33 research outputs found
HECT E3 Ubiquitin Ligase Itch Functions as a Novel Nega
The transcription factor Gli-similar 3 (Glis3) plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the PPxY motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases
The Genome of Deep-Sea Vent Chemolithoautotroph Thiomicrospira crunogena XCL-2
Presented here is the complete genome sequence of Thiomicrospira crunogena XCL-2, representative of ubiquitous chemolithoautotrophic sulfur-oxidizing bacteria isolated from deep-sea hydrothermal vents. This gammaproteobacterium has a single chromosome (2,427,734 base pairs), and its genome illustrates many of the adaptations that have enabled it to thrive at vents globally. It has 14 methyl-accepting chemotaxis protein genes, including four that may assist in positioning it in the redoxcline. A relative abundance of coding sequences (CDSs) encoding regulatory proteins likely control the expression of genes encoding carboxysomes, multiple dissolved inorganic nitrogen and phosphate transporters, as well as a phosphonate operon, which provide this species with a variety of options for acquiring these substrates from the environment. Thiom. crunogena XCL-2 is unusual among obligate sulfur-oxidizing bacteria in relying on the Sox system for the oxidation of reduced sulfur compounds. The genome has characteristics consistent with an obligately chemolithoautotrophic lifestyle, including few transporters predicted to have organic allocrits, and Calvin-Benson-Bassham cycle CDSs scattered throughout the genome
Isolation and functional characterization of a dioxin-inducible CYP1A regulatory region from zebrafish (Danio rerio)
Cytochrome P4501A1 (CYP1A1) is a phase I bio-transformation enzyme involved in the metabolism of xenobiotics via the oxygenation of polycyclic aromatic hydrocarbons (PAHs) including the carcinogen, benzo(a)pyrene. Induction of the CYP1A1 gene is regulated at the transcriptional level and is ligand dependent with the prototypical 2,3,7,8,-tetrachlorodibenzo- p-dioxin (TCDD) being the most potent known inducer of CYP1A1 transcription. This process is mediated by the AHR/ARNT signaling pathway whereby ligand binds AHR in the cytoplasm allowing its translocation to the nucleus where it binds with its hertrodimerization partner, ARNT and subsequently binds DNA at cognate binding sites termed xenobiotic responsive elements (XREs) located in the 5’ flanking region of the CYP1A1 and other genes. The zebrafish (Danio rerio) has recently become an important model system for the study of TCDD-mediated developmental toxicity due to their relative ease of maintaining and breeding, external fertilization, abundant transparent embryos, and sensitivity to TCDD similar to mammalian models. It is therefore essential to characterize the molecular mechanisms of AHR mediated gene regulation in this organism. The upstream flanking region of a putative CYP1A gene from zebrafish was identified by the screening of a PAC genomic library. Sequencing revealed a region which contains 8 putative core xenobiotic response elements (XREs) organized in two distinct clusters. The region between –580 to –187 contains XRE 1-3 while the region between –2608 to –2100 contains XRE 4-8. Only XRE 1, 3, 4, 7, and 8 exhibited TCDD-dependant association of AHR/ARNT complexes when evaluated by gel shift assays. The use of in vitro mutagenesis and Luciferase reporter assays further showed that only XRE’s 4, 7, and 8 were capable of conveying TCDD-mediated gene induction. The role of nucleotides flanking the core XRE was investigated through the use of EMSA and reporter assays. Similar methods were employed on additional transcription factor binding sites identified by in silico analyses revealing two sites conforming to an HNF-3α and CREB motif, respectively, which demonstrate importance to regulation of the gene
Isolation and Functional Characterization of a Dioxin-Inducible CYP1A Regulatory Region From Zebrafish (\u3cem\u3eDanio rerio\u3c/em\u3e)
Cytochrome P4501A1 (CYP1A1) is a phase I bio-transformation enzyme involved in the metabolism of xenobiotics via the oxygenation of polycyclic aromatic hydrocarbons (PAHs) including the carcinogen, benzo(a)pyrene. Induction of the CYP1A1 gene is regulated at the transcriptional level and is ligand dependent with the prototypical 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) being the most potent known inducer of CYP1A1 transcription. This process is mediated by the AHR/ARNT signaling pathway whereby ligand binds AHR in the cytoplasm allowing its translocation to the nucleus where it binds with its hertrodimerization partner, ARNT and subsequently binds DNA at cognate binding sites termed xenobiotic responsive elements (XREs) located in the 5\u27 flanking region of the CYP1A1 and other genes.
The zebrafish (Danio rerio) has recently become an important model system for the study of TCDD-mediated developmental toxicity due to their relative ease of maintaining and breeding, external fertilization, abundant transparent embryos, and sensitivity to TCDD similar to mammalian models. It is therefore essential to vii characterize the molecular mechanisms of AHR mediated gene regulation in this organism.
The upstream flanking region of a putative CYP1A gene from zebrafish was identified by the screening of a PAC genomic library. Sequencing revealed a region which contains 8 putative core xenobiotic response elements (XREs) organized in two distinct clusters. The region between -580 to -187 contains XRE 1-3 while the region between -2608 to -2100 contains XRE 4-8. Only XRE 1, 3, 4, 7, and 8 exhibited TCDD-dependant association of AHR/ARNT complexes when evaluated by gel shift assays. The use of in vitro mutagenesis and Luciferase reporter assays further showed that only XRE\u27s 4, 7, and 8 were capable of conveying TCDD-mediated gene induction. The role of nucleotides flanking the core XRE was investigated through the use of EMSA and reporter assays. Similar methods were employed on additional transcription factor binding sites identified by in silico analyses revealing two sites conforming to an HNF- 3α and CREB motif, respectively, which demonstrate importance to regulation of the gene
Isolation and Functional Characterization of a Dioxin-Inducible CYP1A Regulatory Region From Zebrafish (\u3cem\u3eDanio rerio\u3c/em\u3e)
Cytochrome P4501A1 (CYP1A1) is a phase I bio-transformation enzyme involved in the metabolism of xenobiotics via the oxygenation of polycyclic aromatic hydrocarbons (PAHs) including the carcinogen, benzo(a)pyrene. Induction of the CYP1A1 gene is regulated at the transcriptional level and is ligand dependent with the prototypical 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) being the most potent known inducer of CYP1A1 transcription. This process is mediated by the AHR/ARNT signaling pathway whereby ligand binds AHR in the cytoplasm allowing its translocation to the nucleus where it binds with its hertrodimerization partner, ARNT and subsequently binds DNA at cognate binding sites termed xenobiotic responsive elements (XREs) located in the 5\u27 flanking region of the CYP1A1 and other genes.
The zebrafish (Danio rerio) has recently become an important model system for the study of TCDD-mediated developmental toxicity due to their relative ease of maintaining and breeding, external fertilization, abundant transparent embryos, and sensitivity to TCDD similar to mammalian models. It is therefore essential to vii characterize the molecular mechanisms of AHR mediated gene regulation in this organism.
The upstream flanking region of a putative CYP1A gene from zebrafish was identified by the screening of a PAC genomic library. Sequencing revealed a region which contains 8 putative core xenobiotic response elements (XREs) organized in two distinct clusters. The region between -580 to -187 contains XRE 1-3 while the region between -2608 to -2100 contains XRE 4-8. Only XRE 1, 3, 4, 7, and 8 exhibited TCDD-dependant association of AHR/ARNT complexes when evaluated by gel shift assays. The use of in vitro mutagenesis and Luciferase reporter assays further showed that only XRE\u27s 4, 7, and 8 were capable of conveying TCDD-mediated gene induction. The role of nucleotides flanking the core XRE was investigated through the use of EMSA and reporter assays. Similar methods were employed on additional transcription factor binding sites identified by in silico analyses revealing two sites conforming to an HNF- 3α and CREB motif, respectively, which demonstrate importance to regulation of the gene
Pancreatic beta cell dysfunction in response to chronic hyperglycemia is partially mediated by transcriptional downregulation of Gli-similar 3 (Glis3)
Gli-similar 3 (Glis3) is a Krüppel-like zinc finger protein that plays critical roles in development and in the maintenance of normal physiological functions in a variety of tissues. In humans, GLIS3 deficiency has been linked to a rare syndrome characterized by neonatal diabetes, hypothyroidism, and polycystic kidney disease. In addition, genome-wide association studies (GWAS) have implicated Glis3 as a risk locus for the development of diabetes. While Glis3 is known to play important roles in the specification of endocrine cell fates during pancreatic development, its role in the mature beta cell and the mechanism by which Glis3 dysfunction results in type 2 diabetes remains unclear. We have characterized a rat pancreatic hybridoma cell line (BRIN BD11) that secretes insulin in response to physiologically normal glucose levels but has greatly diminished expression of Glis3. The cell line exhibited significantly decreased levels of known Glis3 target genes, Ins2 and Ccnd2 in addition to the insulin activator, MafA. We investigated the effects of stable Glis3 overexpression on BRIN-BD11 cells and found that Ins2 and Ccnd2 levels were increased in the presence of exogenous Glis3. Interestingly, while chronic exposure of beta cells to high levels of glucose results reduced expression of Ins1/2 and MafA, we found that BRIN BD11 cells were relatively protected from the effects of glucotoxicity and exogenous Glis3 expression partially rescued the phenotype. Collectively, these data suggest that BRIN BD11 cells may be a useful model for the study of beta cell dysfunction preceding the onset of type 2 diabetes
Prey Consumed by Insectivorous Bats After the Occurrence of White-Nose Syndrome
The decline of cave-dwelling bats since the introduction of white-nose syndrome (WNS) to North America led to changes in community interactions as evidenced by spatiotemporal partitioning studies. Indirect effects, such as disease-mediated competition at the community level, can influence the ability of imperiled species to recover because of competitive exclusion. To further investigate community structure following WNS, we assessed the diet of sympatric species with differential WNS susceptibility using molecular techniques. In western Kentucky, Perimyotis subflavus (susceptible) populations declined severely following WNS occurrence. Conversely, Nycticeius humeralis (non-susceptible) populations are increasing. We collected guano from N. humeralis (n=38) and P. subflavus (n=9) during summer 2016. Arthropod DNA was extracted from the guano and a 157 bp target region of insect-COI was amplified. Sequences were analyzed to the lowest taxonomic level provided by the online Barcode of Life Database. Nycticeius humeralis consumed 165 genera belonging to 12 arthropod orders, while P. subflavus ate 92 genera from 8 arthropod orders. All orders consumed by P. subflavus were also eaten by N. humeralis, while 33% percent of all orders occurred exclusively in N. humeralis. Furthermore, N. humeralis consumed 61% of the genera identified in P. subflavus. These data support the potential of increased niche overlap between the two species based on 1) the more generalist habits of N. humeralis and 2) high dietary similarity between N. humeralis and P. subflavus. An increase in niche overlap may suppress the recovery of P. subflavus populations
Generating a transgenic zebrafish line using CRISPR/Cas9 genome editing to determine the spatial and temporal expression of transcription factor glis3 during development
Insulin is a hormone produced by the β-cells of the endocrine pancreas that plays an essential role in blood glucose homeostasis by signaling the uptake of glucose by peripheral tissues to be used for energy or stored as fat. Impairment of the insulin response underlies the development of diabetes mellitus. The transcription factor, Gli-similar 3 (Glis3) is important during the development of the endocrine pancreas and plays additional roles as a transcriptional activator of the insulin gene in the mature organ. Multiple pathologies have been linked to mutations within the GLIS3 locus in humans, including type 1 and type 2 diabetes. Although previous research suggests that Glis3 plays a significant role in the specification of β cells during pancreatic development, the spatio-temporal expression pattern of Glis3, its target genes, and its interacting partners remain enigmatic. The zebrafish (Danio rerio) has emerged as a valuable model organism to study development, largely because of its rapid external development and transparent embryos. In order to visualize the glis3 protein during development, a transgenic zebrafish line is being engineered using the CRISPR/Cas9 system to produce fish that express a chimeric protein consisting of glis3 fused in-frame to enhanced green fluorescent protein (EGFP). This line will provide a better understanding of glis3 expression throughout development and can serve as a tool for future experiments to identify glis3 target genes, protein-protein interactions, and characterize molecular mechanisms that could aid in identifying therapeutic targets for the treatment of Glis3-associated diseases
PIAS-family proteins negatively regulate Glis3 transactivation function through SUMO modification in pancreatic β cells
Gli-similar 3 (Glis3) is Krüppel-like transcription factor associated with the transcriptional regulation of insulin. Mutations within the Glis3 locus have been implicated in a number of pathologies including diabetes mellitus and hypothyroidism. Despite its clinical significance, little is known about the proteins and posttranslational modifications that regulate Glis3 transcriptional activity. In this report, we demonstrate that the SUMO-pathway associated proteins, PIASy and Ubc9 are capable of regulating Glis3 transactivation function through a SUMO-dependent mechanism. We present evidence that SUMOylation of Glis3 by PIAS-family proteins occurs at two conserved lysine residues within the Glis3 N-terminus and modification of Glis3 by SUMO dramatically inhibited insulin transcription. Finally, we provide evidence that Glis3 SUMOylation increases under conditions of chronically elevated glucose and correlates with decreased insulin transcription. Collectively, these results indicate that SUMOylation may serve as a mechanism to regulate Glis3 activity in β cells