 1-3 proteins constitute a subfamily of Krüppel-like zinc-finger proteins that are closely related to members of the Gli family. Glis proteins have been implicated in several pathologies, including cystic kidney disease, diabetes, hypothyroidism, fibrosis, osteoporosis, psoriasis, and cancer. In humans, a mutation in the Glis2 gene has been linked to the development of nephronophthisis (NPHP), a recessive cystic kidney disease, while mutations in Glis3 lead to an extended multisystem phenotype that includes the development of neonatal diabetes, polycystic kidneys, congenital hypothyroidism, and facial dysmorphism. Glis3 has also been identified as a risk locus for type-1 and type-2 diabetes and additional studies have revealed a role for Glis3 in pancreatic endocrine development, ß-cell maintenance, and insulin regulation. Similar to Gli1-3, Glis2 and 3 have been reported to localize to the primary cilium. These studies appear to suggest that Glis proteins are part of a primary cilium-associated signaling pathway(s). It has been hypothesized that Glis proteins are activated through posttranslational modifications and subsequently translocate to the nucleus where they regulate transcription by interacting with Glis-binding sites in the promoter regions of target genes. This chapter summarizes the current state of knowledge regarding mechanisms of action of the Glis family of proteins, their physiological functions, as well as their roles in disease.

Gli-similar (Glis) Krüppel-like zinc finger proteins: insights into their physiological functions and critical roles in neonatal diabetes and cystic renal disease.

GLI-similar (Glis) 1-3 proteins constitute a subfamily of the Krüppel-like zinc finger transcription factors that are closely related to the Gli family. Glis1-3 play critical roles in the regulation of a number of physiological processes and have been implicated in several pathologies. Mutations in GLIS2 have been linked to nephronophthisis, an autosomal recessive cystic kidney disease. Loss of Glis2 function leads to renal atrophy and fibrosis that involves epithelial-mesenchymal transition (EMT) of renal tubule epithelial cells. Mutations in human GLIS3 have been implicated in a syndrome characterized by neonatal diabetes and congenital hypothyroidism (NDH) and in some patients accompanied by polycystic kidney disease, glaucoma, and liver fibrosis. In addition, the GLIS3 gene has been identified as a susceptibility locus for the risk of type 1 and 2 diabetes. Glis3 plays a key role in pancreatic development, particularly in the generation of ß-cells and in the regulation of insulin gene expression. Glis2 and Glis3 proteins have been demonstrated to localize to the primary cilium, a signaling organelle that has been implicated in several pathologies, including cystic renal diseases. This association suggests that Glis2/3 are part of primary cilium-associated signaling pathways that control the activity of Glis proteins. Upon activation in the primary cilium, Glis proteins may translocate to the nucleus where they subsequently regulate gene transcription by interacting with Glis-binding sites in the promoter regulatory region of target genes. In this review, we discuss the current knowledge of the Glis signaling pathways, their physiological functions, and their involvement in several human pathologies.

A systematic analysis of predicted phosphorylation sites within the human pregnane X receptor protein.

The pregnane X receptor (PXR, NR1I2) regulates the expression of genes that encode drug-metabolizing enzymes and drug transporter proteins in liver and intestine. Understanding the molecular mechanisms that modulate PXR activity is therefore critical for the development of effective therapeutic strategies. Several recent studies have implicated the activation of kinase signaling pathways in the regulation of PXR biological activity, although direct evidence and molecular mechanisms are currently lacking. We therefore sought to characterize potential phosphorylation sites within the PXR protein by use of a rational, comprehensive, and systematic site-directed mutagenesis approach to generate phosphomimetic mutations (Ser/Thr --> Asp) and phospho-deficient mutations (Ser/Thr --> Ala) at 18 predicted consensus kinase recognition sequences in the human PXR protein. Here, we identify amino acid residues Ser8, Thr57, Ser208, Ser305, Ser350, and Thr408 as being critical for biological activity of the PXR protein. Mutations at positions 57 and 408 abolish ligand-inducible PXR activity. Mutations in the extreme N terminus and in the PXR ligand-binding domain at positions Ser8, Ser305, Ser350, and Thr408 decrease the ability of PXR to form heterodimers with retinoid X receptor alpha. Mutations at positions Ser208, Ser305, Ser350, and Thr408 alter PXR-protein cofactor interactions. Finally, the subcellular localization of the PXR protein is profoundly affected by mutations at position Thr408. These data suggest that PXR activity can potentially be regulated by phosphorylation at specific amino acid residues within several predicted consensus kinase recognition sequences to differentially affect PXR biological activity.

Cyclic AMP-dependent protein kinase signaling modulates pregnane x receptor activity in a species-specific manner.

Pregnane x receptor is a ligand-activated transcription factor that regulates drug-inducible expression of several key cytochrome P450 enzymes and drug transporter proteins in liver and intestine in a species-specific manner. Activation of this receptor modulates several key biochemical pathways, including gluconeogenesis, beta-oxidation of fatty acids, fatty acid uptake, cholesterol homeostasis, and lipogenesis. It is of current interest to determine whether the interaction between pregnane x receptor and these key biochemical pathways is evolutionarily conserved. We show here that activation of the cyclic AMP-dependent protein kinase signaling pathway synergizes with pregnane x receptor-mediated gene activation in mouse hepatocytes. Conversely, cyclic AMP-dependent protein kinase signaling has a repressive effect upon pregnane x receptor-mediated gene activation in rat and human hepatocytes. We show that the human pregnane x receptor protein can serve as an effective substrate for catalytically active cyclic AMP-dependent protein kinase in vitro. Metabolic labeling of the protein in vivo indicates that human pregnane x receptor exists as a phosphoprotein and that activation of cyclic AMP-dependent protein kinase signaling modulates the phosphorylation status of pregnane x receptor. Activation of cyclic AMP-dependent protein kinase signaling also modulates the interactions of pregnane x receptor with protein cofactors. Our results define the species-specific impact of cyclic AMP-dependent protein kinase signaling on pregnane x receptor and provide a molecular explanation of cyclic AMP-dependent protein kinase-mediated repression of human pregnane x receptor activity. Taken together, our results identify a novel mode of regulation of pregnane x receptor activity and highlight prominent functional differences in the process across species.

The traditional Chinese herbal remedy tian xian activates pregnane X receptor and induces CYP3A gene expression in hepatocytes.

The pregnane X receptor (PXR, NR1I2) is a member of the nuclear receptor superfamily that is activated by a myriad of clinically used compounds and natural products. Activation of PXR in liver regulates the expression genes encoding proteins that are intimately involved in the hepatic uptake, metabolism, and elimination of toxic compounds from our bodies. PXR-mediated herb-drug interactions can have undesirable effects in patients receiving combination therapy. This can be especially important in cancer patients who self-administer over-the-counter herbal remedies together with conventional anticancer chemotherapeutics. Tian xian is a traditional Chinese herbal anticancer remedy that activates human PXR in cell-based reporter gene assays. Moreover, tian xian alters the strength of interaction between the human PXR protein and transcriptional cofactor proteins. A novel line of humanized PXR mice are described and used here to show that tian xian increases expression of Cyp3a11 in primary cultures of rodent hepatocytes. Tian xian also induces expression of CYP3A4 in primary cultures of human hepatocytes. Taken together, these data indicate that coadministration of tian xian is probably contraindicated in patients undergoing anticancer therapy with conventional chemotherapeutic agents. These data are of particular importance due to the fact that this herbal remedy is currently marketed as an adjunct therapy that reduces the side effects of conventional chemotherapy and is available without a prescription. Future studies should be conducted to determine the extent to which coadministration of this Chinese herbal remedy alters the pharmacokinetic and pharmacodynamic properties of conventional anticancer therapy.