Page 47 - 2019_12-Haematologica-web

Red Cell Biology & its Disorders
Disruption of the MBD2-NuRD complex but not MBD3-NuRD induces high level HbF expression in human adult erythroid cells
Xiaofei Yu,1* Alexander Azzo,1,2,3* Stephanie M. Bilinovich,4 Xia Li, 1,5 Mikhail Dozmorov,4 Ryo Kurita,7 Yukio Nakamura,7 David C. Williams Jr.4 and Gordon D. Ginder 1,5,8,9
*XY and AA contributed equally to this work
ABSTRACT
As high fetal hemoglobin levels ameliorate the underlying pathophys- iological defects in sickle cell anemia and beta (β)-thalassemia, understanding the mechanisms that enforce silencing of fetal hemo- globin postnatally offers the promise of effective molecular therapy. Depletion of the Nucleosome Remodeling and Deacetylase complex mem- ber MBD2 causes a 10-20-fold increase in g-globin gene expression in adult β-globin locus yeast artificial chromosome transgenic mice. To determine the effect of MBD2 depletion in human erythroid cells, genome editing technology was utilized to knockout MBD2 in Human Umbilical cord Derived Erythroid Progenitor-2 cells resulting in g/g+β mRNA levels of approximately 50% and approximately 40% fetal hemoglobin by high per- formance liquid chromatography. In contrast, MBD3 knockout had no appreciable effect on g-globin expression. Knockdown of MBD2 in primary adult erythroid cells consistently increased g/g+β mRNA ratios by approxi- mately 10-fold resulting in approximately 30-40% g/g+β mRNA levels and a corresponding increase in g-globin protein. MBD2 exerts its repressive effects through recruitment of the chromatin remodeler CHD4 via a coiled- coil domain, and the histone deacetylase core complex via an intrinsically disordered region. Enforced expression of wild-type MBD2 in MBD2 knockout cells caused a 5-fold decrease in g-globin mRNA while neither the coiled-coil mutant nor the intrinsically disordered region mutant MBD2 proteins had an inhibitory effect. Co-immunoprecipitation assays showed that the coiled-coil and intrinsically disorder region mutations disrupt com- plex formation by dissociating the CHD4 and the histone deacetylase core complex components, respectively. These results establish the MBD2 Nucleosome Remodeling and Deacetylase complex as a major silencer of fetal hemoglobin in human erythroid cells and point to the coiled-coil and intrinsically disordered region of MBD2 as potential therapeutic targets.
Introduction
Both sickle cell disease (SCD) and beta (β)-thalassemia result from genetic defects in β-globin production. SCD, which results from a single glutamic acid to valine substitution in the β-globin chain, is the most common inherited blood disorder in the US, affecting approximately 100,000 Americans, as well as millions of people worldwide, most of whom live in underdeveloped nations.1,2 The vascular seque-
Ferrata Storti Foundation
Haematologica 2019 Volume 104(12):2361-2371
1Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA;
2Center for Clinical and Translational Research, PhD Program in Cancer and Molecular Medicine, Virginia Commonwealth University, Richmond, VA, USA; 3MD-PhD Program, Virginia Commonwealth University, Richmond, VA, USA; 4Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC, USA; 5Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA, USA; 6Department of Biostatistics, Virginia Commonwealth University, Richmond, VA, USA; 7Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan; 8Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA, USA and 9Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA, USA
Correspondence:
GORDON D. GINDER
gdginder@vcu.edu
Received: November 2, 2018. Accepted: April 15, 2019. Pre-published: April 19, 2019.
doi:10.3324/haematol.2018.210963
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/104/12/2361
©2019 Ferrata Storti Foundation
Material published in Haematologica is covered by copyright. All rights are reserved to the Ferrata Storti Foundation. Use of published material is allowed under the following terms and conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode. Copies of published material are allowed for personal or inter- nal use. Sharing published material for non-commercial pur- poses is subject to the following conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode, sect. 3. Reproducing and sharing published material for com- mercial purposes is not allowed without permission in writing from the publisher.
haematologica | 2019; 104(12)
2361
ARTICLE