DOCSLIB.ORG

4036.Full.Pdf

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
4036.Full.Pdf The Journal of Immunology Disruption of Thrombocyte and T Lymphocyte Development by a Mutation in ARPC1B Raz Somech,*,† Atar Lev,*,† Yu Nee Lee,*,† Amos J. Simon,*,†,‡ Ortal Barel,†,x Ginette Schiby,{ Camila Avivi,{ Iris Barshack,{ Michele Rhodes,‖ Jiejing Yin,‖ Minshi Wang,‖ Yibin Yang,‖ Jennifer Rhodes,‖ Nufar Marcus,# Ben-Zion Garty,# Jerry Stein,** Ninette Amariglio,†,‡,x,†† Gideon Rechavi,†,x David L. Wiest,‖,1 and Yong Zhang‖,1 Regulation of the actin cytoskeleton is crucial for normal development and function of the immune system, as evidenced by the severe immune abnormalities exhibited by patients bearing inactivating mutations in the Wiskott–Aldrich syndrome protein (WASP), a key regulator of actin dynamics. WASP exerts its effects on actin dynamics through a multisubunit complex termed Arp2/3. Despite the critical role played by Arp2/3 as an effector of WASP-mediated control over actin polymerization, mutations in protein components of the Arp2/3 complex had not previously been identified as a cause of immunodeficiency. Here, we describe two brothers with hematopoietic and immunologic symptoms reminiscent of Wiskott–Aldrich syndrome (WAS). However, these patients lacked mutations in any of the genes previously associated with WAS. Whole-exome sequencing revealed a homozygous 2 bp deletion, n.c.G623DEL-TC (p.V208VfsX20), in Arp2/3 complex component ARPC1B that causes a frame shift resulting in premature termination. Modeling of the disease in zebrafish revealed that ARPC1B plays a critical role in supporting T cell and thrombocyte development. Moreover, the defects in development caused by ARPC1B loss could be rescued by the intact human ARPC1B ortholog, but not by the p.V208VfsX20 variant identified in the patients. Moreover, we found that the expression of ARPC1B is restricted to hematopoietic cells, potentially explaining why a mutation in ARPC1B has now been observed as a cause of WAS, whereas mutations in other, more widely expressed, components of the Arp2/3 complex have not been observed. The Journal of Immunology, 2017, 199: 4036–4045. he actin cytoskeleton is a network of actin filaments that widespread immunological defects observed upon disruption of are polymerized from actin monomers. A key regulator of this regulation (3). Consequently, it is perhaps not surprising that T this process, in hematopoietic cells, is the Wiskott–Aldrich the molecules regulating this critical process have been linked to syndrome protein (WASP). WASP regulates actin polymerization the etiology of immunodeficiency. The first described and most by activating the Arp2/3 complex, allowing nucleation of new extensively studied actin-related protein causing primary immu- actin filaments and cross-linking them from end to side branch (1). nodeficiency is WASP, resulting in Wiskott–Aldrich syndrome The Arp2/3 complex consists of seven subunits: Arp2, Arp3, Arc-p16, (WAS) (4). WAS is an X-linked immunodeficiency disease Arc-p20, Arc-p21, Arc-p34, and Arc-p41 (2). Among these sub- with a characteristic clinical phenotype that includes micro- units, Arc-p41, also known as ARPC1B, is proposed to have a thrombocytopenia, eczema, combined T and B cell immunodefi- regulatory role, facilitating assembly and maintenance of the whole ciency, and an increased incidence of autoimmune manifestations complex. and malignancies (5). Mutations in WIP, a known stabilizer of The precise regulation of actin cytoskeleton dynamics is critical WASP, were also reported to cause a similar clinical phenotype (6). to nearly every stage of the immune response, as evidenced by the Mutations in the Arp2/3 complex or its activators, have long been *Pediatric Department A and Immunology Service, Jeffrey Modell Foundation Center, Received for publication March 30, 2017. Accepted for publication October 6, 2017. Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Tel Hashomer, This work was supported by National Institutes of Health Grants P30 CA006927 and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 52621, Israel; R21 AI111208 (to D.L.W.), an appropriation from the Commonwealth of Pennsyl- †The Wohl Institute for Translational Medicine, Sheba Medical Center, Tel Hashomer, vania (to D.L.W.), and also by the Bishop Fund (to D.L.W.). Y.Z. was supported by Tel Aviv 52621, Israel; ‡Hematology Laboratory, Sheba Medical Center, Tel Hashomer, x Leukemia Specialized Program of Research Excellence Grant CA100632 from MD Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 52621, Israel; Sheba Cancer Anderson Cancer Center and Institutional Postdoctoral Training Grant T32 Research Center, Sheba Medical Center, Tel Hashomer, Sackler Faculty of Medicine, { CA009035 from Fox Chase Cancer Center. Tel Aviv University, Tel Aviv 52621, Israel; Department of Pathology, Sheba Medical Center, Tel Hashomer, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv Address correspondence and reprint requests to Dr. Yong Zhang and Dr. David 52621, Israel; ‖Blood Cell Development and Function Program, Fox Chase Cancer L. Wiest, Fox Chase Cancer Center, 333 Cottman Avenue, Room 364, Reimann Center, Philadelphia, PA 19111; #Allergy and Immunology Unit, Schneider Children’s Building, Philadelphia, PA 19111. E-mail addresses: [email protected] (Y.Z.) Medical Center of Israel, Felsenstein Medical Research Center, Kipper Institute of and [email protected] (D.L.W.) Immunology, Petach Tikva, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv The online version of this article contains supplemental material. 4920235, Israel; **Bone Marrow Transplantation Unit, Schneider Children’s Medical Center of Israel, Petach Tikva, Sackler Faculty of Medicine, Tel Aviv University, Tel Abbreviations used in this article: dpf, day postfertilization; hpf, hour postfertiliza- Aviv 4920235, Israel; and ††The Mina and Everard Goodman Faculty of Life Sciences, tion; MO, morpholino oligonucleotide; pt1, patient 1; pt2, patient 2; TREC, TCR Bar-Ilan University, Ramat Gan 52900, Israel excision circle; WAS, Wiskott–Aldrich syndrome; WASP, Wiskott–Aldrich syndrome protein; WISH, whole mount in situ hybridization; WT, wild-type. 1D.L.W. and Y.Z. contributed equally to this work. ORCIDs: 0000-0001-8648-5253 (Y.N.L.); 0000-0002-0225-0586 (M.W.); 0000-0001- Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 7298-1213 (J.R.); 0000-0001-6352-6956 (J.S.); 0000-0002-0792-3188 (D.L.W.). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700460 The Journal of Immunology 4037 sought as a cause of immunodeficiency syndromes with WAS-like turing was done using the Illumina Nextera DNA sample preparation pathologies; however, such mutations have not been observed, kit. Overall, ∼50 M sequence reads were produced for each sample. perhaps because many of these genes are essential for normal de- BWA-mem algorithm (11) for alignment versus the hg19 version of the human genome was applied. Around 40 M reads were properly velopment and so the loss of their function would likely result in aligned. The median coverage was around 40 reads per base. GATK early lethality (7). In support, the loss of Arp2/3 function in Arc- (12) version 2.4.7 with the UnifiedGenotyper algorithm was applied p21–deficient mice is embryonic lethal (8). Nevertheless, here we for variant calling including all steps mentioned in the best practice report the finding of two brothers with a WAS-like clinical phe- pipeline. KGG-sEquation (13) was used for annotation of detected variants and for comparing with allele frequency population data- notype that harbor a novel mutation in the ARPC1B gene. A distinct bases. In-house scripts were applied for filtering, based on family mutation in this gene was also recently reported in a patient with pedigrees and intersections. The ARPC1B mutation was validated by defects in platelets and neutrophils (9). We describe here the clinical dideoxy Sanger sequencing in the patients and carriers. PCR ampli- phenotype of the patients and definitively link their developmental fication products were directly sequenced using BigDye 3.1 Termi- defects in platelets and T cells to the ARPC1B mutation by reca- nator chemistry (Applied Biosystems) and separated on an ABI 3500 genetic analyzer (Applied Biosystems). Data were evaluated using pitulating the disease in a zebrafish model. Sequencer v5.0 software (Gene Codes, Ann Arbor, MI). The resulting data were deposited as a batch deposit of “Human; version 1.0” Materials and Methods samples in the National Center for Biotechnology Information BioSample database (http://www.ncbi.nlm.nih.gov/biosample/; Submis- Patients sion SUB2910482; Accession STUDY; PRJNA397356, SRS2412383 and The patients were diagnosed at the Edmond and Lily Safra Children’s SRX3067724). Hospital. The Institutional Review Board (Sheba Medical Center, Tel Identification of zebrafish arpc1b ortholog Hashomer) approved this study and a written informed consent was obtained from their parents. The zebrafish ortholog (NM_213156) of human ARPC1B was identified using the reciprocal “best-hits” basic local alignment search tool Immunological evaluation search strategy (14). Then multiple alignments of amino acid se- Cell surface markers of PBMCs, lymphocyte proliferative responses to quences for human, mouse, and zebrafish ARPC1B were obtained mitogens, and the amount of signal joint TCR excision circles (TRECs) by using the Clustal V algorithm (http://www.ebi.ac.uk/Tools/msa/ were determined as previously described (10). Serum concentration of Igs clustalo/). Synteny analysis
Load more

Recommended publications
  • Regulation of Cdc42 and Its Effectors in Epithelial Morphogenesis Franck Pichaud1,2,*, Rhian F
    © 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs217869. doi:10.1242/jcs.217869 REVIEW SUBJECT COLLECTION: ADHESION Regulation of Cdc42 and its effectors in epithelial morphogenesis Franck Pichaud1,2,*, Rhian F. Walther1 and Francisca Nunes de Almeida1 ABSTRACT An overview of Cdc42 Cdc42 – a member of the small Rho GTPase family – regulates cell Cdc42 was discovered in yeast and belongs to a large family of small – polarity across organisms from yeast to humans. It is an essential (20 30 kDa) GTP-binding proteins (Adams et al., 1990; Johnson regulator of polarized morphogenesis in epithelial cells, through and Pringle, 1990). It is part of the Ras-homologous Rho subfamily coordination of apical membrane morphogenesis, lumen formation and of GTPases, of which there are 20 members in humans, including junction maturation. In parallel, work in yeast and Caenorhabditis elegans the RhoA and Rac GTPases, (Hall, 2012). Rho, Rac and Cdc42 has provided important clues as to how this molecular switch can homologues are found in all eukaryotes, except for plants, which do generate and regulate polarity through localized activation or inhibition, not have a clear homologue for Cdc42. Together, the function of and cytoskeleton regulation. Recent studies have revealed how Rho GTPases influences most, if not all, cellular processes. important and complex these regulations can be during epithelial In the early 1990s, seminal work from Alan Hall and his morphogenesis. This complexity is mirrored by the fact that Cdc42 can collaborators identified Rho, Rac and Cdc42 as main regulators of exert its function through many effector proteins.
    [Show full text]
  • Seq2pathway Vignette
    seq2pathway Vignette Bin Wang, Xinan Holly Yang, Arjun Kinstlick May 19, 2021 Contents 1 Abstract 1 2 Package Installation 2 3 runseq2pathway 2 4 Two main functions 3 4.1 seq2gene . .3 4.1.1 seq2gene flowchart . .3 4.1.2 runseq2gene inputs/parameters . .5 4.1.3 runseq2gene outputs . .8 4.2 gene2pathway . 10 4.2.1 gene2pathway flowchart . 11 4.2.2 gene2pathway test inputs/parameters . 11 4.2.3 gene2pathway test outputs . 12 5 Examples 13 5.1 ChIP-seq data analysis . 13 5.1.1 Map ChIP-seq enriched peaks to genes using runseq2gene .................... 13 5.1.2 Discover enriched GO terms using gene2pathway_test with gene scores . 15 5.1.3 Discover enriched GO terms using Fisher's Exact test without gene scores . 17 5.1.4 Add description for genes . 20 5.2 RNA-seq data analysis . 20 6 R environment session 23 1 Abstract Seq2pathway is a novel computational tool to analyze functional gene-sets (including signaling pathways) using variable next-generation sequencing data[1]. Integral to this tool are the \seq2gene" and \gene2pathway" components in series that infer a quantitative pathway-level profile for each sample. The seq2gene function assigns phenotype-associated significance of genomic regions to gene-level scores, where the significance could be p-values of SNPs or point mutations, protein-binding affinity, or transcriptional expression level. The seq2gene function has the feasibility to assign non-exon regions to a range of neighboring genes besides the nearest one, thus facilitating the study of functional non-coding elements[2]. Then the gene2pathway summarizes gene-level measurements to pathway-level scores, comparing the quantity of significance for gene members within a pathway with those outside a pathway.
    [Show full text]
  • Mouse Wipf1 Knockout Project (CRISPR/Cas9)
    https://www.alphaknockout.com Mouse Wipf1 Knockout Project (CRISPR/Cas9) Objective: To create a Wipf1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Wipf1 gene (NCBI Reference Sequence: NM_153138 ; Ensembl: ENSMUSG00000075284 ) is located on Mouse chromosome 2. 8 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000094681). Exon 3~6 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Homozygous mutants have immunological abnormalities, although lymphocyte development appears normal. Mutants show abnormal B and T cell proliferative responses, high serum immunoglobulin levels and impaired immunological synapse formation. Exon 3 starts from about 3.52% of the coding region. Exon 3~6 covers 85.26% of the coding region. The size of effective KO region: ~9629 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 3 4 5 6 8 Legends Exon of mouse Wipf1 Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of Exon 3 is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis.
    [Show full text]
  • Long Noncoding Intronic Rnas Are Differentially Expressed in Primary
    Tahira et al. Molecular Cancer 2011, 10:141 http://www.molecular-cancer.com/content/10/1/141 RESEARCH Open Access Long noncoding intronic RNAs are differentially expressed in primary and metastatic pancreatic cancer Ana C Tahira1, Márcia S Kubrusly2, Michele F Faria1, Bianca Dazzani1, Rogério S Fonseca1, Vinicius Maracaja-Coutinho1, Sergio Verjovski-Almeida1, Marcel CC Machado2 and Eduardo M Reis1* Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is known by its aggressiveness and lack of effective therapeutic options. Thus, improvement in current knowledge of molecular changes associated with pancreatic cancer is urgently needed to explore novel venues of diagnostics and treatment of this dismal disease. While there is mounting evidence that long noncoding RNAs (lncRNAs) transcribed from intronic and intergenic regions of the human genome may play different roles in the regulation of gene expression in normal and cancer cells, their expression pattern and biological relevance in pancreatic cancer is currently unknown. In the present work we investigated the relative abundance of a collection of lncRNAs in patients’ pancreatic tissue samples aiming at identifying gene expression profiles correlated to pancreatic cancer and metastasis. Methods: Custom 3,355-element spotted cDNA microarray interrogating protein-coding genes and putative lncRNA were used to obtain expression profiles from 38 clinical samples of tumor and non-tumor pancreatic tissues. Bioinformatics analyses were performed to characterize structure and conservation of lncRNAs expressed in pancreatic tissues, as well as to identify expression signatures correlated to tissue histology. Strand-specific reverse transcription followed by PCR and qRT-PCR were employed to determine strandedness of lncRNAs and to validate microarray results, respectively.
    [Show full text]
  • Dual Proteome-Scale Networks Reveal Cell-Specific Remodeling of the Human Interactome
    bioRxiv preprint doi: https://doi.org/10.1101/2020.01.19.905109; this version posted January 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Dual Proteome-scale Networks Reveal Cell-specific Remodeling of the Human Interactome Edward L. Huttlin1*, Raphael J. Bruckner1,3, Jose Navarrete-Perea1, Joe R. Cannon1,4, Kurt Baltier1,5, Fana Gebreab1, Melanie P. Gygi1, Alexandra Thornock1, Gabriela Zarraga1,6, Stanley Tam1,7, John Szpyt1, Alexandra Panov1, Hannah Parzen1,8, Sipei Fu1, Arvene Golbazi1, Eila Maenpaa1, Keegan Stricker1, Sanjukta Guha Thakurta1, Ramin Rad1, Joshua Pan2, David P. Nusinow1, Joao A. Paulo1, Devin K. Schweppe1, Laura Pontano Vaites1, J. Wade Harper1*, Steven P. Gygi1*# 1Department of Cell Biology, Harvard Medical School, Boston, MA, 02115, USA. 2Broad Institute, Cambridge, MA, 02142, USA. 3Present address: ICCB-Longwood Screening Facility, Harvard Medical School, Boston, MA, 02115, USA. 4Present address: Merck, West Point, PA, 19486, USA. 5Present address: IQ Proteomics, Cambridge, MA, 02139, USA. 6Present address: Vor Biopharma, Cambridge, MA, 02142, USA. 7Present address: Rubius Therapeutics, Cambridge, MA, 02139, USA. 8Present address: RPS North America, South Kingstown, RI, 02879, USA. *Correspondence: [email protected] (E.L.H.), [email protected] (J.W.H.), [email protected] (S.P.G.) #Lead Contact: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/2020.01.19.905109; this version posted January 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.
    [Show full text]
  • Advancing a Clinically Relevant Perspective of the Clonal Nature of Cancer
    Advancing a clinically relevant perspective of the clonal nature of cancer Christian Ruiza,b, Elizabeth Lenkiewicza, Lisa Eversa, Tara Holleya, Alex Robesona, Jeffrey Kieferc, Michael J. Demeurea,d, Michael A. Hollingsworthe, Michael Shenf, Donna Prunkardf, Peter S. Rabinovitchf, Tobias Zellwegerg, Spyro Moussesc, Jeffrey M. Trenta,h, John D. Carpteni, Lukas Bubendorfb, Daniel Von Hoffa,d, and Michael T. Barretta,1 aClinical Translational Research Division, Translational Genomics Research Institute, Scottsdale, AZ 85259; bInstitute for Pathology, University Hospital Basel, University of Basel, 4031 Basel, Switzerland; cGenetic Basis of Human Disease, Translational Genomics Research Institute, Phoenix, AZ 85004; dVirginia G. Piper Cancer Center, Scottsdale Healthcare, Scottsdale, AZ 85258; eEppley Institute for Research in Cancer and Allied Diseases, Nebraska Medical Center, Omaha, NE 68198; fDepartment of Pathology, University of Washington, Seattle, WA 98105; gDivision of Urology, St. Claraspital and University of Basel, 4058 Basel, Switzerland; hVan Andel Research Institute, Grand Rapids, MI 49503; and iIntegrated Cancer Genomics Division, Translational Genomics Research Institute, Phoenix, AZ 85004 Edited* by George F. Vande Woude, Van Andel Research Institute, Grand Rapids, MI, and approved June 10, 2011 (received for review March 11, 2011) Cancers frequently arise as a result of an acquired genomic insta- on the basis of morphology alone (8). Thus, the application of bility and the subsequent clonal evolution of neoplastic cells with purification methods such as laser capture microdissection does variable patterns of genetic aberrations. Thus, the presence and not resolve the complexities of many samples. A second approach behaviors of distinct clonal populations in each patient’s tumor is to passage tumor biopsies in tissue culture or in xenografts (4, 9– may underlie multiple clinical phenotypes in cancers.
    [Show full text]
  • Systematic Data-Querying of Large Pediatric Biorepository Identifies Novel Ehlers-Danlos Syndrome Variant Akshatha Desai1, John J
    Desai et al. BMC Musculoskeletal Disorders (2016) 17:80 DOI 10.1186/s12891-016-0936-8 RESEARCH ARTICLE Open Access Systematic data-querying of large pediatric biorepository identifies novel Ehlers-Danlos Syndrome variant Akshatha Desai1, John J. Connolly1, Michael March1, Cuiping Hou1, Rosetta Chiavacci1, Cecilia Kim1, Gholson Lyon1, Dexter Hadley1 and Hakon Hakonarson1,2* Abstract Background: Ehlers Danlos Syndrome is a rare form of inherited connective tissue disorder, which primarily affects skin, joints, muscle, and blood cells. The current study aimed at finding the mutation that causing EDS type VII C also known as “Dermatosparaxis” in this family. Methods: Through systematic data querying of the electronic medical records (EMRs) of over 80,000 individuals, we recently identified an EDS family that indicate an autosomal dominant inheritance. The family was consented for genomic analysis of their de-identified data. After a negative screen for known mutations, we performed whole genome sequencing on the male proband, his affected father, and unaffected mother. We filtered the list of non- synonymous variants that are common between the affected individuals. Results: The analysis of non-synonymous variants lead to identifying a novel mutation in the ADAMTSL2 (p. Gly421Ser) gene in the affected individuals. Sanger sequencing confirmed the mutation. Conclusion: Our work is significant not only because it sheds new light on the pathophysiology of EDS for the affected family and the field at large, but also because it demonstrates the utility of unbiased large-scale clinical recruitment in deciphering the genetic etiology of rare mendelian diseases. With unbiased large-scale clinical recruitment we strive to sequence as many rare mendelian diseases as possible, and this work in EDS serves as a successful proof of concept to that effect.
    [Show full text]
  • The Guanine Nucleotide Exchange Factor Arhgef5 Plays Crucial Roles in Src-Induced Podosome Formation
    1726 Research Article The guanine nucleotide exchange factor Arhgef5 plays crucial roles in Src-induced podosome formation Miho Kuroiwa, Chitose Oneyama, Shigeyuki Nada and Masato Okada* Department of Oncogene Research, Research institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan *Author for correspondence ([email protected]) Accepted 19 January 2011 Journal of Cell Science 124, 1726-1738 © 2011. Published by The Company of Biologists Ltd doi:10.1242/jcs.080291 Summary Podosomes and invadopodia are actin-rich membrane protrusions that play a crucial role in cell adhesion and migration, and extracellular matrix remodeling in normal and cancer cells. The formation of podosomes and invadopodia is promoted by upregulation of some oncogenic molecules and is closely related to the invasive potential of cancer cells. However, the molecular mechanisms underlying the podosome and invadopodium formation still remain unclear. Here, we show that a guanine nucleotide exchange factor (GEF) for Rho family GTPases (Arhgef5) is crucial for Src-induced podosome formation. Using an inducible system for Src activation, we found that Src-induced podosome formation depends upon the Src SH3 domain, and identified Arhgef5 as a Src SH3-binding protein. RNA interference (RNAi)-mediated depletion of Arhgef5 caused robust inhibition of Src-dependent podosome formation. Overexpression of Arhgef5 promoted actin stress fiber remodeling through activating RhoA, and the activation of RhoA or Cdc42 was required for Src-induced podosome formation. Arhgef5 was tyrosine-phosphorylated by Src and bound to Src to positively regulate its activity. Furthermore, the pleckstrin homology (PH) domain of Arhgef5 was required for podosome formation, and Arhgef5 formed a ternary complex with Src and phosphoinositide 3-kinase when Src and/or Arhgef5 were upregulated.
    [Show full text]
  • Supplemental Information
    Supplemental information Dissection of the genomic structure of the miR-183/96/182 gene. Previously, we showed that the miR-183/96/182 cluster is an intergenic miRNA cluster, located in a ~60-kb interval between the genes encoding nuclear respiratory factor-1 (Nrf1) and ubiquitin-conjugating enzyme E2H (Ube2h) on mouse chr6qA3.3 (1). To start to uncover the genomic structure of the miR- 183/96/182 gene, we first studied genomic features around miR-183/96/182 in the UCSC genome browser (http://genome.UCSC.edu/), and identified two CpG islands 3.4-6.5 kb 5’ of pre-miR-183, the most 5’ miRNA of the cluster (Fig. 1A; Fig. S1 and Seq. S1). A cDNA clone, AK044220, located at 3.2-4.6 kb 5’ to pre-miR-183, encompasses the second CpG island (Fig. 1A; Fig. S1). We hypothesized that this cDNA clone was derived from 5’ exon(s) of the primary transcript of the miR-183/96/182 gene, as CpG islands are often associated with promoters (2). Supporting this hypothesis, multiple expressed sequences detected by gene-trap clones, including clone D016D06 (3, 4), were co-localized with the cDNA clone AK044220 (Fig. 1A; Fig. S1). Clone D016D06, deposited by the German GeneTrap Consortium (GGTC) (http://tikus.gsf.de) (3, 4), was derived from insertion of a retroviral construct, rFlpROSAβgeo in 129S2 ES cells (Fig. 1A and C). The rFlpROSAβgeo construct carries a promoterless reporter gene, the β−geo cassette - an in-frame fusion of the β-galactosidase and neomycin resistance (Neor) gene (5), with a splicing acceptor (SA) immediately upstream, and a polyA signal downstream of the β−geo cassette (Fig.
    [Show full text]
  • WO 2019/079361 Al 25 April 2019 (25.04.2019) W 1P O PCT
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization I International Bureau (10) International Publication Number (43) International Publication Date WO 2019/079361 Al 25 April 2019 (25.04.2019) W 1P O PCT (51) International Patent Classification: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, C12Q 1/68 (2018.01) A61P 31/18 (2006.01) DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, C12Q 1/70 (2006.01) HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, (21) International Application Number: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, PCT/US2018/056167 OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (22) International Filing Date: SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, 16 October 2018 (16. 10.2018) TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (26) Publication Language: English GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, (30) Priority Data: UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, 62/573,025 16 October 2017 (16. 10.2017) US TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, ΓΕ , IS, IT, LT, LU, LV, (71) Applicant: MASSACHUSETTS INSTITUTE OF MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TECHNOLOGY [US/US]; 77 Massachusetts Avenue, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, Cambridge, Massachusetts 02139 (US).
    [Show full text]
  • Supp Material.Pdf
    Simon et al. Supplementary information: Table of contents p.1 Supplementary material and methods p.2-4 • PoIy(I)-poly(C) Treatment • Flow Cytometry and Immunohistochemistry • Western Blotting • Quantitative RT-PCR • Fluorescence In Situ Hybridization • RNA-Seq • Exome capture • Sequencing Supplementary Figures and Tables Suppl. items Description pages Figure 1 Inactivation of Ezh2 affects normal thymocyte development 5 Figure 2 Ezh2 mouse leukemias express cell surface T cell receptor 6 Figure 3 Expression of EZH2 and Hox genes in T-ALL 7 Figure 4 Additional mutation et deletion of chromatin modifiers in T-ALL 8 Figure 5 PRC2 expression and activity in human lymphoproliferative disease 9 Figure 6 PRC2 regulatory network (String analysis) 10 Table 1 Primers and probes for detection of PRC2 genes 11 Table 2 Patient and T-ALL characteristics 12 Table 3 Statistics of RNA and DNA sequencing 13 Table 4 Mutations found in human T-ALLs (see Fig. 3D and Suppl. Fig. 4) 14 Table 5 SNP populations in analyzed human T-ALL samples 15 Table 6 List of altered genes in T-ALL for DAVID analysis 20 Table 7 List of David functional clusters 31 Table 8 List of acquired SNP tested in normal non leukemic DNA 32 1 Simon et al. Supplementary Material and Methods PoIy(I)-poly(C) Treatment. pIpC (GE Healthcare Lifesciences) was dissolved in endotoxin-free D-PBS (Gibco) at a concentration of 2 mg/ml. Mice received four consecutive injections of 150 μg pIpC every other day. The day of the last pIpC injection was designated as day 0 of experiment.
    [Show full text]
  • ARPC1A Antibody Cat
    ARPC1A Antibody Cat. No.: 42-980 ARPC1A Antibody 42-980 (0.1ug/ml) staining of fetal Mouse Brain (A) and adult Rat Brain (B) lysates (35ug protein 42-980 (5ug/ml) staining of paraffin embedded Human Heart. in RIPA buffer). Primary incubation was 1 hour. Steamed antigen retrieval with citrate buffer pH 6, AP-staining. Detected by chemiluminescence. Specifications HOST SPECIES: Goat SPECIES REACTIVITY: Human, Mouse, Rat HOMOLOGY: Expected Species Reactivity based on sequence homology: Pig, Cow IMMUNOGEN: The immunogen for this antibody is: C-DFKTLESSIQGLR TESTED APPLICATIONS: ELISA, IHC, WB September 26, 2021 1 https://www.prosci-inc.com/arpc1a-antibody-42-980.html Peptide ELISA: antibody detection limit dilution 1:64000.Western Blot:Approx 45kDa band observed in lysates of cell line HeLa (calculated MW of 41.6kDa according to NP_006400.2). Approx 37kDa band observed in fetal Mouse Brain and adult Rat Brain APPLICATIONS: lysates (calculated MW of 41.6kDa according to Mouse NP_062741.1). Recommended concentration: 0.1-0.3ug/ml.Immunohistochemistry:Paraffin embedded Human Heart. Recommended concentration: 5ug/ml. This antibody is expected to recognize both reported isoforms (NP_006400.2; SPECIFICITY: NP_001177925.1). No cross-reactiviry expected with ARPC1B. PREDICTED MOLECULAR Approx 45 kDa WEIGHT: Properties Purified from goat serum by ammonium sulphate precipitation followed by antigen PURIFICATION: affinity chromatography using the immunizing peptide. CLONALITY: Polyclonal CONJUGATE: Unconjugated PHYSICAL STATE: Liquid Supplied at 0.5 mg/ml in Tris saline, 0.02% sodium azide, pH7.3 with 0.5% bovine serum BUFFER: albumin. Aliquot and store at -20°C. Minimize freezing and thawing. CONCENTRATION: 500 ug/mL STORAGE CONDITIONS: Aliquot and store at -20˚C.
    [Show full text]