DOCSLIB.ORG

Loss of the Repressor REST in Uterine Fibroids Promotes Aberrant G

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Loss of the Repressor REST in Uterine Fibroids Promotes Aberrant G Loss of the repressor REST in uterine fibroids promotes aberrant G protein-coupled receptor 10 expression and activates mammalian target of rapamycin pathway Binny V. Varghesea, Faezeh Koohestania, Michelle McWilliamsa, Arlene Colvina, Sumedha Gunewardenaa, William H. Kinseyb, Romana A. Nowakc, Warren B. Nothnicka, and Vargheese M. Chennathukuzhia,1 Departments of aMolecular and Integrative Physiology and bAnatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160; and cDepartment of Animal Sciences, University of Illinois, Urbana–Champaign, IL 61801 Edited by R. Michael Roberts, University of Missouri, Columbia, MO, and approved November 28, 2012 (received for review September 12, 2012) Uterine fibroids (leiomyomas) are the most common tumors of the differentially activate this pathway in fibroids compared with female reproductive tract, occurring in up to 77% of reproductive- normal myometrium. aged women, yet molecular pathogenesis remains poorly under- In a focused effort aimed at finding putative signaling molecules stood. A role for atypically activated mammalian target of rapamycin upstream of mTOR that may regulate cell growth and tumori- (mTOR) pathway in the pathogenesis of uterine fibroids has been genesis in leiomyomas, we identified GPR10 as the most highly up- suggested in several studies. We identified that G protein-coupled regulated G protein coupled-receptor (GPCR) in human fibroid receptor 10 [GPR10, a putative signaling protein upstream of the samples. GPR10, also known as the prolactin releasing hormone phosphoinositide 3-kinase–protein kinase B/AKT–mammalian target receptor (PRLHR), is the receptor for prolactin releasing peptide fi of rapamycin (PI3K/AKT–mTOR) pathway] is aberrantly expressed in (PrRP), although evidence for its speci c role in pituitary PRL uterine fibroids. The activation of GPR10 by its cognate ligand, pro- production is tenuous (16, 17). Normal expression and function of lactin releasing peptide, promotes PI3K–AKT–mTOR pathways and GPR10 have been shown to be limited to several regions of the cell proliferation specifically in cultured primary leiomyoma cells. hypothalamus (18, 19). The mechanism of hypothalamic GPR10 Additionally, we report that RE1 suppressing transcription factor/ action that evokes stress hormone release (19) from the pituitary is BIOLOGY neuron-restrictive silencing factor (REST/NRSF), a known tumor sup- not well understood and its function in the periphery, either in DEVELOPMENTAL pressor, transcriptionally represses GPR10 in the normal myometrium, normal tissues or in disease, has not been reported. The near- ubiquitous overexpression of GPR10, a normally neuronal specific and that the loss of REST in fibroids permits GPR10 expression. Impor- G protein coupled receptor (GPCR), in human leiomyomas is tantly, mice overexpressing human GPR10 in the myometrium develop unparalleled among genes shown to be dysregulated in fibroids. myometrial hyperplasia with excessive extracellular matrix deposi- fi fi This increased expression of GPR10 in uterine broids is partic- tion, a hallmark of uterine broids. We demonstrate previously un- ularly relevant because its activation by PrRP has been shown to recognized roles for GPR10 and its upstream regulator REST in the promote the proliferation of cultured cells (20). pathogenesis of uterine fibroids. Importantly, we report a unique ge- fi The RE1 silencing transcription factor/neuron-restrictive silenc- netically modi ed mouse model for a gene that is misexpressed in ing factor (REST/NRSF) has been shown to transcriptionally re- fi uterine broids. press GPR10 in cell lines (21). Particularly relevant to the role that PI3K/AKT appears to play in the pathogenesis of leiomyomas, the terine fibroids (leiomyomas) are benign smooth muscle cell activation of GPR10 as well as the loss of REST/NRSF, is shown to U(SMC) tumors of the myometrium. Leiomyomas represent trigger PI3K/AKT signaling and proliferation in tumor cell lines (20, the most frequent clinical indication for hysterectomy that pre- 22–24). REST is an important transcriptional repressor that silences maturely ends a woman’s reproductive capability (1). However, a multitude of genes in the periphery through epigenetic mecha- despite this, there are currently no approved drugs that can nisms (25). The loss of this tumor suppressor through proteasomal provide effective, long-term treatment for these tumors. In the degradation has been shown to result in oncogenic transformation year 2010, the estimated annual cost of uterine fibroid tumors in of human mammary epithelial cells (24). Mutations or alternative the United States was $5.9–34.4 billion (2). Although uterine splicing that result in dominant negative forms of REST have also fibroids are the most common tumors of the female reproductive been associated with lung and colon cancers (24, 26, 27). A tumor tract and a major quality of life issue for a significant percentage suppressor role for REST in the uterus has not been described of women, the mechanisms that initiate leiomyoma growth and previously. We demonstrate that the loss of REST permits over- pathogenesis are still not well understood. expression of GPR10 in leiomyomas and this misexpression func- Several genetic and environmental risk factors have been sug- tionally promotes tumor cell proliferation contributing to the gested to influence the etiology of leiomyomas (1, 3–5). Although, pathogenesis of uterine fibroids. Our study describes unique roles rare germ-line mutations that give rise to heritable leiomyomas are for GPR10 and for the loss of REST in the development of fibroids. known, such mutations being rare do not account for the vast majority of uterine fibroids. The occurrence of high-frequency somatic missense mutations in the second coding exon of the Author contributions: V.M.C. designed research; B.V.V., F.K., M.M., A.C., S.G., W.H.K., R.A.N., mediator gene MED12 was reported in fibroid tumors in a recent W.B.N., and V.M.C. performed research; R.A.N. contributed new reagents/analytic tools; study (6). The functional significance of missense mutations to this B.V.V., F.K., M.M., A.C., S.G., W.H.K., W.B.N., and V.M.C. analyzed data; and B.V.V., F.K., mediator complex protein and its developmental origins are not M.M., S.G., W.H.K., R.A.N., W.B.N., and V.M.C. wrote the paper. currently known. Alternative mechanisms including epigenetic The authors declare no conflict of interest. variations, microRNA-mediated posttranscriptional gene regula- This article is a PNAS Direct Submission. tion, and altered cell signaling have recently gained traction as Freely available online through the PNAS open access option. – putative disease initiating events (7 12). Several recent studies Data deposition: The Affymetrix gene expression data from the siRNA knockdown experi- have demonstrated a central role for a dysregulated phosphoino- ments reported in this paper have been deposited in the Gene Expression Omnibus (GEO) sitide 3-kinase–protein kinase B/AKT (PI3K/AKT) pathway database, www.ncbi.nlm.nih.gov/geo (accession no. GSE41386). leading to the activation of mammalian target of rapamycin 1To whom correspondence should be addressed. E-mail: [email protected]. – (mTOR) in the pathogenesis of leiomyomas (13 15). There is This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. a need to identify signaling molecules upstream of mTOR that 1073/pnas.1215759110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1215759110 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 Results Aberrant Expression of GPR10 in Uterine Fibroids. In an effort to identify drug targetable, signaling molecules upstream of the mTOR pathway that might be aberrantly regulated in uterine fibroids, we analyzed gene expression profiling data available from the GEO database. Analysis of human leiomyoma and normal myometrial samples from dataset GSE13319 revealed that GPR10, a GPCR with known functions in PI3K/AKT pathway activation, was the most highly dysregulated GPCR in fibroids (Fig. 1A). TaqMan RT-PCR was used to confirm the expression of GPR10 mRNA in fibroids (Fig. 1B). Expression of GPR10 was signifi- cantly up-regulated in 13 out of 14 patient samples. The aberrant expression of GPR10 was further confirmed by Western blotting (Fig. 1C). Expression of the receptor protein was evident in all of the fibroid tissue samples tested, whereas expression was either absent or negligible in matched normal myometrial tissues (Fig. 1C). Because fibroid tumor growth is known to be associated with activation of the PI3K/AKT pathway leading to the acti- vation of mTOR, we compared the expression of GPR10 to the status of AKT phosphorylation. The AKT phosphorylation lev- els in individual patient samples reflected the level of expression of GPR10 in those samples (Fig. 1D), indicating that the aber- rant expression of GPR10 and PI3K/AKT pathway activation Fig. 2. Cultured primary leiomyoma cells express functional GPR10. (A) may be related. GPR10 expression in primary myometrial (MC1–3) and autologous leiomyoma SMCs (LC1–3). β-Actin was used as loading control. (B) Anti–phospho-AKT μ Activation of GPR10 Leads to PI3K/AKT–mTOR Pathway Activation and (s473) Western blot showing that the activation of GPR10 using PrRP (1 M) leads to the phosphorylation of AKT in leiomyoma SMCs. Total AKT was used Leiomyoma Cell Proliferation. Next we tested whether the activa- – tion of GPR10 leads to regulation of the PI3K/AKT–mTOR as loading control. (C and D) Representative anti p-AKT (s473) Western blot and densitometric analysis of Western blots from three independent pathways in primary myometrial and leiomyoma SMCs cultured experiments, respectively, showing that siRNA knockdown of GPR10 leads to in vitro. Abundant expression of GPR10 was maintained in – a loss of PrRP response on AKT phosphorylation. (E) PrRP treatment of cultured leiomyoma cells (Fig. 2A, LC1 LC3), whereas in nor- leiomyoma SMCs leads to mTOR pathway activation. β-Actin was used as mal myometrial SMCs GPR10 expression was negligible (Fig.
Load more

Recommended publications
  • 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.
    [Show full text]
  • Histone Deacetylases 1 and 2 Regulate the Transcriptional Programs Of
    © 2018. Published by The Company of Biologists Ltd | Development (2018) 145, dev153619. doi:10.1242/dev.153619 STEM CELLS AND REGENERATION RESEARCH ARTICLE Histone deacetylases 1 and 2 regulate the transcriptional programs of nephron progenitors and renal vesicles Hongbing Liu*, Shaowei Chen, Xiao Yao, Yuwen Li, Chao-Hui Chen, Jiao Liu, Zubaida Saifudeen and Samir S. El-Dahr ABSTRACT Six2, a homeodomain transcription factor, is a key factor within Nephron progenitor cells (NPCs) are Six2-positive metanephric the kidney metanephric mesenchyme that maintains the NPC pool mesenchyme cells, which undergo self-renewal and differentiation (Kobayashi et al., 2008; Self et al., 2006). In Six2 null mice, ectopic to give rise to nephrons until the end of nephrogenesis. Histone renal vesicles (the earliest epithelialized forms of nascent nephrons) deacetylases (HDACs) are a group of epigenetic regulators that develop at the onset of nephrogenesis and the progenitor pool is – control cell fate, but their role in balancing NPC renewal and rapidly lost (Self et al., 2006). Many transcriptional regulators differentiation is unknown. Here, we report that NPC-specific such as Osr1, WT1 and Sall1/Mi-2b (Chd4)/nucleosome deletion of Hdac1 and Hdac2 genes in mice results in early remodeling and deacetylase (NuRD), which function to maintain – postnatal lethality owing to renal hypodysplasia and loss of NPCs. the NPC pool display genetic interactions with Six2 (Basta et al., HDAC1/2 interact with the NPC renewal regulators Six2, Osr1 and 2014; Denner and Rauchman, 2013; Hartwig et al., 2010; Kanda Sall1, and are co-bound along with Six2 on the Six2 enhancer. et al., 2014; Xu et al., 2014).
    [Show full text]
  • Transcriptional Control of Microglia Phenotypes in Health and Disease
    REVIEW SERIES: GLIA AND NEURODEGENERATION The Journal of Clinical Investigation Series Editors: Marco Colonna and David Holtzmann Transcriptional control of microglia phenotypes in health and disease Inge R. Holtman,1,2 Dylan Skola,1 and Christopher K. Glass1,3 1Department of Cellular and Molecular Medicine, UCSD, San Diego, California, USA. 2Department of Medical Physiology, University of Groningen, University Medical Center Groningen, Groningen, Netherlands. 3Department of Medicine, UCSD, San Diego, California, USA. Microglia are the main resident macrophage population of the CNS and perform numerous functions required for CNS development, homeostasis, immunity, and repair. Many lines of evidence also indicate that dysregulation of microglia contributes to the pathogenesis of neurodegenerative and behavioral diseases. These observations provide a compelling argument to more clearly define the mechanisms that control microglia identity and function in health and disease. In this Review, we present a conceptual framework for how different classes of transcription factors interact to select and activate regulatory elements that control microglia development and their responses to internal and external signals. We then describe functions of specific transcription factors in normal and pathological contexts and conclude with a consideration of open questions to be addressed in the future. Introduction regulatory control necessary to generate cell type–specific programs Microglia are tissue-resident macrophages that perform CNS- of gene expression. This additional information is provided by distal specific functions (1). They derive from a unique lineage of eryth- regulatory elements called enhancers (12). Enhancers represent the romyeloid precursors (EMPs) in the yolk sac and fetal liver (2). most numerous binding sites for LDTFs and signal-dependent tran- EMPs infiltrate the brain during early development, differentiate scription factors (SDTFs), and are major sites for the integration of into microglia, and maintain their population by self-renewal (3).
    [Show full text]
  • Transitions in Cell Potency During Early Mouse Development Are Driven by Notch
    bioRxiv preprint doi: https://doi.org/10.1101/451922; this version posted October 24, 2018. 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. Menchero et al. Transitions in cell potency during early mouse development are driven by Notch Sergio Menchero1, Antonio Lopez-Izquierdo1, Isabel Rollan1, Julio Sainz de Aja1, Ϯ, Maria Jose Andreu1, Minjung Kang2, Javier Adan1, Rui Benedito3, Teresa Rayon1, †, Anna-Katerina Hadjantonakis2 and Miguel Manzanares1,* 1 Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Melchor Fernández Almagro 3, 28029 Madrid, Spain. 2 Developmental Biology Program, Sloan Kettering Institute, New York NY 10065, USA. 3 Molecular Genetics of Angiogenesis Group, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Melchor Fernández Almagro 3, 28029 Madrid, Spain. Ϯ Current address: Children's Hospital, Stem Cell Program, 300 Longwood Ave, Boston MA 02115, USA. † Current address: The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. * Corresponding author ([email protected]) 1 bioRxiv preprint doi: https://doi.org/10.1101/451922; this version posted October 24, 2018. 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. Menchero et al. Abstract The Notch signalling pathway plays fundamental roles in diverse developmental processes in metazoans, where it is important in driving cell fate and directing differentiation of various cell types.
    [Show full text]
  • Genetic Analysis of the Sall Transcription Factor Family in Murine Development
    Genetic Analysis of the Sall Transcription Factor Family in Murine Development DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER NATURWISSENSCHAFTEN (DR. RER. NAT.) DER NATURWISSENSCHAFTLICHEN FAKULTÄT III – BIOLOGIE UND VORKLINISCHE MEDIZIN DER UNIVERSITÄT REGENSBURG vorgelegt von Ulrich Elling aus Freising 2005 Promotionsgesuch eingereicht am: 21. November 2005 Die Arbeit wurde angeleitet von: Dr. M. Treier Doktorvater: Prof. Dr. Schneuwly Thesis advisory committee, EMBL: Dr. S. Cohen Dr. C. Nerlov Prüfungsausschuß: Vorsitzender: Prof. Dr. Kunzelmann 1. Gutachter Prof. Dr. Schneuwly 2. Gutachter Prof. Dr. Witzgall 3. Prüfer: Prof. Dr. Seufert Ersatzperson: Prof. Dr. Warth Zusammenfassung Zusammenfassung Spalt Proteine bilden eine Familie von Transkriptionsfaktoren, die von Caenorhabdits elegans über Drosophila melanogaster bis hin zu Vertebraten konserviert sind. In Säugetiergenomen findet man jeweils vier Gene, die als Spalt-like (SALL1 bis SALL4) bezeichnet werden. Mutationen in SALL1 und SALL4 verursachen im Menschen Townes- Brocks-Syndrom (TBS) beziehungsweise Okihiro-/Duane Radial Ray-Syndrom (DRRS). Beide Syndrome variieren stark in ihrem klinischen Befund, beschrieben sind unter anderem Verdopplung oder Fehlen von Fingern und Zehen, Schäden im zentralen Nervensystem sowie Missbildungen im Urogenitalsystem. Wie es zu diesen multiplen Fehlbildungen kommt ist nicht verstanden. Aufbauend auf eine systematische, vergleichende Untersuchung der Expressionsmuster wurden in der vorliegenden Arbeit die Auswirkungen von Mutationen in Sall1 bis Sall4 auf die Embryonalentwicklung von Mäusen einzeln oder in Kombination untersucht. Bisher wurde nur gezeigt, dass Verlust von Sall1 zur Nierenagenese führt. Das Ziel war deshalb, durch Untersuchung der verschiedenen Sall Mutationen in unterschiedlichen Organsystemen zugrunde liegende Gemeinsamkeiten herauszuarbeiten, die eine generelle Rolle von Sall Genen während der Embryonalentwicklung aufzeigen. Sall4 ist der erste Sall Transkriptionsfaktor, der während der Embryonalentwicklung exprimiert wird.
    [Show full text]
  • Bioinformatic Analysis Reveals the Importance of Epithelial-Mesenchymal Transition in the Development of Endometriosis
    www.nature.com/scientificreports OPEN Bioinformatic analysis reveals the importance of epithelial- mesenchymal transition in the development of endometriosis Meihong Chen1,6, Yilu Zhou2,3,6, Hong Xu4, Charlotte Hill2, Rob M. Ewing2,3, Deming He1, Xiaoling Zhang1 ✉ & Yihua Wang2,3,5 ✉ Background: Endometriosis is a frequently occurring disease in women, which seriously afects their quality of life. However, its etiology and pathogenesis are still unclear. Methods: To identify key genes/ pathways involved in the pathogenesis of endometriosis, we recruited 3 raw microarray datasets (GSE11691, GSE7305, and GSE12768) from Gene Expression Omnibus database (GEO), which contain endometriosis tissues and normal endometrial tissues. We then performed in-depth bioinformatic analysis to determine diferentially expressed genes (DEGs), followed by gene ontology (GO), Hallmark pathway enrichment and protein-protein interaction (PPI) network analysis. The fndings were further validated by immunohistochemistry (IHC) staining in endometrial tissues from endometriosis or control patients. Results: We identifed 186 DEGs, of which 118 were up-regulated and 68 were down-regulated. The most enriched DEGs in GO functional analysis were mainly associated with cell adhesion, infammatory response, and extracellular exosome. We found that epithelial-mesenchymal transition (EMT) ranked frst in the Hallmark pathway enrichment. EMT may potentially be induced by infammatory cytokines such as CXCL12. IHC confrmed the down-regulation of E-cadherin (CDH1) and up-regulation of CXCL12 in endometriosis tissues. Conclusions: Utilizing bioinformatics and patient samples, we provide evidence of EMT in endometriosis. Elucidating the role of EMT will improve the understanding of the molecular mechanisms involved in the development of endometriosis. Endometriosis is a frequently occurring gynaecological disease characterised by chronic pelvic pain, dysmenor- rhea and infertility1.
    [Show full text]
  • High Incidence of the R276X SALL1 Mutation in Sporadic but Not Familial
    1of4 J Med Genet: first published as 10.1136/jmg.40.11.e127 on 19 November 2003. Downloaded from J Med Genet: first published as 10.1136/jmg.40.11.e127 on 19 November 2003. Downloaded from ONLINE MUTATION REPORT High incidence of the R276X SALL1 mutation in sporadic but not familial Townes–Brocks syndrome and report of the first familial case J Kohlhase, M Liebers, J Backe, A Baumann-Mu¨ller, M Bembea, A Destre´e, M Gattas, S Gru¨ßner, TMu¨ller, G Mortier, C Skrypnyk, S Yano, J Wirbelauer, R C Michaelis ............................................................................................................................... J Med Genet 2003;40:127 (http://www.jmedgenet.com/cgi/content/full/40/11/127) ownes–Brocks syndrome (TBS, OMIM #104780) is a rare autosomal dominant malformation syndrome charac- KEY POINTS Tterised by anal, renal, limb, and ear anomalies.1 TBS is caused by mutations in SALL1, a human putative zinc finger N The mutation (c.826CRT; Arg276X) is the most transcription factor gene related to the developmental common SALL1 mutation in Townes–Brocks syndrome regulator sal of Drosophila melanogaster.2 The SALL1 gene (TBS), being previously detected in seven unrelated product is a zinc finger protein thought to act as a patients. We report eight additional cases and provide transcription factor. It contains four highly conserved C2H2 a genotype–phenotype correlation for this mutation. double zinc finger domains that are evenly distributed. A N Arg276X has been demonstrated as the cause of TBS single C2H2 motif is attached to the second domain, and at in 15 of 29 cases of sporadic TBS with detectable the amino terminus SALL1 contains a C2HC motif.3 The SALL1 mutations but has not been observed up to now protein is exclusively found in the nucleus and localises to pericentromeric heterochromatin, acting as a transcriptional in familial cases.
    [Show full text]
  • Genetic Screening of 202 Individuals with Congenital Limb Malformations
    Original article Genetic screening of 202 individuals with congenital J Med Genet: first published as 10.1136/jmg.2009.066027 on 7 May 2009. Downloaded from limb malformations and requiring reconstructive surgery D Furniss,1,2 S-h Kan,1 I B Taylor,1 D Johnson,2 P S Critchley,2 H P Giele,2 A O M Wilkie1 c Additional tables and figure ABSTRACT Previous investigations into the genetics of are published online only at Background: Congenital limb malformations (CLMs) are human CLM have taken two approaches. First, http://jmg.bmj.com/content/ vol46/issue11 common and present to a variety of specialties, notably positional candidate methods have been used to plastic and orthopaedic surgeons, and clinical geneticists. identify mutated genes in affected families follow- 1 Weatherall Institute of The authors aimed to characterise causative mutations in ing linkage analysis, or in individuals harbouring Molecular Medicine, University 34 of Oxford, Oxford, UK; an unselected cohort of patients with CLMs requiring chromosome abnormalities. Second, genetic 2 Department of Plastic and reconstructive surgery. mutations causing CLM have been identified in Reconstructive Surgery, John Methods: 202 patients presenting with CLM were model organisms such as the mouse, and the Radcliffe Hospital, Oxford, UK recruited. The authors obtained G-banded karyotypes and orthologous gene in humans has been screened for 5 Correspondence to: screened EN1, GLI3, HAND2, HOXD13, ROR2, SALL1, mutations in patients with a similar phenotype. Professor A O M Wilkie, SALL4, ZRS of SHH, SPRY4, TBX5, TWIST1 and WNT7A for Although these approaches have yielded many Weatherall Institute of Molecular point mutations using denaturing high performance liquid important gene discoveries, they also have inherent Medicine, University of Oxford, chromatography (DHPLC) and direct sequencing.
    [Show full text]
  • Hox5 Interacts with Plzf to Restrict Shh Expression in the Developing Forelimb
    Hox5 interacts with Plzf to restrict Shh expression in the developing forelimb Ben Xua,1, Steven M. Hrycaja, Daniel C. McIntyrea,2, Nicholas C. Bakera, Jun K. Takeuchib, Lucie Jeannottec, Zachary B. Gaberd, Bennett G. Novitchd, and Deneen M. Wellika,3 aDepartment of Internal Medicine, Division of Molecular Medicine and Genetics, University of Michigan, Ann Arbor, MI 48109; bCardiovascular Regeneration Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan; cCentre de Recherche en Cancérologie de l’Université Laval, Centre Hospitalier Universitaire de Québec, Québec, Canada G1R 2J6; and dDepartment of Neurobiology, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 Edited by Clifford J. Tabin, Harvard Medical School, Boston, MA, and approved October 18, 2013 (received for review August 8, 2013) To date, only the five most posterior groups of Hox genes, Hox9– expression in limb AP patterning (12, 13). Although misexpression Hox13, have demonstrated loss-of-function roles in limb pattern- of more anterior Hox genes in mice reportedly affects limb patterning ing. Individual paralog groups control proximodistal patterning of (16), no loss-of-function mutants of anterior, non–abdominal B the limb skeletal elements. Hox9 genes also initiate the onset of (AbdB)-related genes have demonstrated defects in the patterning of Hand2 expression in the posterior forelimb compartment, and col- limb skeletal elements. Moreover, no HoxB or HoxC group genes had lectively, the posterior HoxA/D genes maintain posterior Sonic been shown to play a role in forelimb development until a report by Hedgehog (Shh) expression.
    [Show full text]
  • SALL1 Gene Spalt Like Transcription Factor 1
    SALL1 gene spalt like transcription factor 1 Normal Function The SALL1 gene is part of a group of related genes that provide instructions for making proteins involved in the formation of tissues and organs before birth. These proteins are transcription factors, which means they attach (bind) to specific regions of DNA and help control the activity of particular genes. The SALL1 protein helps turn off (repress) gene activity by interacting with other proteins that alter how tightly regions of DNA are packaged. This process, known as chromatin remodeling, is one way gene expression is regulated during development; typically, when DNA is tightly packed, gene expression is lower than when DNA is loosely packed. By controlling gene activity, the SALL1 protein plays an important role in development of the hands (particularly the thumbs), ears, anus, kidneys, and other parts of the body before birth. Health Conditions Related to Genetic Changes Townes-Brocks Syndrome At least 75 mutations in the SALL1 gene have been identified in people with Townes- Brocks syndrome, which is typically characterized by a malformation of the anal opening (imperforate anus), abnormally shaped ears, and hand malformations. It is uncertain how these mutations result in the specific features of the condition. Most SALL1 gene mutations involved in this disorder lead to the production of an abnormally short version of the SALL1 protein that malfunctions within cells. The malfunctioning protein is thought to interfere with normal copies of the SALL1 protein, which are produced from the other copy of the SALL1 gene that does not have a mutation. This interference prevents the normal proteins from entering the nucleus to regulate gene activity.
    [Show full text]
  • Supplementary Information Table of Content Supplementary Figures P. 2 Figure S1 P. 2 Figure S2 P. 3 Figure S3 P. 5 Figure S4 P
    Supplementary Information Pokhilko A., et al. Targeted single-cell RNA sequencing of transcription factors enhances the identification of cell types and trajectories Table of content Supplementary Figures p. 2 Figure S1 p. 2 Figure S2 p. 3 Figure S3 p. 5 Figure S4 p. 7 Figure S5 p. 8 Figure S6 p. 15 Figure S7 p. 16 Figure S8 p. 17 Figure S9 p. 19 Figure S10 p. 21 Figure S11 p. 22 Supplementary Tables p. 23 Table S1 p. 23 Table S2 p. 39 Table S3 p. 43 Table S4 p. 44 References p. 44 1 Figure S1. Capture-Seq improves the quality of scRNA-seq libraries and enriches for targeted TFs. a, Post-capture enrichment of 585 captured TFs detected pre-capture. For each gene the enrichment was calculated a ratio between the average CPMs in the post- and pre-capture libraries, plotted in log2 scale after adding a pseudocount of 1(Curion et al. 2020). b, Linear correlation between the pre- and post-capture averaged expression (raw counts) of 585 common TFs. ERCC spike-ins targeted by scCapture-seq are shown in red. a b 2 Figure S2. Characterization of cell clusters in pre- and post-capture libraries. a,b, Heat maps of cell type TF markers, which were differentially expressed post-capture (b), and their pre-capture expression (a). c,d, Heat maps of TF DEGs, which were differentially expressed post-capture (d,e) and their expression in pre-capture (c). Cells are ordered by post-capture clusters on a-d, as annotated above the heatmaps.
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2009/0269772 A1 Califano Et Al
    US 20090269772A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0269772 A1 Califano et al. (43) Pub. Date: Oct. 29, 2009 (54) SYSTEMS AND METHODS FOR Publication Classification IDENTIFYING COMBINATIONS OF (51) Int. Cl. COMPOUNDS OF THERAPEUTIC INTEREST CI2O I/68 (2006.01) CI2O 1/02 (2006.01) (76) Inventors: Andrea Califano, New York, NY G06N 5/02 (2006.01) (US); Riccardo Dalla-Favera, New (52) U.S. Cl. ........... 435/6: 435/29: 706/54; 707/E17.014 York, NY (US); Owen A. (57) ABSTRACT O'Connor, New York, NY (US) Systems, methods, and apparatus for searching for a combi nation of compounds of therapeutic interest are provided. Correspondence Address: Cell-based assays are performed, each cell-based assay JONES DAY exposing a different sample of cells to a different compound 222 EAST 41ST ST in a plurality of compounds. From the cell-based assays, a NEW YORK, NY 10017 (US) Subset of the tested compounds is selected. For each respec tive compound in the Subset, a molecular abundance profile from cells exposed to the respective compound is measured. (21) Appl. No.: 12/432,579 Targets of transcription factors and post-translational modu lators of transcription factor activity are inferred from the (22) Filed: Apr. 29, 2009 molecular abundance profile data using information theoretic measures. This data is used to construct an interaction net Related U.S. Application Data work. Variances in edges in the interaction network are used to determine the drug activity profile of compounds in the (60) Provisional application No. 61/048.875, filed on Apr.
    [Show full text]