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Chromatin Remodelling Factor SMARCD2 Regulates Transcriptional Networks Controlling Differentiation of Neutrophil Granulocytes DOI: 10.1038/Ng.3833

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Chromatin Remodelling Factor SMARCD2 Regulates Transcriptional Networks Controlling Differentiation of Neutrophil Granulocytes DOI: 10.1038/Ng.3833 The University of Manchester Research Chromatin remodelling factor SMARCD2 regulates transcriptional networks controlling differentiation of neutrophil granulocytes DOI: 10.1038/ng.3833 Document Version Accepted author manuscript Link to publication record in Manchester Research Explorer Citation for published version (APA): Witzel, M., Petersheim, D., Fan, Y., Bahrami, E., Racek, T., Rohlfs, M., Puchalka, J., Mertes, C., Gagneur, J., Ziegenhain, C., Enard, W., Stray-Pedersen, A., Arkwright, P., Abboud, M. R., Pazhakh, V., Lieschke, G. J., Krawitz, P. M., Dahlhoff, M., Schneider, M. R., ... Klein, C. (2017). Chromatin remodelling factor SMARCD2 regulates transcriptional networks controlling differentiation of neutrophil granulocytes. Nature Genetics, 49(5), 742-752. https://doi.org/10.1038/ng.3833 Published in: Nature Genetics Citing this paper Please note that where the full-text provided on Manchester Research Explorer is the Author Accepted Manuscript or Proof version this may differ from the final Published version. If citing, it is advised that you check and use the publisher's definitive version. General rights Copyright and moral rights for the publications made accessible in the Research Explorer are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Takedown policy If you believe that this document breaches copyright please refer to the University of Manchester’s Takedown Procedures [http://man.ac.uk/04Y6Bo] or contact [email protected] providing relevant details, so we can investigate your claim. Download date:10. Oct. 2021 Chromatin remodelling factor SMARCD2 regulates transcriptional networks controlling differentiation of neutrophil granulocytes 1,2 1 1 1 1 Maximilian Witzel , Daniel Petersheim , Yanxin Fan , Ehsan Bahrami , Tomas Racek , Meino 1 1§ 2 2,3 4 Rohlfs , Jacek Puchałka , Christian Mertes , Julien Gagneur , Christoph Ziegenhain , Wolfgang 4 5 6 7 8 Enard , Asbjørg Stray-Pedersen , Peter D. Arkwright , Miguel R. Abboud , Vahid Pazhakh , Graham 8 9 10 10 10 J. Lieschke , Peter M. Krawitz , Maik Dahlhoff , Marlon R. Schneider , Eckhard Wolf , Hans-Peter Horny11, Heinrich Schmidt1, Alejandro A. Schäffer12 and Christoph Klein1,2 1Department of Pediatrics, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-Universität München, Munich, Germany 2Gene Center Munich, Ludwig-Maximilians-Universität München, Munich, Germany 3Department of Informatics, Technical University of Munich, Munich, Germany 4Anthropology and Human Genomics, Department Biology II, Faculty of Biology, Ludwig-Maximilians- Universität München, Munich, Germany 5Norwegian National Unit for Newborn Screening, Oslo University Hospital, Oslo, Norway 6 University of Manchester, Department of Paediatric Allergy & Immunology, Royal Manchester Children's Hospital, Manchester, UK 7Department of Pediatrics and Adolescent Medicine, American University of Beirut Medical Center, Beirut, Lebanon 8Australian Regenerative Medicine Institute, Monash University, Clayton, Australia 9Medical Genetics and Human Genetic, Charite University Hospital, Berlin, Germany 10Molecular Animal Breeding and Biotechnology, Gene Center Ludwig-Maximilians-Universität München, Munich, Germany 11Pathology Institute, Faculty of Medicine, Ludwig-Maximilians-Universität München, Munich, Germany 12National Center for Biotechnology Information, National Institutes of Health, Department of Health and Human Services, Bethesda, MD USA §Deceased (in September 2015) 1 Correspondence: Christoph Klein MD PhD Department of Pediatrics Dr. von Hauner Children’s Hospital Ludwig-Maximilians-Universität München Lindwurmstrasse 4 D-80337 Munich Tel +49-89-4400 5 7700 Fax +49-89-4400 5 7703 Email [email protected] Key words: Hematopoiesis, primary immunodeficiency disorder, SWI/SNF complex, specific granule deficiency, leukemogenesis, congenital neutropenia 2 1 Abstract 2 Differentiation of hematopoietic stem cells follows a hierarchical program of 3 transcription factor regulated events1-3. Early myeloid cell differentiation is dependent 4 on PU.1 and CEBPA (CCAAT/enhancer binding protein alpha), late myeloid 5 differentiation is orchestrated by CEBPE (CCAAT/enhancer binding protein epsilon)4. 6 The influence of SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin 7 remodelling factors as novel master regulators of hematopoietic differentiation is only 8 beginning to be explored3,5,6. Here, we identify SMARCD2 (SWI/SNF related, matrix 9 associated, actin dependent regulator of chromatin, subfamily d, member 2) as a 10 critical regulator of myeloid differentiation in humans, mice, and zebrafish. Studying 11 patients from three unrelated pedigrees characterized by neutropenia, specific granule 12 deficiency, myelodysplasia with excess of blast cells and various developmental 13 aberrations, we identified three loss-of-function mutations in SMARCD2. Using mice 14 and zebrafish as model systems, we showed that SMARCD2 controls early steps in the 15 differentiation of myeloid-erythroid progenitor cells. In vitro, SMARCD2 interacts 16 with the transcription factor CEBPE and controls expression of neutrophil proteins 17 stored in specific granules. Defective expression of SMARCD2 leads to transcriptional 18 and chromatin changes in acute myeloid leukemia (AML) human promyelocytic cells. 19 In summary, SMARCD2 is a key factor controlling myelopoiesis and a potential 20 tumor suppressor in leukemia. 21 22 Article 23 24 Differentiation of hematopoietic cells is controlled by transcription-factor mediated 25 instructive events and less well-defined permissive events orchestrated by a variety of 26 epigenetic modulators7,8. Dynamic chromatin remodelling adds another level of complexity. 3 27 Embedding of promoter DNA into nucleosome landscapes restricts accessibility of cognate 28 binding sites to transcription factors and restricts gene expression 9-11. The SWI/SNF complex 29 is composed of multimeric units that use energy derived from ATP hydrolysis to unwrap or 30 restructure nucleosomes12. SMARCD2 is a component of the SWI/SNF complex in 31 hematopoietic stem cells (HSC) and other hematopoietic cells6,13,14. The two paralogous 32 proteins, SMARCD1 (BAF60A) and SMARCD3 (BAF60C), control embryonic stem cell15 33 and heart muscle cell differentiation16, respectively. 34 Here, we investigated three independent pedigrees with four patients who presented as 35 neonates with delayed separation of umbilical cord and subsequently developed severe 36 bacterial infections associated with neutropenia, parasitosis or chronic diarrhea (Table S1). 37 Extrahematopoietic findings included mild to moderate developmental delay and dysmorphic 38 features (Figure S1, Table S1). The bone marrow of patients showed hypercellularity, paucity 39 of neutrophil granulocytes, dysplastic features (Figure 1), and progressive development of 40 myelodysplasia (Figure 1, S2). Neutrophil granulocytes were characterized by absence of 41 granules (Figure S3). 42 In search of the underlying genetic defect, we performed homozygosity mapping and whole 43 exome sequencing (WES), followed by Sanger sequencing of patients and family members 44 (see Supplementary Materials & Methods for details). Homozygosity mapping identified an 45 especially large perfect marker interval of over 50Mbp in family A on chromosome 17; 46 within this interval, family B had two non-adjacent perfect intervals spanning 1.8Mbp and 47 0.5Mbp. The asymptotic LOD scores for these intervals are +4.2 (+1.8 for A and +2.4 for B) 48 and peak observed LOD scores with a more realistic disease haplotype frequency of 0.05, are 49 3.0 (+1.2, +1.8). There were approximately 36 genes located in the two shared intervals, 50 including SMARCD2. 51 4 52 We identified distinct segregating homozygous mutations in SMARCD2 in all three pedigrees 53 (Figure 2a, b, c). Mutations are described by their putative effect on transcript SMARCD2-001 54 (ENST00000448276). Effects on hypothetical transcripts are shown in Table S2. At the DNA 55 level, the mutations in pedigrees A and C affect splice sites, while the mutation in pedigree B 56 is a duplication of 25bp, leading to a frameshift and premature termination (Table S2). 57 Western blot analyses showed an absence of SMARCD2 protein in patient cells (Figure 2d). 58 To confirm that the SMARCD2 mutations lead to a loss of function, we sequenced reverse 59 transcribed mRNA from patient cells (Figure 2e) and determined their putatively encoded 60 proteins. We then cloned 2 isoforms of patient AII.1 (AII.1a: p.Ile362Cysfs*3 and AII1b: 61 p.Ser394Argfs*1), one isoform of patient BII.1. (BII.1: p.Gln147Glufs*5) and one isoform of 62 patient CII.1 (CII.1: p.Arg73Valfs*8). FLAG-tagged expression vectors carrying mutated 63 SMARCD2 versions and a red fluorescence protein gene separated by an Internal Ribosomal 64 Entry Sequence (IRES.RFP) were transfected into 293T cells and were investigated for co- 65 immunoprecipitation with native SWI/SNF core members. As shown in Figure 2f, only the 66 wild-type version of SMARCD2 was able to co-precipitate with SMARCA4 (BRG1), 67 SMARCC2 (BAF170), SMARCC1 (BAF155), and SMARCB1 (BAF47); none of the mutant 68 versions were able to co-precipitate with any of these proteins, suggesting that the mutations 69 constitute loss-of-function alleles. 70 71 Since all SMARCD2-deficient patients had either been subjected to allogeneic HSCT or had
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