DOCSLIB.ORG

The Regulation of Lunatic Fringe During Somitogenesis

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

THE REGULATION OF LUNATIC FRINGE DURING SOMITOGENESIS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Emily T. Shifley ***** The Ohio State University 2009 Dissertation Committee: Approved by Professor Susan Cole, Advisor Professor Christine Beattie _________________________________ Professor Mark Seeger Advisor Graduate Program in Molecular Genetics Professor Michael Weinstein ABSTRACT Somitogenesis is the morphological hallmark of vertebrate segmentation. Somites bud from the presomitic mesoderm (PSM) in a sequential, periodic fashion and give rise to the rib cage, vertebrae, and dermis and muscles of the back. The regulation of somitogenesis is complex. In the posterior region of the PSM, a segmentation clock operates to organize cohorts of cells into presomites, while in the anterior region of the PSM the presomites are patterned into rostral and caudal compartments (R/C patterning). Both of these stages of somitogenesis are controlled, at least in part, by the Notch pathway and Lunatic fringe (Lfng), a glycosyltransferase that modifies the Notch receptor. To dissect the roles played by Lfng during somitogenesis, we created a novel allele that lacks cyclic Lfng expression within the segmentation clock, but that maintains expression during R/C somite patterning (Lfng∆FCE1). Lfng∆FCE1/∆FCE1 mice have severe defects in their anterior vertebrae and rib cages, but relatively normal sacral and tail vertebrae, unlike Lfng knockouts. Segmentation clock function is differentially affected by the ∆FCE1 deletion; during anterior somitogenesis the expression patterns of many clock genes are disrupted, while during posterior somitogenesis, certain clock components have recovered. R/C patterning occurs relatively normally in Lfng∆FCE1/∆FCE1 embryos, likely contributing to the partial phenotype rescue, and confirming that Lfng ii plays separate roles in the two regions of the PSM. These results reveal that the oscillatory regulation of the Notch pathway plays an important role in the segmentation clock during the development of the anterior skeleton. As part of the segmentation clock, Lfng mRNA is periodically transcribed and LFNG protein levels have also been observed to cycle. Lfng mRNA and LFNG protein molecules must therefore have rapid turnover rates in the PSM. We hypothesize that cyclic Lfng transcription is coupled with signals in the Lfng 3’UTR that confer a short half-life on Lfng mRNA. To test this, we examined the expression patterns of transgenes containing conserved sections of the Lfng 3’UTR and determined which sections cooperatively confer a short RNA half-life in the PSM. LFNG protein acts in the Golgi, but is also cleaved and released into the extracellular space. We hypothesized that this cleavage/secretion contributes to short LFNG intracellular half-life, facilitating its rapid oscillations. To test this, we localized N-terminal protein sequences that control the secretory behavior of the fringe proteins and found that LFNG processing is promoted by furin-like protein convertases. Mutations that alter LFNG processing increase its intracellular half-life in vitro without affecting the specificity of its function in the Notch pathway. To determine the importance of LFNG cleavage/secretion in vivo, we created a novel allele that tethers LFNG to the Golgi (LfngRL). LfngRL/+ mice show significant segmentation and patterning defects suggesting that the short intracellular half-life of LFNG is important for somitogenesis. Thus, the cyclic activity of Lfng in the segmentation clock is achieved through multiple mechanisms, including tight regulation of mRNA and protein levels. Overall, we find that Lfng plays an important role in spatially and temporally regulating Notch signaling during vertebrate segmentation. iii DEDICATION This work is dedicated to my parents, to my siblings, and to my husband for all of their love and support. iv ACKNOWLEDGMENTS I would like to acknowledge my advisor, Dr. Susan Cole, for all of her guidance and encouragement during my graduate studies. I am grateful for the opportunity to have been a part of her research and truly appreciate all of the mentoring and training she has given me. I could not have asked for a more supportive advisor. I would also like to thank my committee members, Dr. Beattie, Dr. Chamberlin, Dr. Seeger, and Dr. Weinstein for their time and guidance during my graduate studies. I am especially grateful to Dr. Weinstein for helping with knock-in mice. I would like to acknowledge current and previous members of the lab for their contributions to this work and their support. Kellie contributed some of the data included in this thesis, as indicated, and was a wonderful co-worker and friend. To the other Cole lab graduate students, Ariadna, Maurisa, and Dustin, thank you for making the lab a supportive and fun place to work, I wish you all the best. To Dawn and Jorge, thank you for your contributions to the GFP transgenic mice and all of the work you do in the lab. To all of the undergraduates who have come through the Cole lab, thank you for your enthusiasm. v I would like to thank my classmates for their camaraderie as we have worked through graduate school together. I have truly enjoyed our friendship and time spent together. I also wish to thank the editors of Birth Defects Research (Part C), Development, and Biochimica et Biophysica Acta: Molecular Cell Research for the use of the previously published text and figures in this dissertation. vi VITA September 29, 1981………………..….Born –Cincinnati, OH. 2003……………………………………Bachelor of Science. Ohio University. 2003 – 2004………………………...….University Fellow The Ohio State University. 2004 – 2009…………………………....Research and Teaching Assistant The Ohio State University. PUBLICATIONS 1. E.T. Shifley and S.E. Cole. (2008) Lunatic fringe protein processing by proprotein convertases may contribute to the short protein half-life in the segmentation clock. Biochim Biophys Acta, Mol Cell Res., 1783.2384-2390. 2. E.T. Shifley, K.M. VanHorn, A. Pérez-Balaguer, J.D. Franklin, M. Weinstein, and S.E. Cole. (2008). Oscillatory Lunatic Fringe activity is crucial for segmentation of anterior but not posterior skeleton. Development, 135.899-908. 3. E.T. Shifley and S.E. Cole. (2007). The vertebrate segmentation clock and its role in skeletal birth defects. Birth Defects Part C, 81.121-33. FIELDS OF STUDY Major Field: Molecular Genetics vii TABLE OF CONTENTS Page Abstract……………………………………………………………………………... ii Dedication………………………………………………………………………...... iv Acknowledgements………………………………………………………………… v Vita…………………………………………………………………………………. vii List of Tables……………………………………………………………………….. xii List of Figures……………………………………………………………………… xiii List of Abbreviations……………………………………………………………….. xvi Chapters: 1. Introduction…………………………………………………………………... 1 1.1 Introduction…………………………………………………………….… 1 1.2 Somitogenesis………………………………………………………….… 2 1.3 The Segmentation Clock…………………………………………………. 5 1.3.1 Cycling genes……………………………………………………… 6 1.3.2 Segmentation Clock Variations…………………………………… 9 1.3.3 Recent Clock Models……………………………………………… 10 1.4 The Wavefront…………………………………………………………… 12 1.5 The Notch Signaling Pathway…………………………………………… 13 1.5.1 Notch signaling plays multiple roles during somitogenesis………. 15 1.5.2 Notch pathway knockouts…………………………………………. 16 1.6 Human Disease…………………………………………………………... 18 1.6.1 Spondylocostal Dysostosis………………………………………... 18 1.7 The regulation of Lfng during somitogenesis……………………………. 23 viii 2. Lunatic Fringe is important for segmentation clock function during primary not secondary body formation………...……………………………………... 32 2.1 Introduction………………………………………………………………. 32 2.2 Materials and Methods………………………………………………….... 34 2.2.1 Targeted deletion of FCE1………………………………………… 34 2.2.2 Genotyping……………………………………………………….... 34 2.2.3 Whole mount in situ hybridization……………………………….... 35 2.2.4 Skeletal preparations………………………………………………. 36 2.2.5 Whole mount immunohistochemistry……………………………... 36 2.3 Results……………………………………………………………………. 36 2.3.1 Deletion of FCE1 from the Lfng locus perturbs clock-linked Lfng RNA expression……………………………………………………….. 36 2.3.2 The loss of Lfng expression in the segmentation clock perturbs normal skeletal development…………………………………………….. 37 2.3.3 Rostral-caudal somite patterning is partially rescued in Lfng∆FCE1/∆FCE1 embryos………………………………………………….. 39 2.3.4 Somites differentiate properly in Lfng∆FCE1/∆FCE1 embryos………... 41 2.3.5 The loss of cyclic Lfng expression in the posterior PSM perturbs oscillatory NOTCH1 activity……………………………………………. 41 2.3.6 Expression of oscillatory genes is differentially affected during primary and secondary body formation in Lfng∆FCE1/∆FCE1 embryos…….. 42 2.3.7 Wnt targets continue to cycle, and Brachyury expression is not disrupted in Lfng∆FCE1/∆FCE1 embryos……………………………………. 44 2.4 Discussion………………………………………………………………... 44 2.4.1 The FCE1 enhancer is necessary for cyclic expression of Lfng in Region I of the PSM………...…………………………………………... 44 2.4.2 Oscillatory Lfng expression and Notch signaling are critical for the proper segmentation during primary, but not secondary, body formation………………………………………………………………… 45 2.4.3 Lfng plays separable roles in the segmentation clock and R/C patterning………………………………………………………………... 47 2.4.4 Differential segmentation clock regulation at distinct levels of the axial skeleton?…………………………………………………………... 48 2.4.5 R/C patterning of anterior somites may affect ongoing segmentation during secondary body formation…………………………
Recommended publications
  • Anti-Lunatic Fringe Antibody (ARG40136)

    Anti-Lunatic Fringe Antibody (ARG40136)

    Product datasheet [email protected] ARG40136 Package: 100 μl anti-Lunatic Fringe antibody Store at: -20°C Summary Product Description Rabbit Polyclonal antibody recognizes Lunatic Fringe Tested Reactivity Hu Tested Application FACS, IHC-P, WB Host Rabbit Clonality Polyclonal Isotype IgG Target Name Lunatic Fringe Antigen Species Human Immunogen KLH-conjugated synthetic peptide corresponding to aa. 86-114 of Human Lunatic Fringe. Conjugation Un-conjugated Alternate Names O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase; EC 2.4.1.222; SCDO3; Beta-1,3-N- acetylglucosaminyltransferase lunatic fringe Application Instructions Application table Application Dilution FACS 1:10 - 1:50 IHC-P 1:50 - 1:100 WB 1:1000 Application Note * The dilutions indicate recommended starting dilutions and the optimal dilutions or concentrations should be determined by the scientist. Positive Control K562 Calculated Mw 42 kDa Properties Form Liquid Purification Purification with Protein A and immunogen peptide. Buffer PBS and 0.09% (W/V) Sodium azide. Preservative 0.09% (W/V) Sodium azide. Storage instruction For continuous use, store undiluted antibody at 2-8°C for up to a week. For long-term storage, aliquot and store at -20°C or below. Storage in frost free freezers is not recommended. Avoid repeated freeze/thaw cycles. Suggest spin the vial prior to opening. The antibody solution should be gently mixed before use. www.arigobio.com 1/3 Note For laboratory research only, not for drug, diagnostic or other use. Bioinformation Gene Symbol LFNG Gene Full Name LFNG O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase Background This gene is a member of the fringe gene family which also includes radical and manic fringe genes.
  • Regulation of Cardiac Progenitors by Combination of Mesp1 and ETS

    Regulation of Cardiac Progenitors by Combination of Mesp1 and ETS

    REGULATION OF CARDIAC PROGENITORS BY COMBINATION OF MESP1 AND ETS TRANSCRIPTION FACTORS A Dissertation by KUO-CHAN WENG Submitted to the Office of Graduate and Professional Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Chair of Committee, Fen Wang Co-Chair of Committee, Robert J. Schwartz Committee Members, James F. Martin Jiang Chang Head of Department, Fen Wang May 2014 Major Subject: Medical Science Copyright 2014 Kuo-Chan Weng ABSTRACT Heart disease remains the leading cause of death worldwide. By understanding the regulating networks during cardiac development we can exploit those networks to manipulate adult cells into cardiac progenitors and provide an alternative for repairing diseased hearts. Mesp1 is considered to have critical roles during cardiac development but the molecular mechanisms need to be further studied. The roles of ETS transcription factors have been primarily limited to hematopoietic differentiation and cancer progression. The ETS transcription factors are known to have proliferating roles and were hypothesized to also be involved in cardiac differentiation and may potentially be used for cell reprogramming. The first part of this study characterizes the expression pattern of Mesp1 protein in early mouse embryo from E6.5 to E9.5 and provides a full expression profile in differentiating embryoid bodies in vitro from the undifferentiated stage to Day10. Our work showed Mesp1 expresses in the posterior region of E6.5 embryo then starts migrating through the primitive streak toward anterior mesoderm and endoderm in E7.5. A Mesp1 linage tracing ES cell line was established, and it allowed us to trace the Mesp1 derived cell population.
  • Segmentation in Vertebrates: Clock and Gradient Finally Joined

    Segmentation in Vertebrates: Clock and Gradient Finally Joined

    Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Segmentation in vertebrates: clock and gradient finally joined Alexander Aulehla1 and Bernhard G. Herrmann2 Max-Planck-Institute for Molecular Genetics, Department of Developmental Genetics, 14195 Berlin, Germany The vertebral column is derived from somites formed by terior (A–P) axis. Somite formation takes place periodi- segmentation of presomitic mesoderm, a fundamental cally in a fixed anterior-to-posterior sequence. process of vertebrate embryogenesis. Models on the In the chick embryo, a new somite is formed approxi- mechanism controlling this process date back some mately every 90 min, whereas in the mouse embryo, the three to four decades. Access to understanding the mo- periodicity varies, dependent on the axial position (Tam lecular control of somitogenesis has been gained only 1981). Classical embryology experiments revealed that recently by the discovery of molecular oscillators (seg- periodicity and directionality of somite formation are mentation clock) and gradients of signaling molecules, controlled by an intrinsic program set off in the cells as as predicted by early models. The Notch signaling path- they are recruited into the psm. For instance, when the way is linked to the oscillator and plays a decisive role in psm is inverted rostro–caudally, somite formation in the inter- and intrasomitic boundary formation. An Fgf8 sig- inverted region proceeds from caudal to rostral, main- naling gradient is involved in somite size control. And taining the original direction (Christ et al. 1974). More- the (canonical) Wnt signaling pathway, driven by Wnt3a, over, neither the transversal bisection nor the isolation appears to integrate clock and gradient in a global of the psm from all surrounding tissues stops the seg- mechanism controlling the segmentation process.
  • Detailed Review Paper on Retinoid Pathway Signalling

    Detailed Review Paper on Retinoid Pathway Signalling

    1 1 Detailed Review Paper on Retinoid Pathway Signalling 2 December 2020 3 2 4 Foreword 5 1. Project 4.97 to develop a Detailed Review Paper (DRP) on the Retinoid System 6 was added to the Test Guidelines Programme work plan in 2015. The project was 7 originally proposed by Sweden and the European Commission later joined the project as 8 a co-lead. In 2019, the OECD Secretariat was added to coordinate input from expert 9 consultants. The initial objectives of the project were to: 10 draft a review of the biology of retinoid signalling pathway, 11 describe retinoid-mediated effects on various organ systems, 12 identify relevant retinoid in vitro and ex vivo assays that measure mechanistic 13 effects of chemicals for development, and 14 Identify in vivo endpoints that could be added to existing test guidelines to 15 identify chemical effects on retinoid pathway signalling. 16 2. This DRP is intended to expand the recommendations for the retinoid pathway 17 included in the OECD Detailed Review Paper on the State of the Science on Novel In 18 vitro and In vivo Screening and Testing Methods and Endpoints for Evaluating 19 Endocrine Disruptors (DRP No 178). The retinoid signalling pathway was one of seven 20 endocrine pathways considered to be susceptible to environmental endocrine disruption 21 and for which relevant endpoints could be measured in new or existing OECD Test 22 Guidelines for evaluating endocrine disruption. Due to the complexity of retinoid 23 signalling across multiple organ systems, this effort was foreseen as a multi-step process.
  • 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.
  • Watsonjn2018.Pdf (1.780Mb)

    Watsonjn2018.Pdf (1.780Mb)

    UNIVERSITY OF CENTRAL OKLAHOMA Edmond, Oklahoma Department of Biology Investigating Differential Gene Expression in vivo of Cardiac Birth Defects in an Avian Model of Maternal Phenylketonuria A THESIS SUBMITTED TO THE GRADUATE FACULTY In partial fulfillment of the requirements For the degree of MASTER OF SCIENCE IN BIOLOGY By Jamie N. Watson Edmond, OK June 5, 2018 J. Watson/Dr. Nikki Seagraves ii J. Watson/Dr. Nikki Seagraves Acknowledgements It is difficult to articulate the amount of gratitude I have for the support and encouragement I have received throughout my master’s thesis. Many people have added value and support to my life during this time. I am thankful for the education, experience, and friendships I have gained at the University of Central Oklahoma. First, I would like to thank Dr. Nikki Seagraves for her mentorship and friendship. I lucked out when I met her. I have enjoyed working on this project and I am very thankful for her support. I would like thank Thomas Crane for his support and patience throughout my master’s degree. I would like to thank Dr. Shannon Conley for her continued mentorship and support. I would like to thank Liz Bullen and Dr. Eric Howard for their training and help on this project. I would like to thank Kristy Meyer for her friendship and help throughout graduate school. I would like to thank my committee members Dr. Robert Brennan and Dr. Lilian Chooback for their advisement on this project. Also, I would like to thank the biology faculty and staff. I would like to thank the Seagraves lab members: Jailene Canales, Kayley Pate, Mckayla Muse, Grace Thetford, Kody Harvey, Jordan Guffey, and Kayle Patatanian for their hard work and support.
  • 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.
  • BMP Signaling Orchestrates a Transcriptional Network to Control the Fate of Mesenchymal Stem Cells (Mscs)

    BMP Signaling Orchestrates a Transcriptional Network to Control the Fate of Mesenchymal Stem Cells (Mscs)

    bioRxiv preprint doi: https://doi.org/10.1101/104927; this version posted February 1, 2017. 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 4.0 International license. BMP signaling orchestrates a transcriptional network to control the fate of mesenchymal stem cells (MSCs) Jifan Feng1, Junjun Jing1,2, Jingyuan Li1, Hu Zhao1, Vasu Punj3, Tingwei Zhang1,2, Jian Xu1 and Yang Chai1,* 1Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, CA 90033, USA 2State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China 3Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA *Corresponding author: Yang Chai 2250 Alcazar Street – CSA 103 Center for Craniofacial Molecular Biology University of Southern California Los Angeles, CA Phone number: 323-442-3480 [email protected] Running Title: BMP regulates cell fate of MSCs Key Words: BMP, mesenchymal stem cells (MSCs), odontogenesis, Gli1 Summary Statement: BMP signaling activity is required for the lineage commitment of MSCs and transcription factors downstream of BMP signaling may determine distinct cellular identities within the dental mesenchyme. 1 bioRxiv preprint doi: https://doi.org/10.1101/104927; this version posted February 1, 2017. 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 4.0 International license.
  • Genome-Wide DNA Methylation Analysis of KRAS Mutant Cell Lines Ben Yi Tew1,5, Joel K

    Genome-Wide DNA Methylation Analysis of KRAS Mutant Cell Lines Ben Yi Tew1,5, Joel K

    www.nature.com/scientificreports OPEN Genome-wide DNA methylation analysis of KRAS mutant cell lines Ben Yi Tew1,5, Joel K. Durand2,5, Kirsten L. Bryant2, Tikvah K. Hayes2, Sen Peng3, Nhan L. Tran4, Gerald C. Gooden1, David N. Buckley1, Channing J. Der2, Albert S. Baldwin2 ✉ & Bodour Salhia1 ✉ Oncogenic RAS mutations are associated with DNA methylation changes that alter gene expression to drive cancer. Recent studies suggest that DNA methylation changes may be stochastic in nature, while other groups propose distinct signaling pathways responsible for aberrant methylation. Better understanding of DNA methylation events associated with oncogenic KRAS expression could enhance therapeutic approaches. Here we analyzed the basal CpG methylation of 11 KRAS-mutant and dependent pancreatic cancer cell lines and observed strikingly similar methylation patterns. KRAS knockdown resulted in unique methylation changes with limited overlap between each cell line. In KRAS-mutant Pa16C pancreatic cancer cells, while KRAS knockdown resulted in over 8,000 diferentially methylated (DM) CpGs, treatment with the ERK1/2-selective inhibitor SCH772984 showed less than 40 DM CpGs, suggesting that ERK is not a broadly active driver of KRAS-associated DNA methylation. KRAS G12V overexpression in an isogenic lung model reveals >50,600 DM CpGs compared to non-transformed controls. In lung and pancreatic cells, gene ontology analyses of DM promoters show an enrichment for genes involved in diferentiation and development. Taken all together, KRAS-mediated DNA methylation are stochastic and independent of canonical downstream efector signaling. These epigenetically altered genes associated with KRAS expression could represent potential therapeutic targets in KRAS-driven cancer. Activating KRAS mutations can be found in nearly 25 percent of all cancers1.
  • Supplementary Figure S4

    Supplementary Figure S4

    18DCIS 18IDC Supplementary FigureS4 22DCIS 22IDC C D B A E (0.77) (0.78) 16DCIS 14DCIS 28DCIS 16IDC 28IDC (0.43) (0.49) 0 ADAMTS12 (p.E1469K) 14IDC ERBB2, LASP1,CDK12( CCNE1 ( NUTM2B SDHC,FCGR2B,PBX1,TPR( CD1D, B4GALT3, BCL9, FLG,NUP21OL,TPM3,TDRD10,RIT1,LMNA,PRCC,NTRK1 0 ADAMTS16 (p.E67K) (0.67) (0.89) (0.54) 0 ARHGEF38 (p.P179Hfs*29) 0 ATG9B (p.P823S) (0.68) (1.0) ARID5B, CCDC6 CCNE1, TSHZ3,CEP89 CREB3L2,TRIM24 BRAF, EGFR (7p11); 0 ABRACL (p.R35H) 0 CATSPER1 (p.P152H) 0 ADAMTS18 (p.Y799C) 19q12 0 CCDC88C (p.X1371_splice) (0) 0 ADRA1A (p.P327L) (10q22.3) 0 CCNF (p.D637N) −4 −2 −4 −2 0 AKAP4 (p.G454A) 0 CDYL (p.Y353Lfs*5) −4 −2 Log2 Ratio Log2 Ratio −4 −2 Log2 Ratio Log2 Ratio 0 2 4 0 2 4 0 ARID2 (p.R1068H) 0 COL27A1 (p.G646E) 0 2 4 0 2 4 2 EDRF1 (p.E521K) 0 ARPP21 (p.P791L) ) 0 DDX11 (p.E78K) 2 GPR101, p.A174V 0 ARPP21 (p.P791T) 0 DMGDH (p.W606C) 5 ANP32B, p.G237S 16IDC (Ploidy:2.01) 16DCIS (Ploidy:2.02) 14IDC (Ploidy:2.01) 14DCIS (Ploidy:2.9) -3 -2 -1 -3 -2 -1 -3 -2 -1 -3 -2 -1 -3 -2 -1 -3 -2 -1 Log Ratio Log Ratio Log Ratio Log Ratio 12DCIS 0 ASPM (p.S222T) Log Ratio Log Ratio 0 FMN2 (p.G941A) 20 1 2 3 2 0 1 2 3 2 ERBB3 (p.D297Y) 2 0 1 2 3 20 1 2 3 0 ATRX (p.L1276I) 20 1 2 3 2 0 1 2 3 0 GALNT18 (p.F92L) 2 MAPK4, p.H147Y 0 GALNTL6 (p.E236K) 5 C11orf1, p.Y53C (10q21.2); 0 ATRX (p.R1401W) PIK3CA, p.H1047R 28IDC (Ploidy:2.0) 28DCIS (Ploidy:2.0) 22IDC (Ploidy:3.7) 22DCIS (Ploidy:4.1) 18IDC (Ploidy:3.9) 18DCIS (Ploidy:2.3) 17q12 0 HCFC1 (p.S2025C) 2 LCMT1 (p.S34A) 0 ATXN7L2 (p.X453_splice) SPEN, p.P677Lfs*13 CBFB 1 2 3 4 5 6 7 8 9 10 11
  • Transcription Factors That Govern Development and Disease: an Achilles Heel in Cancer

    Transcription Factors That Govern Development and Disease: an Achilles Heel in Cancer

    G C A T T A C G G C A T genes Review Transcription Factors That Govern Development and Disease: An Achilles Heel in Cancer Dhananjay Huilgol , Prabhadevi Venkataramani , Saikat Nandi * and Sonali Bhattacharjee * Bungtown Road, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, NY 11724, USA; [email protected] (D.H.); [email protected] (P.V.) * Correspondence: [email protected] (S.N.); [email protected] (S.B.) Received: 15 September 2019; Accepted: 8 October 2019; Published: 12 October 2019 Abstract: Development requires the careful orchestration of several biological events in order to create any structure and, eventually, to build an entire organism. On the other hand, the fate transformation of terminally differentiated cells is a consequence of erroneous development, and ultimately leads to cancer. In this review, we elaborate how development and cancer share several biological processes, including molecular controls. Transcription factors (TF) are at the helm of both these processes, among many others, and are evolutionarily conserved, ranging from yeast to humans. Here, we discuss four families of TFs that play a pivotal role and have been studied extensively in both embryonic development and cancer—high mobility group box (HMG), GATA, paired box (PAX) and basic helix-loop-helix (bHLH) in the context of their role in development, cancer, and their conservation across several species. Finally, we review TFs as possible therapeutic targets for cancer and reflect on the importance of natural resistance against cancer in certain organisms, yielding knowledge regarding TF function and cancer biology. Keywords: transcription factors; embryonic development; evolution; cancer; clinical trials; high mobility group box (HMG); basic helix loop helix (bHLH); paired box (Pax); GATA 1.
  • Evidence That the WNT-Inducible Growth Arrest- Specific Gene 1 Encodes an Antagonist of Sonic Hedgehog Signaling in the Somite

    Evidence That the WNT-Inducible Growth Arrest- Specific Gene 1 Encodes an Antagonist of Sonic Hedgehog Signaling in the Somite

    Evidence that the WNT-inducible growth arrest- specific gene 1 encodes an antagonist of sonic hedgehog signaling in the somite Catherine S. Lee*, Laura Buttitta*, and Chen-Ming Fan† Department of Embryology, Carnegie Institution of Washington, 115 West University Parkway, Baltimore, MD 21210 Communicated by Donald D. Brown, Carnegie Institution of Washington, Baltimore, MD, August 8, 2001 (received for review July 16, 2001) The dorsal–ventral polarity of the somite is controlled by antago- Materials and Methods ͞ nistic signals from the dorsal neural tube surface ectoderm, me- cDNA Library Screen. A cDNA library was constructed in pMT21 diated by WNTs, and from the ventral notochord, mediated by with the use of poly(A)ϩ RNA isolated from the caudal quarter sonic hedgehog (SHH). Each factor can act over a distance greater of 180 embryonic day (E)9.5 CD1 mouse embryos. COS cells than a somite diameter in vitro, suggesting they must limit each were transfected with 174 pools (1,000 cDNAs per pool) of the other’s actions within their own patterning domains in vivo.We library with Lipofectamine (GIBCO͞BRL) and screened for show here that the growth-arrest specific gene 1 (Gas1), which is SHH-N-alkaline phosphatase (SHH-N-AP) binding activity expressed in the dorsal somite, is induced by WNTs and encodes a (14). Positive pools were rescreened to obtain single clones. protein that can bind to SHH. Furthermore, ectopic expression of SHH-N-AP and SHH-N-Fc were fusions of SHH-N (amino acids Gas1 in presomitic cells attenuates the response of these cells to 1–198) to AP and to Ig constant region (Fc) (15).