DOCSLIB.ORG

Enhancer of WT1 Gene Is Essential for Its Transcription in Acute Leukemia and Solid Tumor Cell Lines

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Enhancer of WT1 Gene Is Essential for Its Transcription in Acute Leukemia and Solid Tumor Cell Lines Leukemia (2009) 23, 1270–1277 & 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00 www.nature.com/leu ORIGINAL ARTICLE GATA-1 and GATA-2 binding to 30 enhancer of WT1 gene is essential for its transcription in acute leukemia and solid tumor cell lines A Furuhata1, M Murakami1, H Ito1, S Gao1, K Yoshida1, S Sobue2, R Kikuchi3, T Iwasaki3, A Takagi1, T Kojima1, M Suzuki4, A Abe5, T Naoe5 and T Murate1 1Department of Medical Technology, Nagoya University Graduate School of Health Sciences, Nagoya, Japan; 2Department of Pathology, College of Life and Health Sciences, Chubu University, Kasugai, Japan; 3Department of Laboratory Medicine, Nagoya University School of Medicine, Nagoya, Japan; 4Division of Molecular Carcinogenesis, Nagoya University Graduate School of Medicine, Nagoya, Japan and 5Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya, Japan Although oncogenic functions and the clinical significance of WT1 have been well documented, its gene-expression mecha- Wilms tumor 1 (WT1) have been extensively studied in acute nism remains undetermined. leukemia, the regulatory mechanism of its transcription still 13 remains to be determined. We found a significant correlation In the promoter analysis of WT1 gene, Cohen et al. reported among the amounts of WT1, GATA-1 and GATA-2 mRNAs from the importance of Sp1 for mRNA expression, although the leukemia and solid tumor cell lines. Overexpression and small promoter region may not determine the tissue-specific manner interfering RNA (siRNA) transfection experiments of GATA-1 of WT1 expression.13,14 Regarding this point, the 30 enhancer and GATA-2 showed that these GATA transcription factors located 450 kb downstream of the promoter was identified as could induce WT1 expression. Promoter analysis showed that 0 increasing the basal transcription rate of the WT1 promoter in the 5 promoter did not explain the different WT1 mRNA levels 14 15 between cell lines. The 30 enhancer, especially the distal sites the erythroleukemia cell line K562. Subsequently, Wu et al. out of six putative GATA binding sites located within the region, showed that GATA-1 may bind to this enhancer, which is 16 but not the intron 3 enhancer, were essential for the WT1 mRNA reported to be specific in hematopoietic cells. To date, in level. Electrophoretic mobility shift assay (EMSA) showed both addition to Sp1 and GATA-1, PAX2 and PAX8 are also reported GATA-1 and GATA-2 bound to these GATA sites. Besides acute to be the transcription factors that regulate WT1 gene expres- leukemia cell lines, solid tumor cell lines including, TYK-nu-cPr sion.17–20 Dehbi et al.17 reported the binding sites of PAX2 and also showed a high level of WT1 mRNA. We showed that GATA- 0 20 2 expression is a determinant of WT1 mRNA expression in both PAX8 in the 5 promoter of WT1 gene. Zhang et al. indicated TYK-nu-cPr cells and HL60 cells without GATA-1 expression. the importance of the intron 3 enhancer of WT1 gene, which Taken together, these results suggest that GATA-1 and/or was activated by GATA-1 and Myb. GATA-2 binding to a GATA site of the 30 enhancer of WT1 These earlier results suggest that between the cells analyzed, the played an important role in WT1 gene expression. regulation of WT1 gene expression might be complex and Leukemia (2009) 23, 1270–1277; doi:10.1038/leu.2009.13; heterogeneous. Furthermore, it was not clear what the major published online 12 February 2009 determinant of WT1 mRNA level is either in leukemia or in solid Keywords: WT1 message; GATA-1 and 2; 50 promoter; 30 enhancer; EMSA; CHIP assay tumors. Although the GATA family has been shown to play critical roles in erythroid and other hematopoietic lineages,21,22 GATA-1 expression is low in M3, M4 and M5 of acute myelogeneous leukemia,23 in which WT1 expression has been observed. Introduction Therefore, GATA-1 might not be sufficient to maintain full WT1 gene expression. For another transcription factor candidate, we Wilms tumor 1 (WT1) gene was isolated as a tumor suppressor focused on GATA-2, which is presented in cells at the early- gene responsible for Wilm’s tumor, a kidney neoplasm of differentiated stage of hematopoietic lineages. We examined the childhood. The gene product, WT1, represses the transcription relationship between WT1, GATA-1 and GATA-2 mRNA from 20 of growth factors, growth factor receptors and other genes.1–3 leukemia cell lines and 26 acute leukemia bone marrows, as well On the other hand, WT1 has been regarded as an oncogene for as purified stem cell and progenitor cell fractions from two normal leukemia and some solid tumors because high expression levels bone marrows. We also examined solid tumor cell lines with high of WT1 mRNA were observed in these diseases, and in vivo and WT1 expressions. There are no reports describing the regulatory in vitro studies showed the positive roles it plays in cell growth mechanism of WT1 gene expression in these cell lines. and transformation.4–8 WT1 mRNA is reportedly a good clinical On the basis of our results, we discussed the functional marker for disease progression, diagnosis and detection of the relationship between Sp1 and WT1 expression and that between minimal residual diseases of myelodysplastic syndromes and GATA and WT1 expression in leukemia cell lines and several leukemia.9–11 Thus, depending on the cells and situations solid tumor cell lines. expressing WT1, it displays both anti-oncogenic and oncogenic characters.12 Despite the fact that the oncogenic functions of Materials and methods Correspondence: Professor T Murate, Department of Medical Technology, Nagoya University Graduate School of Health Sciences, Daiko-minami, 1-1-20 Higashi-ku, Nagoya, Aichi 461-8673, Japan. Clinical samples and cell lines E-mail: [email protected] After obtaining informed consent, bone marrow cells were Received 28 June 2008; revised 28 November 2008; accepted 19 collected from 23 acute myeloid leukemia (AML) patients and December 2008; published online 12 February 2009 three ALL patients without Ph1 chromosome. Normal control GATA-1/2-induced WT1 gene expression through a 30-enhancer GATA site A Furuhata et al 1271 samples were derived from malignant lymphoma patients without plasmid containing various lengths of the 50 promoter and/or bone marrow involvement as described earlier.24,25 Our analysis enhancer region of WT1 gene and 1 mgofb-galactosidase of WT1, GATA-1 and GATA-2 was approved by IRB (approved expression vector (Promega). The lipofectin reagent (Invitrogen, number 99) of the Nagoya University School of Medicine. Some Carlsbad, CA, USA) was used for K562. The calcium precipita- leukemia cell lines were provided by Dr Hirokazu Nagai tion method was used for TYK-nu-cPr and HepG2. Trans (National Hospital Organization, Nagoya Medical Center, IT-Jurkat (Mirus Bio Co., Madison, WI, USA) was used for Jurkat Nagoya, Japan). Solid tumor cell lines with high WT1 mRNA cells. After 48 h, cell lysates were prepared. Luciferase activity levels8 were the generous gifts of Dr Haruo Sugiyama (Osaka was normalized with the b-galactosidase activity. University Graduate School of Medicine, Osaka, Japan). DNA transfection Quantitative reverse transcriptase-PCR Transfection of the expression vector was carried out using Trans Quantitative reverse transcriptase-PCR was performed as IT-Jurkat, for Jurkat, and the calcium precipitation method for described.24,25 WT1 mRNA was measured using the Light Cycler TYK-nu-cPr cells. GATA-1 and GATA-2 expression vectors were system (Roche Diagnostics, Mannheim, Germany). Each primer obtained from Dr Ritsuko Shimizu (Tsukuba University, Tochigi, set and PCR conditions have been described before.24,25 GATA-1, Japan) and Dr Haruhiko Asano (Chubu University, Kasugai, GATA-2 and glyceraldehyde-3-phosphate dehydrogenase Aichi, Japan), respectively. GATA-1 cDNA or GATA-2 cDNA (GAPDH) mRNAs were measured with a Power SYBR Green was transferred into pcDNA3.1 vector (Invitrogen). master mix using the ABI PRISM 7000 sequence detection systems (Applied Biosystems Japan Ltd, Tokyo, Japan). After producing first-strand cDNA, quantitative PCR was carried out using the 0 RNA interference following primer sets: GATA-1, forward 5 -CCCAAGAAGCGCCT 5 GATTGT-30; reverse 50-GTGTAGCTTGTAGTAGAGGCCGC-30. K562 cells (5 Â 10 ) were transfected with small interfering RNA GATA-2, forward 50-CGTTCCTGTTCAGAAGGC-30; reverse 50-G (siRNA) (final 100 nM) using Oligofectamine reagent TTCTGCCCATTCATCTTGT-30. GAPDH, forward 50-CAGGAGC (Invitrogen). GATA-1 siRNA (ID no. 3197) and GATA-2 siRNA GAGATCCCTCCAA-30; reverse 50-CCCCCTGCAAATGAGCCC-30. (ID no. 145419) were purchased from Ambion (Austin, TX, PCR conditions were GATA-1, 95 1C for 10 s followed by 56 1Cfor USA). Scrambled siRNA was purchased from Dharmacon (GE 15 s; GATA-2, 95 1C for 10 s followed by 56 1C for 15 s; and Healthcare Sciences, Tokyo, Japan). GAPDH, 95 1C for 10 s followed by 63 1C for 15 s. Forty cycles were used for WT1, GATA-1, GATA-2 and GAPDH. GAPDH was used as the internal control for both clinical samples and cell lines. Electrophoresis mobility shift assay Relative mRNA was calculated as the respective mRNA/GAPDH Nuclear extract was prepared from K562, Jurkat, Daudi, HL-60, mRNA in the log scale. TYK-nu-cPr, Sw480, Az521 and HepG2 cells. Electrophoretic mobility shift assay (EMSA) was performed as described earlier.26 For the supershift experiment, anti-GATA-1 or anti- Western blotting GATA-2 antibody (Santa Cruz Inc.) was added to the nuclear Western blotting of WT1, GATA-1, GATA-2 and Sp1 was carried extract for 30 min at room temperature before mixing with out using the anti-WT1 antibody (clone 6F-H2, Dakocytomation, biotin-labeled probes as described below.
Load more

Recommended publications
  • The Role of PU.1 and GATA-1 Transcription Factors During Normal and Leukemogenic Hematopoiesis
    Leukemia (2010) 24, 1249–1257 & 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 www.nature.com/leu REVIEW The role of PU.1 and GATA-1 transcription factors during normal and leukemogenic hematopoiesis P Burda1, P Laslo2 and T Stopka1,3 1Department of Pathophysiology and Center of Experimental Hematology, First Faculty of Medicine, Charles University, Prague, Czech Republic; 2Section of Experimental Haematology, Leeds Institute of Molecular Medicine, University of Leads, St James’s University Hospital, Leeds, UK and 31st Department of Medicine-Hematology, General University Hospital, Prague, Czech Republic Hematopoiesis is coordinated by a complex regulatory network Additional domains include an N-terminal acidic domain and a of transcription factors and among them PU.1 (Spi1, Sfpi1) glutamine-rich domain, both involved in transcriptional activa- represents a key molecule. This review summarizes the tion, as well as a PEST domain involved in protein–protein indispensable requirement of PU.1 during hematopoietic cell fate decisions and how the function of PU.1 can be modulated interactions. PU.1 protein can be modified post-translationally by protein–protein interactions with additional factors. The by phosporylation at serines 41 (N-terminal acidic domain) and mutual negative regulation between PU.1 and GATA-1 is 142 and 148 (PEST domain), which results in augmented detailed within the context of normal and leukemogenic activity. hematopoiesis and the concept of ‘differentiation therapy’ to The PU.1 protein can physically interact with a variety of restore normal cellular differentiation of leukemic cells is regulatory factors including (i) general transcription factors discussed. Leukemia (2010) 24, 1249–1257; doi:10.1038/leu.2010.104; (TFIID, TBP), (ii) early hematopoietic transcription factors published online 3 June 2010 (GATA-2 and Runx-1), (iii) erythroid factor (GATA-1) and (iv) Keywords: PU.1; leukemia differentiation; GATA-1; chromatin; non-erythroid factors (C/EBPa, C/EBPb, IRF4/8 and c-Jun).
    [Show full text]
  • 5045.Full.Pdf
    IFN Consensus Sequence Binding Protein (Icsbp) Is Critical for Eosinophil Development This information is current as Maja Milanovic, Grzegorz Terszowski, Daniela Struck, of September 28, 2021. Oliver Liesenfeld and Dirk Carstanjen J Immunol 2008; 181:5045-5053; ; doi: 10.4049/jimmunol.181.7.5045 http://www.jimmunol.org/content/181/7/5045 Downloaded from References This article cites 47 articles, 33 of which you can access for free at: http://www.jimmunol.org/content/181/7/5045.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 28, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology IFN Consensus Sequence Binding Protein (Icsbp) Is Critical for Eosinophil Development1 Maja Milanovic,2* Grzegorz Terszowski,2† Daniela Struck,2‡ Oliver Liesenfeld,‡ and Dirk Carstanjen3* IFN consensus sequence binding protein (Icsbp) (IFN response factor-8) is a hematopoietic transcription factor with dual functions in myelopoiesis and immunity.
    [Show full text]
  • Transcriptome Analysis of Gravitational Effects on Mouse Skeletal Muscles Under Microgravity and Artificial 1 G Onboard Environm
    www.nature.com/scientificreports OPEN Transcriptome analysis of gravitational efects on mouse skeletal muscles under microgravity and artifcial 1 g onboard environment Risa Okada1,2, Shin‑ichiro Fujita3,4, Riku Suzuki5,6, Takuto Hayashi3,5, Hirona Tsubouchi5, Chihiro Kato5,7, Shunya Sadaki5, Maho Kanai5,6, Sayaka Fuseya3,5, Yuri Inoue3,5, Hyojung Jeon5, Michito Hamada5, Akihiro Kuno5,6, Akiko Ishii8, Akira Tamaoka8, Jun Tanihata9, Naoki Ito10, Dai Shiba1,2, Masaki Shirakawa1,2, Masafumi Muratani1,4, Takashi Kudo1,5* & Satoru Takahashi1,5* Spacefight causes a decrease in skeletal muscle mass and strength. We set two murine experimental groups in orbit for 35 days aboard the International Space Station, under artifcial earth‑gravity (artifcial 1 g; AG) and microgravity (μg; MG), to investigate whether artifcial 1 g exposure prevents muscle atrophy at the molecular level. Our main fndings indicated that AG onboard environment prevented changes under microgravity in soleus muscle not only in muscle mass and fber type composition but also in the alteration of gene expression profles. In particular, transcriptome analysis suggested that AG condition could prevent the alterations of some atrophy‑related genes. We further screened novel candidate genes to reveal the muscle atrophy mechanism from these gene expression profles. We suggest the potential role of Cacng1 in the atrophy of myotubes using in vitro and in vivo gene transductions. This critical project may accelerate the elucidation of muscle atrophy mechanisms. Gravity is the most constant factor afecting the entire process of evolution of organisms on Earth. As adapting to a changing environment is key for any organism’s survival, the constant mechanical stimulus of gravitational force has been shared by all organisms on Earth through evolution 1.
    [Show full text]
  • Expression of Wilms Tumor 1 Gene Distinguishes Vascular Malformations from Proliferative Endothelial Lesions
    OBSERVATION Expression of Wilms Tumor 1 Gene Distinguishes Vascular Malformations From Proliferative Endothelial Lesions Leslie P. Lawley, MD; Francesca Cerimele, MD, PhD; Sharon W. Weiss, MD; Paula North, MD; Cynthia Cohen, MD; Harry P. W. Kozakewich, MD; John B. Mulliken, MD; Jack L. Arbiser, MD, PhD Background: Vascular malformations and hemangio- Observations: The biochemical differences between mas, which are endothelial lesions of childhood, may re- hemangiomas, which involute, and vascular malforma- sult in considerable morbidity because they can cause dis- tions, which do not involute, are not well understood. comfort and functional impairment and have a negative We found that the transcription factor encoded by the affect on the patient’s appearance. Although vascular mal- Wilms tumor 1 (WT1) gene is expressed in the endothe- formations may initially appear very similar to heman- lium of hemangiomas but not in vascular malforma- giomas, they have distinct clinical courses. Infantile hem- tions. angiomas progress through 3 stages: proliferative, involuting, and involuted. The proliferative phase is char- Conclusions: Defects in WT1 signaling may underlie the acterized by clinical growth. Once hemangiomas reach inability of malformation endothelial cells to undergo their maximum size, they begin to regress or involute. physiologic apoptosis and remodeling. The availability Histologically, this stage is characterized by endothelial of WT1 staining in hospital laboratories may allow the apoptosis. Finally, the involuted stage of the heman- clinician to distinguish hemangiomas from vascular mal- gioma occurs when the original lesion is replaced by a formations and thus to give appropriate therapy to the connective tissue remnant. In contrast to hemangio- patient. mas, vascular malformations do not involute but con- tinue to enlarge as the patient grows.
    [Show full text]
  • Letters to the Editor
    LETTERS TO THE EDITOR The significant upregulation of Gata1 and EpoR mRNA Mice over-expressing human erythropoietin expression in hEPO over-expressing tg6 mice was con - indicate that erythropoietin enhances expression firmed by analyzing the spleen as a major source of of its receptor via up-regulated Gata1 and Tal1 hematopoiesis (Figure 2A and B). To further dissect the complexity of changes in the transcriptional network, the analysis of Myb mRNA expression served as marker for The development of medullary hematopoiesis is char - adult definitive erythroblasts, 10 showing significantly acterized by a specific expression profile of hematopoiet - ic transcription factors, including GATA transcription fac - tors. At mid-gestation, when hematopoiesis is newly A GATA 1 2 established in the bone marrow of human fetuses, initial - wt ly high GATA2 expression becomes subsequently down- regulated, while GATA1 expression increases in parallel. 1 1.5 Both transcription factors bind to overlapping sets of tg6 hematopoietic downstream target genes, often at distinct 1 sites, to regulate the balance between proliferation and differentiation. Chromatin occupancy by GATA1 and 0.5 GATA2 can change in the course of hematopoietic differ - entiation, leading to the so-called GATA switch. 2 Thus, a 0 n d7 d21 d49 i spatio-temporal regulation of GATA1 or GATA2 activities t c is required within lineage-specific differentiation. During a - erythroid differentiation GATA1 expression peaks at the b B GATA 2 3 o level of colony-forming units (CFU-E), where erythro - t 2 e poietin (Epo) exerts most specifically its effects, but v i t blocks terminal maturation if constitutively over- a 1.5 l 4 e expressed.
    [Show full text]
  • GATA1 Gene GATA Binding Protein 1
    GATA1 gene GATA binding protein 1 Normal Function The GATA1 gene provides instructions for making a protein that attaches (binds) to specific regions of DNA and helps control the activity of many other genes. On the basis of this action, the GATA1 protein is known as a transcription factor. The GATA1 protein is involved in the specialization (differentiation) of immature blood cells. To function properly, these immature cells must differentiate into specific types of mature blood cells. By binding to DNA and interacting with other proteins, the GATA1 protein regulates the growth and division (proliferation) of immature red blood cells and platelet-precursor cells (megakaryocytes) to facilitate their differentiation. Red blood cells help carry oxygen to various tissues throughout the body and platelets aid in blood clotting. The GATA1 protein is also important for the maturation of several types of white blood cells that help fight infection, including eosinophils, mast cells, and dendritic cells. Two versions of the GATA1 protein are produced from the GATA1 gene: a regular length protein and a shorter version called GATA1s. The GATA1s protein lacks a specific region called the transactivation domain. Although the specific function of this region is unclear, researchers believe that it interacts with other proteins to modify GATA1 protein function. Health Conditions Related to Genetic Changes Dyserythropoietic anemia and thrombocytopenia At least eight different mutations in the GATA1 gene have been found to cause dyserythropoietic anemia and thrombocytopenia. Most of these mutations change a single protein building block (amino acid) in the GATA1 protein. GATA1 gene mutations disrupt the protein's ability to bind with DNA or interact with other proteins.
    [Show full text]
  • Germline Mutations in Etv6 Result in Autosomal Dominant
    GERMLINE MUTATIONS IN ETV6 RESULT IN AUTOSOMAL DOMINANT THROMBOCYTOPENIA By LEILA J. NOETZLI B.S., University of California San Diego, 2007 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Human Medical Genetics and Genomics Program 2017 This thesis for the Doctor of Philosophy degree by Leila J. Noetzli has been approved for the Human Medical Genetics and Genomics Program by Robert Sclafani, Chair Jorge Di Paola, Advisor Jay Hesselberth Richard Spritz Rytis Prekeris Date: May 19, 2017 ii Noetzli, Leila J. (Ph.D., Human Medical Genetics and Genomics) Germline mutations in ETV6 result in autosomal dominant thrombocytopenia Thesis directed by Associate Professor Jorge Di Paola ABSTRACT Whole exome sequencing on a family with autosomal dominant thrombocytopenia and two occurrences of acute lymphoblastic leukemia (ALL) of unknown cause identified a heterozygous single nucleotide change in the gene ETV6, encoding a p.Pro214Leu amino acid substitution. We screened families with the same phenotype and found mutations in ETV6 in two additional families: one with the identical p.Pro214Leu mutation and one with a p.Arg418Gly substitution. ETV6 is a transcriptional repressor that functions through self-oligomerization and is essential for hematopoietic stem cell proliferation and platelet production in mice. Our report was among the first to describe pathogenic germline mutations in ETV6. Functional characterization of the corresponding ETV6 protein mutations showed aberrant cytoplasmic localization, impaired transcriptional repression, and delayed megakaryocyte maturation. Furthermore, we found that mutant ETV6 protein oligomerizes with WT ETV6 and sequesters it from the nucleus suggesting a dominant negative mechanism.
    [Show full text]
  • Global Analysis of Estrogen Receptor Beta Binding to Breast Cancer Cell Genome Reveals an Extensive Interplay with Estrogen Rece
    Grober et al. BMC Genomics 2011, 12:36 http://www.biomedcentral.com/1471-2164/12/36 RESEARCHARTICLE Open Access Global analysis of estrogen receptor beta binding to breast cancer cell genome reveals an extensive interplay with estrogen receptor alpha for target gene regulation Oli MV Grober1†, Margherita Mutarelli1†, Giorgio Giurato1, Maria Ravo1,2, Luigi Cicatiello1, Maria Rosaria De Filippo1, Lorenzo Ferraro1, Giovanni Nassa1,2, Maria Francesca Papa1, Ornella Paris1, Roberta Tarallo1,2, Shujun Luo3, Gary P Schroth3, Vladimir Benes4, Alessandro Weisz1,2* Abstract Background: Estrogen receptors alpha (ERa) and beta (ERb) are transcription factors (TFs) that mediate estrogen signaling and define the hormone-responsive phenotype of breast cancer (BC). The two receptors can be found co-expressed and play specific, often opposite, roles, with ERb being able to modulate the effects of ERa on gene transcription and cell proliferation. ERb is frequently lost in BC, where its presence generally correlates with a better prognosis of the disease. The identification of the genomic targets of ERb in hormone-responsive BC cells is thus a critical step to elucidate the roles of this receptor in estrogen signaling and tumor cell biology. Results: Expression of full-length ERb in hormone-responsive, ERa-positive MCF-7 cells resulted in a marked reduction in cell proliferation in response to estrogen and marked effects on the cell transcriptome. By ChIP-Seq we identified 9702 ERb and 6024 ERa binding sites in estrogen-stimulated cells, comprising sites occupied by either ERb,ERa or both ER subtypes. A search for TF binding matrices revealed that the majority of the binding sites identified comprise one or more Estrogen Response Element and the remaining show binding matrixes for other TFs known to mediate ER interaction with chromatin by tethering, including AP2, E2F and SP1.
    [Show full text]
  • A Core Erythroid Transcriptional Network Is Repressed by a Master Regulator of Myelo-Lymphoid Differentiation
    A core erythroid transcriptional network is repressed by a master regulator of myelo-lymphoid differentiation Sandeep N. Wontakala,1, Xingyi Guob,1, Cameron Smithc,2, Thomas MacCarthyc,d,2, Emery H. Bresnicke, Aviv Bergmanc, Michael P. Snyderf,g, Sherman M. Weissmanh,3, Deyou Zhengb,i,3, and Arthur I. Skoultchia,3 Departments of aCell Biology, bNeurology, cSystems and Computational Biology, and iGenetics and Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461; dDepartment of Applied Mathematics and Statistics, State University of New York, Stony Brook, NY 11794; eWisconsin Institutes for Medical Research, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705; hDepartment of Genetics, Yale University School of Medicine, New Haven, CT 06520; fDepartment of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520; and gDepartment of Genetics, Stanford University, Stanford, CA 94305 Contributed by Sherman M. Weissman, December 29, 2011 (sent for review October 10, 2011) Two mechanisms that play important roles in cell fate decisions are PU.1, is a negative regulator of terminal erythroid differentiation control of a “core transcriptional network” and repression of alter- (16–19). Surprisingly, PU.1 was found to occupy more genes in native transcriptional programs by antagonizing transcription fac- erythroid progenitors than the three erythroid-promoting factors tors. Whether these two mechanisms operate together is not (20). However, the extent of overlap between the genes bound by known. Here we report that GATA-1, SCL, and Klf1 form an erythroid PU.1 and the three erythroid factors is not known. core transcriptional network by co-occupying >300 genes.
    [Show full text]
  • Roles of Krüppel Like Factors Klf1, Klf2, and Klf4 in Embryonic Beta-Globin Gene Expression
    Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2009 ROLES OF KRÜPPEL LIKE FACTORS KLF1, KLF2, AND KLF4 IN EMBRYONIC BETA-GLOBIN GENE EXPRESSION Yousef Alhashem Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Medical Genetics Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/1880 This Thesis is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. Virginia Commonwealth University School of Medicine This is to certify that the thesis prepared by Yousef N. Alhashem entitled “Roles Of Krüppel Like Factors KLF1, KLF2, And KLF4 In Embryonic Beta-Globin Gene Expression” has been approved by the student advisory committee as a satisfactory for the completion of the thesis reqirement for the degree of Master of Science. _____________________________________________ Joyce A. Lloyd, Ph.D., Director of Thesis, School of Medicine _____________________________________________ Rita Shiang, Ph.D., School of Medicine _____________________________________________ David C. Williams Jr., Ph.D., School of Medicine _____________________________________________ Paul B. Fisher, Ph.D., Chair, Department of Human and Molecular Genetics _____________________________________________ Jerome F. Strauss, III, M.D., Ph.D., Dean, School of Medicine _____________________________________________ F. Douglas Boudinot, Ph.D., Dean, Graduate School _____________________________________________ Date © Yousef Nassir Alhashem 2009 ROLES OF KRÜPPEL LIKE FACTORS KLF1, KLF2, AND KLF4 IN EMBRYONIC BETA-GLOBIN GENE EXPRESSION A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University By Yousef Nassir Alhashem, B.Sc.
    [Show full text]
  • Protective Role of Estrogen Against Excessive Erythrocytosis in Monge's
    Azad et al. Experimental & Molecular Medicine (2021) 53:125–135 https://doi.org/10.1038/s12276-020-00550-2 Experimental & Molecular Medicine ARTICLE Open Access Protective role of estrogen against excessive erythrocytosis in Monge’s disease Priti Azad1, Francisco C. Villafuerte2, Daniela Bermudez2,GargiPatel1 and Gabriel G. Haddad1,3,4 Abstract Monge’s disease (chronic mountain sickness (CMS)) is a maladaptive condition caused by chronic (years) exposure to high-altitude hypoxia. One of the defining features of CMS is excessive erythrocytosis with extremely high hematocrit levels. In the Andean population, CMS prevalence is vastly different between males and females, being rare in females. Furthermore, there is a sharp increase in CMS incidence in females after menopause. In this study, we assessed the role of sex hormones (testosterone, progesterone, and estrogen) in CMS and non-CMS cells using a well-characterized in vitro erythroid platform. While we found that there was a mild (nonsignificant) increase in RBC production with testosterone, we observed that estrogen, in physiologic concentrations, reduced sharply CD235a+ cells (glycophorin A; a marker of RBC), from 56% in the untreated CMS cells to 10% in the treated CMS cells, in a stage-specific and dose- responsive manner. At the molecular level, we determined that estrogen has a direct effect on GATA1, remarkably decreasing the messenger RNA (mRNA) and protein levels of GATA1 (p < 0.01) and its target genes (Alas2, BclxL, and Epor, p < 0.001). These changes result in a significant increase in apoptosis of erythroid cells. We also demonstrate that estrogen regulates erythropoiesis in CMS patients through estrogen beta signaling and that its inhibition can diminish the effects of estrogen by significantly increasing HIF1, VEGF, and GATA1 mRNA levels.
    [Show full text]
  • Baicalin Inhibits Recruitment of GATA1 to the HSD3B2 Promoter and Reverses Hyperandrogenism of PCOS
    240 3 Journal of J Yu, Y Liu et al. Baicalin for PCOS 240:3 497–507 Endocrinology RESEARCH Baicalin inhibits recruitment of GATA1 to the HSD3B2 promoter and reverses hyperandrogenism of PCOS Jin Yu1,*, Yuhuan Liu2,*, Danying Zhang1, Dongxia Zhai1, Linyi Song1, Zailong Cai3 and Chaoqin Yu1 1Department of Gynecology of Traditional Chinese Medicine, Changhai Hospital, Naval Medical University, Shanghai, China 2Department of Gynecology and Obstetrics, Changhai Hospital, Naval Medical University, Shanghai, China 3Department of Biochemistry and Molecular Biology, Naval Medical University, Shanghai, China Correspondence should be addressed to C Yu or Z Cai: [email protected] or [email protected] *(J Yu and Y Liu contributed equally to this work) Abstract High androgen levels in patients suffering from polycystic ovary syndrome (PCOS) Key Words can be effectively reversed if the herbScutellaria baicalensis is included in traditional f baicalin Chinese medicine prescriptions. To characterize the effects of baicalin, extracted from f polycystic ovary syndrome S. baicalensis, on androgen biosynthesis in NCI-H295R cells and on hyperandrogenism f hyperandrogenism in PCOS model rats and to elucidate the underlying mechanisms. The optimum f HSD3B2 concentration and intervention time for baicalin treatment of NCI-H295R cells were f GATA1 determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and ELISA. The functional genes affected by baicalin were studied by gene expression profiling (GEP), and the key genes were identified using a dual luciferase assay, RNA interference technique and genetic mutations. Besides, hyperandrogenic PCOS model rats were induced and confirmed before and after baicalin intervention. As a result, baicalin decreased the testosterone concentrations in a dose- and time-dependent manner in NCI-H295R cells.
    [Show full text]