Browsing by Subject "Homeodomain Proteins"

Now showing 1 - 7 of 7
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    Maintenance of Pdx1 mRNA translation in islet β-cells during the unfolded protein response
    (The Endocrine Society, 2014-11) Templin, Andrew T.; Maier, Bernhard; Tersey, Sarah A.; Hatanaka, Masayuki; Mirmira, Raghavendra G.; Department of Pediatrics, IU School of Medicine
    In type 1 diabetes, proinflammatory cytokines secreted by infiltrating immune cells activate the unfolded protein response (UPR) in islet β-cells, which leads to attenuation of global mRNA translation. Under such conditions, privileged mRNAs required for adaptation to the prevailing stress are maintained in an actively translated state. Pdx1 is a β-cell transcription factor that is required for the adaptive UPR, but it is not known how translation of its mRNA is maintained under these conditions. To study translation, we established conditions in vitro with MIN6 cells and mouse islets and a mixture of proinflammatory cytokines (IL-1β, TNF-α, and IFN-γ) that mimicked the UPR conditions seen in type 1 diabetes. Cell extracts were then subjected to polyribosome profiling to monitor changes to mRNA occupancy by ribosomes. Similar to other privileged mRNAs (Atf4 and Chop), Pdx1 mRNA remained partitioned in actively translating polyribosomes under the UPR, whereas the mRNA encoding a proinsulin-processing enzyme (Cpe) and others partitioned into inactively translating monoribosomes. Bicistronic luciferase reporter analyses revealed that the distal portion of the 5'-untranslated region of mouse Pdx1 (between bp -105 to -280) contained elements that promoted translation under both normal and UPR conditions, and this region exhibited conserved sequences and secondary structure similar to those of other known internal ribosome entry sites. Our findings suggest that Pdx1 protein levels are maintained in the setting of the UPR, in part, through elements in the 5'-untranslated region that confer privileged mRNA translation in a 5'-7-methylguanylate cap-independent manner.
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    Pancreatic and duodenal homeobox protein 1 (Pdx-1) maintains endoplasmic reticulum calcium levels through transcriptional regulation of sarco-endoplasmic reticulum calcium ATPase 2b (SERCA2b) in the islet β cell
    (American Society for Biochemistry and Molecular Biology, 2014-11-21) Johnson, Justin S.; Kono, Tatsuyoshi; Tong, Xin; Yamamoto, Wataru R.; Zarain-Herzberg, Angel; Merrins, Matthew J.; Satin, Leslie S.; Gilon, Patrick; Evans-Molina, Carmella; Department of Medicine, IU School of Medicine
    Although the pancreatic duodenal homeobox 1 (Pdx-1) transcription factor is known to play an indispensable role in β cell development and secretory function, recent data also implicate Pdx-1 in the maintenance of endoplasmic reticulum (ER) health. The sarco-endoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) gradient between the cytosol and ER lumen. In models of diabetes, our data demonstrated loss of β cell Pdx-1 that occurs in parallel with altered SERCA2b expression, whereas in silico analysis of the SERCA2b promoter revealed multiple putative Pdx-1 binding sites. We hypothesized that Pdx-1 loss under inflammatory and diabetic conditions leads to decreased SERCA2b levels and activity with concomitant alterations in ER health. To test this, siRNA-mediated knockdown of Pdx-1 was performed in INS-1 cells. The results revealed reduced SERCA2b expression and decreased ER Ca(2+), which was measured using fluorescence lifetime imaging microscopy. Cotransfection of human Pdx-1 with a reporter fused to the human SERCA2 promoter increased luciferase activity 3- to 4-fold relative to an empty vector control, and direct binding of Pdx-1 to the proximal SERCA2 promoter was confirmed by chromatin immunoprecipitation. To determine whether restoration of SERCA2b could rescue ER stress induced by Pdx-1 loss, Pdx1(+/-) mice were fed a high-fat diet. Isolated islets demonstrated an increased spliced-to-total Xbp1 ratio, whereas SERCA2b overexpression reduced the Xbp1 ratio to that of wild-type controls. Together, these results identify SERCA2b as a novel transcriptional target of Pdx-1 and define a role for altered ER Ca(2+) regulation in Pdx-1-deficient states.
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    Safety Outcomes and Near-Adult Height Gain of Growth Hormone-Treated Children with SHOX Deficiency: Data from an Observational Study and a Clinical Trial
    (S. Karger AG, 2017) Benabbad, Imane; Rosilio, Myriam; Child, Christopher J.; Carel, Jean-Claude; Ross, Judith L.; Deal, Cheri L.; Drop, Stenvert L. S.; Zimmermann, Alan G.; Jia, Nan; Quigley, Charmian A.; Blum, Werner F.; Pediatrics, School of Medicine
    BACKGROUND/AIMS: To assess auxological and safety data for growth hormone (GH)-treated children with SHOX deficiency. METHODS: Data were examined for GH-treated SHOX-deficient children (n = 521) from the observational Genetics and Neuroendocrinology of Short Stature International Study (GeNeSIS). For patients with near-adult height information, GeNeSIS results (n = 90) were compared with a clinical trial (n = 28) of SHOX-deficient patients. Near-adult height was expressed as standard deviation score (SDS) for chronological age, potentially increasing the observed effect of treatment. RESULTS: Most SHOX-deficient patients in GeNeSIS had diagnoses of Leri-Weill syndrome (n = 292) or non-syndromic short stature (n = 228). For GeNeSIS patients with near-adult height data, mean age at GH treatment start was 11.0 years, treatment duration 4.4 years, and height SDS gain 0.83 (95% confidence interval 0.49-1.17). Respective ages, GH treatment durations and height SDS gains for GeNeSIS patients prepubertal at baseline (n = 42) were 9.2 years, 6.0 years and 1.19 (0.76-1.62), and for the clinical trial cohort they were 9.2 years, 6.0 years and 1.25 (0.92-1.58). No new GH-related safety concerns were identified. CONCLUSION: Patients with SHOX deficiency who had started GH treatment before puberty in routine clinical practice had a similar height gain to that of patients in the clinical trial on which approval for the indication was based, with no new safety concerns.
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    A systematic analysis of recombination activity and genotype-phenotype correlation in human recombination-activating gene 1 deficiency
    (Elsevier, 2014-04) Lee, Yu Nee; Frugoni, Francesco; Dobbs, Kerry; Walter, Jolan E.; Giliani, Silvia; Gennery, Andrew R.; Al-Herz, Waleed; Haddad, Elie; LeDeist, Francoise; Bleesing, Jack H.; Henderson, Lauren A.; Pai, Sung-Yun; Nelson, Robert P.; El-Ghoneimy, Dalia H.; El-Feky, Reem A.; Reda, Shereen M.; Hossny, Elham; Soler-Palacin, Pere; Fuleihan, Ramsay L.; Patel, Niraj C.; Massaad, Michel J.; Geha, Raif S.; Puck, Jennifer M.; Palma, Paolo; Cancrini, Caterina; Chen, Karin; Vihinen, Mauno; Alt, Frederick W.; Notarangelo, Luigi D.; Department of Medicine, Division of Hematology and Oncology, IU School of Medicine
    Background The recombination-activating gene (RAG) 1/2 proteins play a critical role in the development of T and B cells by initiating the VDJ recombination process that leads to generation of a broad T-cell receptor (TCR) and B-cell receptor repertoire. Pathogenic mutations in the RAG1/2 genes result in various forms of primary immunodeficiency, ranging from T−B− severe combined immune deficiency to delayed-onset disease with granuloma formation, autoimmunity, or both. It is not clear what contributes to such heterogeneity of phenotypes. Objective We sought to investigate the molecular basis for phenotypic diversity presented in patients with various RAG1 mutations. Methods We have developed a flow cytometry–based assay that allows analysis of RAG recombination activity based on green fluorescent protein expression and have assessed the induction of the Ighc locus rearrangements in mouse Rag1−/− pro-B cells reconstituted with wild-type or mutant human RAG1 (hRAG1) using deep sequencing technology. Results Here we demonstrate correlation between defective recombination activity of hRAG1 mutant proteins and severity of the clinical and immunologic phenotype and provide insights on the molecular mechanisms accounting for such phenotypic diversity. Conclusions Using a sensitive assay to measure the RAG1 activity level of 79 mutations in a physiologic setting, we demonstrate correlation between recombination activity of RAG1 mutants and the severity of clinical presentation and show that RAG1 mutants can induce specific abnormalities of the VDJ recombination process.
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    Tlx3 promotes glutamatergic neuronal subtype specification through direct interactions with the chromatin modifier CBP
    (Public Library of Science, 2015) Shimomura, Atsushi; Patel, Dharmeshkumar; Wilson, Sarah M.; Koehler, Karl R.; Khanna, Rajesh; Hashino, Eri; Department of Otolaryngology Head and Neck Surgery, IU School of Medicine
    Nervous system development relies on the generation of precise numbers of excitatory and inhibitory neurons. The homeodomain transcription factor, T-cell leukemia 3 (Tlx3), functions as the master neuronal fate regulator by instructively promoting the specification of glutamatergic excitatory neurons and suppressing the specification of gamma-aminobutyric acid (GABAergic) neurons. However, how Tlx3 promotes glutamatergic neuronal subtype specification is poorly understood. In this study, we found that Tlx3 directly interacts with the epigenetic co-activator cyclic adenosine monophosphate (cAMP)-response element-binding protein (CREB)-binding protein (CBP) and that the Tlx3 homeodomain is essential for this interaction. The interaction between Tlx3 and CBP was enhanced by the three amino acid loop extension (TALE)-class homeodomain transcription factor, pre-B-cell leukemia transcription factor 3 (Pbx3). Using mouse embryonic stem (ES) cells stably expressing Tlx3, we found that the interaction between Tlx3 and CBP became detectable only after these Tlx3-expressing ES cells were committed to a neural lineage, which coincided with increased Pbx3 expression during neural differentiation from ES cells. Forced expression of mutated Tlx3 lacking the homeodomain in ES cells undergoing neural differentiation resulted in significantly reduced expression of glutamatergic neuronal subtype markers, but had little effect on the expression on pan neural markers. Collectively, our results strongly suggest that functional interplay between Tlx3 and CBP plays a critical role in neuronal subtype specification, providing novel insights into the epigenetic regulatory mechanism that modulates the transcriptional efficacy of a selective set of neuronal subtype-specific genes during differentiation.
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    The Transcription Factor Bhlhe40 Programs Mitochondrial Regulation of Resident CD8+ T Cell Fitness and Functionality
    (Elsevier, 2019-09-17) Li, Chaofan; Zhu, Bibo; Son, Young Min; Wang, Zheng; Jiang, Li; Xiang, Min; Ye, Zhenqing; Beckermann, Kathryn E.; Wu, Yue; Jenkins, James W.; Siska, Peter J.; Vincent, Benjamin G.; Prakash, Y. S.; Peikert, Tobias; Edelson, Brian T.; Taneja, Reshma; Kaplan, Mark H.; Rathmell, Jeffrey C.; Dong, Haidong; Hitosugi, Taro; Sun, Jie; Microbiology and Immunology, School of Medicine