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

Cytochrome P450 1 genes in birds : evolutionary relationships and transcription profiles in chicken and Japanese quail embryos

© The Author(s), 2011. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS One 6 (2011): e28257, doi:10.1371/journal.pone.0028257.Cytochrome P450 1 (CYP1) genes are biomarkers for aryl hydrocarbon receptor (AHR) agonists and may be involved in some of their toxic effects. CYP1s other than the CYP1As are poorly studied in birds. Here we characterize avian CYP1B and CYP1C genes and the expression of the identified CYP1 genes and AHR1, comparing basal and induced levels in chicken and quail embryos. We cloned cDNAs of chicken CYP1C1 and quail CYP1B1 and AHR1. CYP1Cs occur in several bird genomes, but we found no CYP1C gene in quail. The CYP1C genomic region is highly conserved among vertebrates. This region also shares some synteny with the CYP1B region, consistent with CYP1B and CYP1C genes deriving from duplication of a common ancestor gene. Real-time RT-PCR analyses revealed similar tissue distribution patterns for CYP1A4, CYP1A5, CYP1B1, and AHR1 mRNA in chicken and quail embryos, with the highest basal expression of the CYP1As in liver, and of CYP1B1 in eye, brain, and heart. Chicken CYP1C1 mRNA levels were appreciable in eye and heart but relatively low in other organs. Basal transcript levels of the CYP1As were higher in quail than in chicken, while CYP1B1 levels were similar in the two species. 3,3′,4,5,5′-Pentachlorobiphenyl induced all CYP1s in chicken; in quail a 1000-fold higher dose induced the CYP1As, but not CYP1B1. The apparent absence of CYP1C1 in quail, and weak expression and induction of CYP1C1 in chicken suggest that CYP1Cs have diminishing roles in tetrapods; similar tissue expression suggests that such roles may be met by CYP1B1. Tissue distribution of CYP1B and CYP1C transcripts in birds resembles that previously found in zebrafish, suggesting that these genes serve similar functions in diverse vertebrates. Determining CYP1 catalytic functions in different species should indicate the evolving roles of these duplicated genes in physiological and toxicological processes.Funding to MEJ and BB was from the Carl Tryggers Stiftelse and The Swedish Research Council Formas. Funding for BRW and JJS was from the United States National Institutes of Health (National Institute of Environmental Health Sciences), grants R01ES015912 and P42ES007381 to JJS

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

HECT E3 Ubiquitin Ligase Itch Functions as a Novel Negative Regulator of Gli-Similar 3 (Glis3) Transcriptional Activity

<div><p>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 <i>PPxY</i> 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 <i>PPxY</i> 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 <i>Ins2</i> 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.</p></div

Itch inhibits Glis3-mediated transactivation of target genes.

<p>A. INS1 832/13 cells were transfected with pGL4.27 or p3xGlisBS-Luc, FLAG-Glis3, the <i>PY</i>^<i>461</i> mutant, or empty vector, and Myc-Itch, the C832G mutant, or empty vector as indicated. After 48 h, cells were assayed for luciferase and β-galactosidase activity and the normalized relative luciferase activity (nRLU) was calculated and plotted. Each bar represents the mean +/- SEM. * Indicates statistically different value from corresponding Myc empty vector control p < 0.02. B. HEK293T cells were transfected with p-mIP-696-Luc, FLAG-Glis3, the <i>PY</i>^<i>461</i> mutant, or empty vector, and Myc-Itch, the C832G mutant, or empty vector as indicated. After 48 h cells were assayed as described for A. Each bar represents the mean +/- SEM. * Indicates statistically different value from corresponding Myc empty vector control p < 0.02. C. INS1 832/13 cells were transfected with Myc-Itch, the C832G mutant, or empty vector as indicated. After 48 h, RNA was collected and rIns2 mRNA was measured by qRT-PCR analysis. Each bar represents relative <i>Ins2</i> mRNA normalized to 18s rRNA +/- SEM. * Indicates statistically different value compared to empty vector control p < 0.02.</p

Itch, Smurf2, and NEDD4 polyubiquitinate Glis3.

<p>A-B. HEK293T cells were transfected with CMV-HA-Ubiquitin, FLAG-Glis3 or FLAG-Glis3-ΔC480 or their respective <i>PY</i>^<i>461</i> mutants, and Myc-Itch, Smurf2, NEDD4, or empty vector as indicated. Cells were treated with 10 μM MG132 for 6 h prior to harvest. Co-immunoprecipitation was performed using an anti-M2 FLAG antibody and immunoprecipitated proteins were analysed by Western blot using a high affinity anti-HA, anti-M2 FLAG-HRP, anti-Myc or goat anti-mouse-HRP antibodies. C. HEK293T cells were transfected with FLAG Glis3, CMV-HA-Ubiquitin, and Myc-Itch or its catalytically inactive mutant as indicated. Co-IP was performed as described in A-B. D. HEK293T cells were transfected with FLAG-Glis3, Myc Itch, and HA-Ubiquitin or the K48R or K63R ubiquitin mutants as indicated. Co-IP was performed as described in A-B.</p

Itch targets Glis3 for degradation.

<p>A. HEK293T cells were transfected with FLAG-Glis3 and Myc-Itch, or empty vector as indicated. Prior to harvest, cells were treated with 10 μg/ml cycloheximide for the indicated duration. Proteins were analysed by Western blotting using anti-M2 FLAG-HRP antibody, anti-Myc and goat anti-mouse-HRP antibodies, or anti-β actin and goat anti-rabbit-HRP antibodies. Bands were quantified as described in Materials and Methods. B. HEK293T cells were transfected with mouse, human, or zebrafish Glis3 and co-transfected with Myc-Empty vector or Myc-Itch as indicated. Glis3 protein levels were assessed by Western blot using anti-M2 FLAG-HRP antibody. C-D. HEK293T cells were transfected with FLAG-Glis3 and Myc-Smurf2, -NEDD4, or empty vector as indicated. Cells were treated and harvested and proteins analysed as described in A.</p

Itch directly interacts with Glis3 through its WW-domains.

<p>A. HEK293T cells were transfected with FLAG-Glis3 and Myc empty vector, Itch, Itch-Δ-C2, Itch-Δ-HECT, or Itch-WW_I containing only the four WW-domains. Co-immunoprecipitation was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131303#pone.0131303.g002" target="_blank">Fig 2A–2C</a>. B. FLAG-Glis3 or the <i>PY</i>^<i>461</i> mutant and Myc Empty or Myc-Itch were transcribed and translated <i>in vitro</i> and an <i>in vitro</i> pulldown assay was performed using anti-Myc antibody as described in Methods and Materials. G. Schematic showing the interaction between Itch with the <i>PY</i>^<i>461</i> motif of Glis3.</p

The <i>PY</i>^<i>461</i> motif and the ZFD are required for Itch-mediated degradation of Glis3.

<p>A. HEK293T cells were transfected with the indicated FLAG-Glis3 construct and Myc-Itch or empty vector. After 48 h, the cells were harvested and proteins examined by Western blot analysis using an anti-M2 FLAG-HRP or anti-GAPDH antibody. Bands were quantified as described in Materials and Methods. The average intensity of the bands of the Itch plus samples (n = 3) were normalized against that of GAPDH and plotted relative to the average intensity of the bands of the Itch minus samples. Representative images are shown below the histogram. B. HEK293T cells were transfected with FLAG Glis3 or the indicated zinc finger mutant and Myc-Itch or empty vector. After 48 h, cells were harvested and proteins examined by Western blot analysis with anti-M2 FLAG-HRP, anti-Myc, and goat anti-mouse-HRP antibodies. C. HEK293T cells were transfected with FLAG-Glis3 or the indicated ZF mutant along with p-mIP-696-Luc luciferase reporter. After 48 h cells were harvested and assayed for luciferase and ß-galactosidase activity and the normalized relative luciferase values (nRLU) were plotted. Each bar represents the mean +/- SEM. * indicates statistically different value from corresponding Myc empty vector control p < 0.02. # indicates statistically different value from corresponding WT Glis3 control p < 0.02. D. HEK293T cells were transfected with FLAG-Glis3 or the indicated zinc finger mutant along with CMV-HA-Ubiquitin and Myc Itch or empty vector. Cells were treated with 10 μM MG132 for 7 h prior to harvest. Co-immunoprecipitation was performed using an anti-M2 FLAG antibody and immunoprecipitated proteins examined by Western blot analysis using anti-M2 FLAG-HRP, anti-HA, and anti-Myc, and goat anti-mouse-HRP antibodies. E. HEK293T cells were transfected with FLAG Glis3 or the indicated zinc finger mutant and Myc Itch or empty vector. Cells were fixed in 4% paraformaldehyde, permeabilized, and stained with anti-M2 FLAG antibody followed by staining with anti-mouse Alexa 488. Protein localization was examined by fluorescence microscopy.</p

Glis3 associates with Itch, Smurf2, and NEDD4.

<p>A-C. HEK293T cells were transfected with FLAG-Glis3 or the FLAG-Glis3-<i>PY</i>^<i>461</i> mutant and Myc empty vector, Myc-Itch-C832G, Myc-Smurf2-C716G, or Myc-NEDD4-C867G as indicated. Co-immunoprecipitation was performed using a mouse anti-Myc antibody and immunoprecipitated proteins were examined by Western blot analysis using anti-M2 FLAG-HRP or anti-Myc and goat anti-mouse-HRP antibodies. D. HEK293T cells were transfected with FLAG-Glis3-ΔC480 or its respective <i>PY</i>^<i>461</i> mutant and Myc empty vector, Myc-Itch-C832G, Myc Smurf2-C716G, or Myc-NEDD4-C867G and co-IPs performed as described for A-C. E. HEK293T cells were transfected with FLAG-Glis3-ΔN496 and Myc empty vector or Myc-Itch-C832G and co-IPs performed as described in A-C. F. HEK293T cells were transfected with FLAG empty vector, FLAG-Glis3 or the FLAG-Glis3-<i>PY</i>^<i>461</i> mutant as indicated. After 48 h co-immunoprecipitation was performed using a mouse anti-M2 FLAG antibody and immunoprecipitated proteins were examined by Western blot analysis using mouse anti-ITCH primary and goat anti-mouse-HRP antibodies.</p