DOCSLIB.ORG

Open Dogan Phdthesis Final.Pdf

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

The Pennsylvania State University The Graduate School Eberly College of Science ELUCIDATING BIOLOGICAL FUNCTION OF GENOMIC DNA WITH ROBUST SIGNALS OF BIOCHEMICAL ACTIVITY: INTEGRATIVE GENOME-WIDE STUDIES OF ENHANCERS A Dissertation in Biochemistry, Microbiology and Molecular Biology by Nergiz Dogan © 2014 Nergiz Dogan Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2014 ii The dissertation of Nergiz Dogan was reviewed and approved* by the following: Ross C. Hardison T. Ming Chu Professor of Biochemistry and Molecular Biology Dissertation Advisor Chair of Committee David S. Gilmour Professor of Molecular and Cell Biology Anton Nekrutenko Professor of Biochemistry and Molecular Biology Robert F. Paulson Professor of Veterinary and Biomedical Sciences Philip Reno Assistant Professor of Antropology Scott B. Selleck Professor and Head of the Department of Biochemistry and Molecular Biology *Signatures are on file in the Graduate School iii ABSTRACT Genome-wide measurements of epigenetic features such as histone modifications, occupancy by transcription factors and coactivators provide the opportunity to understand more globally how genes are regulated. While much effort is being put into integrating the marks from various combinations of features, the contribution of each feature to accuracy of enhancer prediction is not known. We began with predictions of 4,915 candidate erythroid enhancers based on genomic occupancy by TAL1, a key hematopoietic transcription factor that is strongly associated with gene induction in erythroid cells. Seventy of these DNA segments occupied by TAL1 (TAL1 OSs) were tested by transient transfections of cultured hematopoietic cells, and 56% of these were active as enhancers. Sixty-six TAL1 OSs were evaluated in transgenic mouse embryos, and 65% of these were active enhancers in various tissues. Inclusion of additional epigenetic features improved the prediction accuracy, with combinations of TAL1, GATA1, EP300, H3K4me1, and H3K27ac giving high accuracy of enhancer prediction (70%-75% success depending on method of clustering) while having strong discriminatory power maintaining good sensitivity (Sn, up to 84%) and specificity (Sp, up to 80%). Importantly, it was shown that activating histone marks in the absence of key transcription factors or open chromatin profile is a weak predictor of enhancer activity, and had no discriminatory power to predict enhancers. Motifs that distinguish active from inactive TAL1 OSs implicate IRFs, STATs, and FOX protein families as candidate positive co-factors with TAL1, while REST (NRSF) and HOX family proteins are implicated in inactivity. While signals for evolutionary constraint were weak over the entire TAL1-bound DNA segments regardless of activity in either assay, phylogenetic preservation of a TF-binding site motif was associated with enhancer activity. Additionally, we reported that the conservation of GATA1 occupancy is linked to iv pleiotropic functions, meaning they are enhancers in multiple tissues, including non- hematopoietic tissues. Furthermore, the TAL1-bound enhancers validated in enhancer assays were assigned to their target genes and they include not only erythroid but also non-erythroid genes. v TABLE OF CONTENTS LIST OF FIGURES ........................................................................................................ vii LIST OF TABLES ........................................................................................................... ix ACKNOWLEDGEMENTS................................................................................................ x Chapter-1 Introduction to Regulation of Transcription by Cis-Regulatory Modules (CRMs)............................................................................................................................ 1 1.1 Regulation of transcription .................................................................................. 1 1.2 Cis-regulatory modules (CRMs) .......................................................................... 2 1.2.1 Enhancers ................................................................................................... 4 1.2.2 Identify enhancers ....................................................................................... 5 1.2.3 How do enhancers influence human disease? ............................................. 7 1.2.4 How important are enhancers for evolution ................................................ 10 1.3 Regulation of erythropoiesis via transcriptional enhancers and histone modifications ............................................................................................................. 12 1.3.1 Transcription factors ..................................................................................... 13 1.3.2 Histone modifications ................................................................................. 14 1.4 Statement of Thesis .......................................................................................... 17 Chapter-2 Epigenetic and Genetic Features that Lead To Discovery of Enhancer Function ........................................................................................................................ 19 2.1 Introduction.......................................................................................................... 20 2.2 Results ................................................................................................................ 22 2.2.1 Genome-wide prediction of regulated erythroid CRMs by TAL1 occupancy .. 22 2.2.2 Occupancy by TAL1 is a strong predictor of enhancer activity ...................... 26 2.2.3 Impact of additional epigenetic features in predicting enhancer activity in the presence of TAL1 binding ...................................................................................... 33 2.2.4 Confirmation of predictive power of EP300 occupancy, H3K4monomethylation and H3K27 acetylation in enhancer prediction ....................................................... 41 2.2.5 Effective combinations of epigenetic features for prediction of enhancers .... 42 2.2.6 Motifs that distinguish TAL1-bound enhancers from inactive TAL1 OSs ....... 49 2.2.7 Conservation as an illuminator, not a predictor ............................................. 52 2.3 Discussion ........................................................................................................... 56 2.4 Methods .............................................................................................................. 59 2.4.1 ChIP-seq data for epigenetic features ........................................................... 59 2.4.2 Enhancer assays by transient transfection for K562 cells ............................. 59 vi 2.4.3 Transgenic mouse assays (VISTA Enhancer Browser) ................................. 60 2.4.4 Clustering algorithms .................................................................................... 61 2.4.5 Measuring discriminatory power of transcription factors and histone modifications to identify enhancers ........................................................................ 62 2.4.6 Identification of significantly enriched motifs by employing the computer program, Discriminating Matrix Enumerator ........................................................... 62 2.4.7 Analyses of sequence conservation and motif preservation .......................... 63 2.5 Data access ......................................................................................................... 64 Chapter-3 Developing transfection methods for cell lines that allow interrogation of different aspects of erythroid differentiation ................................................................... 65 3.1 Transient transfections to test CRMs ................................................................... 66 3.2 Transcription factors present in different cell line models of erythroid cells .......... 68 3.3 Erythroid enhancement with HBG1 promoter in K562 cells .................................. 69 3.4 GATA1 responsive expression from HBG1 promoter in G1E-ER4 system ........... 71 3.5 GATA1-dependent enhancement with Vav2 promoter in G1E-ER4 system ......... 73 3.6 Potential repressor effect of GATA1-ER on expression in G1E-ER4 system ....... 74 3.7 Discussion ........................................................................................................... 74 3.8 Methods .............................................................................................................. 74 3.8.1 Enhancer assays by transient transfection for G1E-ER4 cell system ............ 75 Chapter-4 Conserved Transcription Factor Occupancy and Enhancer Usage in Different Cell Systems and Multiple Tissues ................................................................................ 77 4.1 Introduction.......................................................................................................... 78 4.2 Contribution of conserved GATA1 occupancy to enhancer activity in different cell systems ..................................................................................................................... 80 4.3 Conserved occupancy is associated with enhancer activity in multiple tissues .... 84 4.4 Discussion ........................................................................................................... 88 4.5 Methods .............................................................................................................
Recommended publications
  • View of HER2: Human Epidermal Growth Factor Receptor 2; TNBC: Triple-Negative Breast Resistance to Systemic Therapy in Patients with Breast Cancer

    View of HER2: Human Epidermal Growth Factor Receptor 2; TNBC: Triple-Negative Breast Resistance to Systemic Therapy in Patients with Breast Cancer

    Wen et al. Cancer Cell Int (2018) 18:128 https://doi.org/10.1186/s12935-018-0625-9 Cancer Cell International PRIMARY RESEARCH Open Access Sulbactam‑enhanced cytotoxicity of doxorubicin in breast cancer cells Shao‑hsuan Wen1†, Shey‑chiang Su2†, Bo‑huang Liou3, Cheng‑hao Lin1 and Kuan‑rong Lee1* Abstract Background: Multidrug resistance (MDR) is a major obstacle in breast cancer treatment. The predominant mecha‑ nism underlying MDR is an increase in the activity of adenosine triphosphate (ATP)-dependent drug efux trans‑ porters. Sulbactam, a β-lactamase inhibitor, is generally combined with β-lactam antibiotics for treating bacterial infections. However, sulbactam alone can be used to treat Acinetobacter baumannii infections because it inhibits the expression of ATP-binding cassette (ABC) transporter proteins. This is the frst study to report the efects of sulbactam on mammalian cells. Methods: We used the breast cancer cell lines as a model system to determine whether sulbactam afects cancer cells. The cell viabilities in the present of doxorubicin with or without sulbactam were measured by MTT assay. Protein identities and the changes in protein expression levels in the cells after sulbactam and doxorubicin treatment were determined using LC–MS/MS. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) was used to analyze the change in mRNA expression levels of ABC transporters after treatment of doxorubicin with or without sulbactam. The efux of doxorubicin was measures by the doxorubicin efux assay. Results: MTT assay revealed that sulbactam enhanced the cytotoxicity of doxorubicin in breast cancer cells. The results of proteomics showed that ABC transporter proteins and proteins associated with the process of transcription and initiation of translation were reduced.
  • Dynamics of Gene Silencing During X Inactivation Using Allele-Specific RNA-Seq Hendrik Marks1*, Hindrik H

    Dynamics of Gene Silencing During X Inactivation Using Allele-Specific RNA-Seq Hendrik Marks1*, Hindrik H

    Marks et al. Genome Biology (2015) 16:149 DOI 10.1186/s13059-015-0698-x RESEARCH Open Access Dynamics of gene silencing during X inactivation using allele-specific RNA-seq Hendrik Marks1*, Hindrik H. D. Kerstens1, Tahsin Stefan Barakat3, Erik Splinter4, René A. M. Dirks1, Guido van Mierlo1, Onkar Joshi1, Shuang-Yin Wang1, Tomas Babak5, Cornelis A. Albers2, Tüzer Kalkan6, Austin Smith6, Alice Jouneau7, Wouter de Laat4, Joost Gribnau3 and Hendrik G. Stunnenberg1* Abstract Background: During early embryonic development, one of the two X chromosomes in mammalian female cells is inactivated to compensate for a potential imbalance in transcript levels with male cells, which contain a single X chromosome. Here, we use mouse female embryonic stem cells (ESCs) with non-random X chromosome inactivation (XCI) and polymorphic X chromosomes to study the dynamics of gene silencing over the inactive X chromosome by high-resolution allele-specific RNA-seq. Results: Induction of XCI by differentiation of female ESCs shows that genes proximal to the X-inactivation center are silenced earlier than distal genes, while lowly expressed genes show faster XCI dynamics than highly expressed genes. The active X chromosome shows a minor but significant increase in gene activity during differentiation, resulting in complete dosage compensation in differentiated cell types. Genes escaping XCI show little or no silencing during early propagation of XCI. Allele-specific RNA-seq of neural progenitor cells generated from the female ESCs identifies three regions distal to the X-inactivation center that escape XCI. These regions, which stably escape during propagation and maintenance of XCI, coincide with topologically associating domains (TADs) as present in the female ESCs.
  • Crystal Structure of Human Gadd45 Reveals an Active Dimer

    Crystal Structure of Human Gadd45 Reveals an Active Dimer

    Protein Cell 2011, 2(10): 814–826 Protein & Cell DOI 10.1007/s13238-011-1090-6 RESEARCH ARTICLE Crystal structure of human Gadd45 reveals an active dimer 1,2,3* 1* 4 4 1 1 3 Wenzheng Zhang , Sheng Fu , Xuefeng Liu , Xuelian Zhao✉ , Wenchi Zhang✉ , Wei Peng , Congying Wu , Yuanyuan Li3, Xuemei Li1, Mark Bartlam1,2, Zong-Hao Zeng1 , Qimin Zhan4 , Zihe Rao1,2,3 1 National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 2 Tianjin Key Laboratory of Protein Science, College of Life Sciences, Nankai University, Tianjin 300071, China 3 Laboratory of Structural Biology, Tsinghua University, Beijing 100084, China 4 State Key Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China ✉ Correspondence: [email protected] (Z.-H. Zeng), [email protected] (Q. Zhan) Received August 23, 2011 Accepted September 19, 2011 ABSTRACT INTRODUCTION The human Gadd45 protein family plays critical roles in The human Gadd45 family of growth arrest and DNA DNA repair, negative growth control, genomic stability, damage-inducible proteins has three members, Gadd45α, cell cycle checkpoints and apoptosis. Here we report the -β, and -γ, grouped together on the basis of their amino acid crystal structure of human Gadd45, revealing a unique sequences and functional similarities (Vairapandi et al., 1996; dimer formed via a bundle of four parallel helices, Takekawa and Saito, 1998; Nakayama et al., 1999). The involving the most conserved residues among the genes encoding the α, β and γ isoforms of Gadd45 are Gadd45 isoforms.
  • Table 2. Significant

    Table 2. Significant

    Table 2. Significant (Q < 0.05 and |d | > 0.5) transcripts from the meta-analysis Gene Chr Mb Gene Name Affy ProbeSet cDNA_IDs d HAP/LAP d HAP/LAP d d IS Average d Ztest P values Q-value Symbol ID (study #5) 1 2 STS B2m 2 122 beta-2 microglobulin 1452428_a_at AI848245 1.75334941 4 3.2 4 3.2316485 1.07398E-09 5.69E-08 Man2b1 8 84.4 mannosidase 2, alpha B1 1416340_a_at H4049B01 3.75722111 3.87309653 2.1 1.6 2.84852656 5.32443E-07 1.58E-05 1110032A03Rik 9 50.9 RIKEN cDNA 1110032A03 gene 1417211_a_at H4035E05 4 1.66015788 4 1.7 2.82772795 2.94266E-05 0.000527 NA 9 48.5 --- 1456111_at 3.43701477 1.85785922 4 2 2.8237185 9.97969E-08 3.48E-06 Scn4b 9 45.3 Sodium channel, type IV, beta 1434008_at AI844796 3.79536664 1.63774235 3.3 2.3 2.75319499 1.48057E-08 6.21E-07 polypeptide Gadd45gip1 8 84.1 RIKEN cDNA 2310040G17 gene 1417619_at 4 3.38875643 1.4 2 2.69163229 8.84279E-06 0.0001904 BC056474 15 12.1 Mus musculus cDNA clone 1424117_at H3030A06 3.95752801 2.42838452 1.9 2.2 2.62132809 1.3344E-08 5.66E-07 MGC:67360 IMAGE:6823629, complete cds NA 4 153 guanine nucleotide binding protein, 1454696_at -3.46081884 -4 -1.3 -1.6 -2.6026947 8.58458E-05 0.0012617 beta 1 Gnb1 4 153 guanine nucleotide binding protein, 1417432_a_at H3094D02 -3.13334396 -4 -1.6 -1.7 -2.5946297 1.04542E-05 0.0002202 beta 1 Gadd45gip1 8 84.1 RAD23a homolog (S.
  • Itcs) in the Mouse Amygdala of Tshz1 Mutants Correlates with Fear, Depression, and Social Interaction Phenotypes

    Itcs) in the Mouse Amygdala of Tshz1 Mutants Correlates with Fear, Depression, and Social Interaction Phenotypes

    1160 • The Journal of Neuroscience, January 31, 2018 • 38(5):1160–1177 Development/Plasticity/Repair Loss of Intercalated Cells (ITCs) in the Mouse Amygdala of Tshz1 Mutants Correlates with Fear, Depression, and Social Interaction Phenotypes X Jeffrey Kuerbitz,1 Melinda Arnett,5 Sarah Ehrman,1 XMichael T. Williams,3 XCharles V. Vorhees,3 X Simon E. Fisher,6,7 Alistair N. Garratt,8 XLouis J. Muglia,5 Ronald R. Waclaw,1,4 and XKenneth Campbell1,2 Divisions of 1Developmental Biology, 2Neurosurgery, 3Neurology, 4Experimental Hematology and Cancer Biology, 5Center for Prevention of Preterm Birth, Perinatal Institute, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229, 6Language and Genetics Department, Max Planck Institute for Psycholinguistics, 6500 AH Nijmegen, The Netherlands, 7Donders Institute for Brain, Cognition and Behaviour, Radboud University, Nijmegen, The Netherlands, and 8Institute of Cell Biology and Neurobiology, Center for Anatomy, Charite´ University Hospital Berlin, 10117 Berlin, Germany The intercalated cells (ITCs) of the amygdala have been shown to be critical regulatory components of amygdalar circuits, which control appropriate fear responses. Despite this, the molecular processes guiding ITC development remain poorly understood. Here we establish the zinc finger transcription factor Tshz1 as a marker of ITCs during their migration from the dorsal lateral ganglionic eminence through maturity. Using germline and conditional knock-out (cKO) mouse models, we show that Tshz1 is required for the proper migration and differentiation of ITCs. In the absence of Tshz1, migrating ITC precursors fail to settle in their stereotypical locations encapsulating the lateral amygdala and BLA. Furthermore, they display reductions in the ITC marker Foxp2 and ectopic persistence of the dorsal lateral ganglionic eminence marker Sp8.
  • DNA·RNA Triple Helix Formation Can Function As a Cis-Acting Regulatory

    DNA·RNA Triple Helix Formation Can Function As a Cis-Acting Regulatory

    DNA·RNA triple helix formation can function as a cis-acting regulatory mechanism at the human β-globin locus Zhuo Zhoua, Keith E. Gilesa,b,c, and Gary Felsenfelda,1 aLaboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892; bUniversity of Alabama at Birmingham Stem Cell Institute, University of Alabama at Birmingham, Birmingham, AL 35294; and cDepartment of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294 Contributed by Gary Felsenfeld, February 4, 2019 (sent for review January 4, 2019; reviewed by James Douglas Engel and Sergei M. Mirkin) We have identified regulatory mechanisms in which an RNA tran- of the criteria necessary to establish the presence of a triplex script forms a DNA duplex·RNA triple helix with a gene or one of its structure, we first describe and characterize triplex formation at regulatory elements, suggesting potential auto-regulatory mecha- the FAU gene in human erythroid K562 cells. FAU encodes a nisms in vivo. We describe an interaction at the human β-globin protein that is a fusion containing fubi, a ubiquitin-like protein, locus, in which an RNA segment embedded in the second intron of and ribosomal protein S30. Although fubi function is unknown, the β-globin gene forms a DNA·RNA triplex with the HS2 sequence posttranslational processing produces S30, a component of the within the β-globin locus control region, a major regulator of glo- 40S ribosome. We used this system to refine methods necessary bin expression. We show in human K562 cells that the triplex is to detect triplex formation and to distinguish it from R-loop stable in vivo.
  • Mediator of DNA Damage Checkpoint 1 (MDC1) Is a Novel Estrogen Receptor Co-Regulator in Invasive 6 Lobular Carcinoma of the Breast 7 8 Evelyn K

    Mediator of DNA Damage Checkpoint 1 (MDC1) Is a Novel Estrogen Receptor Co-Regulator in Invasive 6 Lobular Carcinoma of the Breast 7 8 Evelyn K

    bioRxiv preprint doi: https://doi.org/10.1101/2020.12.16.423142; this version posted December 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 Running Title: MDC1 co-regulates ER in ILC 2 3 Research article 4 5 Mediator of DNA damage checkpoint 1 (MDC1) is a novel estrogen receptor co-regulator in invasive 6 lobular carcinoma of the breast 7 8 Evelyn K. Bordeaux1+, Joseph L. Sottnik1+, Sanjana Mehrotra1, Sarah E. Ferrara2, Andrew E. Goodspeed2,3, James 9 C. Costello2,3, Matthew J. Sikora1 10 11 +EKB and JLS contributed equally to this project. 12 13 Affiliations 14 1Dept. of Pathology, University of Colorado Anschutz Medical Campus 15 2Biostatistics and Bioinformatics Shared Resource, University of Colorado Comprehensive Cancer Center 16 3Dept. of Pharmacology, University of Colorado Anschutz Medical Campus 17 18 Corresponding author 19 Matthew J. Sikora, PhD.; Mail Stop 8104, Research Complex 1 South, Room 5117, 12801 E. 17th Ave.; Aurora, 20 CO 80045. Tel: (303)724-4301; Fax: (303)724-3712; email: [email protected]. Twitter: 21 @mjsikora 22 23 Authors' contributions 24 MJS conceived of the project. MJS, EKB, and JLS designed and performed experiments. JLS developed models 25 for the project. EKB, JLS, SM, and AEG contributed to data analysis and interpretation. SEF, AEG, and JCC 26 developed and performed informatics analyses. MJS wrote the draft manuscript; all authors read and revised the 27 manuscript and have read and approved of this version of the manuscript.
  • Ldb1 Is Essential for Development of Nkx2.1 Lineage Derived Gabaergic and Cholinergic Neurons in the Telencephalon

    Ldb1 Is Essential for Development of Nkx2.1 Lineage Derived Gabaergic and Cholinergic Neurons in the Telencephalon

    Developmental Biology 385 (2014) 94–106 Contents lists available at ScienceDirect Developmental Biology journal homepage: www.elsevier.com/locate/developmentalbiology Ldb1 is essential for development of Nkx2.1 lineage derived GABAergic and cholinergic neurons in the telencephalon Yangu Zhao a,n,1, Pierre Flandin b,2, Daniel Vogt b, Alexander Blood a, Edit Hermesz a,3, Heiner Westphal a, John L. R. Rubenstein b,nn a Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, USA b Department of Psychiatry and the Nina Ireland Laboratory of Developmental Neurobiology, University of California San Francisco, San Francisco, CA 94143, USA article info abstract Article history: The progenitor zones of the embryonic mouse ventral telencephalon give rise to GABAergic and cholinergic Received 1 June 2013 neurons. We have shown previously that two LIM-homeodomain (LIM-HD) transcription factors, Lhx6 and Received in revised form Lhx8, that are downstream of Nkx2.1, are critical for the development of telencephalic GABAergic and 8 October 2013 cholinergic neurons. Here we investigate the role of Ldb1, a nuclear protein that binds directly to all LIM-HD Accepted 9 October 2013 factors, in the development of these ventral telencephalon derived neurons. We show that Ldb1 is expressed Available online 21 October 2013 in the Nkx2.1 cell lineage during embryonic development and in mature neurons. Conditional deletion of Ldb1 Keywords: causes defects in the expression of a series of genes in the ventral telencephalon and severe impairment in the Differentiation tangential migration of cortical interneurons from the ventral telencephalon.
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus

    A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus

    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
  • Identifying Glaucoma Susceptibility Genes in Extended Families

    Identifying Glaucoma Susceptibility Genes in Extended Families

    Identifying Glaucoma Susceptibility Genes in Extended Families by Patricia Stacey Graham BSc(Hons), GradDipEd Menzies Institute for Medical Research | College of Health and Medicine Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy (Medical Studies) University of Tasmania, March 2020 Declaration of Originality This thesis contains no material which has been accepted for a degree or diploma by the University or any other institution, except by way of background information and duly acknowledged in the thesis, and to the best of my knowledge and belief no material previously published or written by another person except where due acknowledgement is made in the text of the thesis, nor does the thesis contain any material that infringes copyright. 13/3/2020 i Authority of access This thesis may be made available for loan and limited copying and communication in accordance with the Copyright Act 1968. 13/3/2020 ii Statement of ethical conduct The research associated with this thesis abides by the international and Australian codes on human experimentation and the guidelines and the rulings of the Ethics Committee of the University. Ethics Approval Numbers: University of Tasmania Human Research Ethics Committee H0014085 Oregon Health and Science University Institutional Review Board #1306 13/3/2020 iii Dedication To my parents Susie and Micky Lowry Who instilled in me the love of learning and who would have been so proud iv Acknowledgements What a rollercoaster the last four years have been …. it’s been an amazing ride. Rollercoasters are no fun alone, and I would sincerely like to thank the following people who kept the ride rolling and to those who sat with me in the carriage and didn’t let me fall out.
  • 4-6 Weeks Old Female C57BL/6 Mice Obtained from Jackson Labs Were Used for Cell Isolation

    4-6 Weeks Old Female C57BL/6 Mice Obtained from Jackson Labs Were Used for Cell Isolation

    Methods Mice: 4-6 weeks old female C57BL/6 mice obtained from Jackson labs were used for cell isolation. Female Foxp3-IRES-GFP reporter mice (1), backcrossed to B6/C57 background for 10 generations, were used for the isolation of naïve CD4 and naïve CD8 cells for the RNAseq experiments. The mice were housed in pathogen-free animal facility in the La Jolla Institute for Allergy and Immunology and were used according to protocols approved by the Institutional Animal Care and use Committee. Preparation of cells: Subsets of thymocytes were isolated by cell sorting as previously described (2), after cell surface staining using CD4 (GK1.5), CD8 (53-6.7), CD3ε (145- 2C11), CD24 (M1/69) (all from Biolegend). DP cells: CD4+CD8 int/hi; CD4 SP cells: CD4CD3 hi, CD24 int/lo; CD8 SP cells: CD8 int/hi CD4 CD3 hi, CD24 int/lo (Fig S2). Peripheral subsets were isolated after pooling spleen and lymph nodes. T cells were enriched by negative isolation using Dynabeads (Dynabeads untouched mouse T cells, 11413D, Invitrogen). After surface staining for CD4 (GK1.5), CD8 (53-6.7), CD62L (MEL-14), CD25 (PC61) and CD44 (IM7), naïve CD4+CD62L hiCD25-CD44lo and naïve CD8+CD62L hiCD25-CD44lo were obtained by sorting (BD FACS Aria). Additionally, for the RNAseq experiments, CD4 and CD8 naïve cells were isolated by sorting T cells from the Foxp3- IRES-GFP mice: CD4+CD62LhiCD25–CD44lo GFP(FOXP3)– and CD8+CD62LhiCD25– CD44lo GFP(FOXP3)– (antibodies were from Biolegend). In some cases, naïve CD4 cells were cultured in vitro under Th1 or Th2 polarizing conditions (3, 4).
  • RNF11 at the Crossroads of Protein Ubiquitination

    RNF11 at the Crossroads of Protein Ubiquitination

    biomolecules Review RNF11 at the Crossroads of Protein Ubiquitination Anna Mattioni, Luisa Castagnoli and Elena Santonico * Department of Biology, University of Rome Tor Vergata, Via della ricerca scientifica, 00133 Rome, Italy; [email protected] (A.M.); [email protected] (L.C.) * Correspondence: [email protected] Received: 29 September 2020; Accepted: 8 November 2020; Published: 11 November 2020 Abstract: RNF11 (Ring Finger Protein 11) is a 154 amino-acid long protein that contains a RING-H2 domain, whose sequence has remained substantially unchanged throughout vertebrate evolution. RNF11 has drawn attention as a modulator of protein degradation by HECT E3 ligases. Indeed, the large number of substrates that are regulated by HECT ligases, such as ITCH, SMURF1/2, WWP1/2, and NEDD4, and their role in turning off the signaling by ubiquitin-mediated degradation, candidates RNF11 as the master regulator of a plethora of signaling pathways. Starting from the analysis of the primary sequence motifs and from the list of RNF11 protein partners, we summarize the evidence implicating RNF11 as an important player in modulating ubiquitin-regulated processes that are involved in transforming growth factor beta (TGF-β), nuclear factor-κB (NF-κB), and Epidermal Growth Factor (EGF) signaling pathways. This connection appears to be particularly significant, since RNF11 is overexpressed in several tumors, even though its role as tumor growth inhibitor or promoter is still controversial. The review highlights the different facets and peculiarities of this unconventional small RING-E3 ligase and its implication in tumorigenesis, invasion, neuroinflammation, and cancer metastasis. Keywords: Ring Finger Protein 11; HECT ligases; ubiquitination 1.