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 (\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

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

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

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