Arid2 regulates hematopoietic stem cell differentiation in normal hematopoiesis

Abstract

•Loss of Arid2 has largely no effect on steady-state hematopoiesis.•Arid2 loss leads to impairment in lymphoid lineage regeneration.•Arid2 null HSPCs exhibit enrichment of myeloid-biased MPP signature.•Loss of Arid2 leads to upregulation of inflammatory pathways.•Arid2 null cells have proliferation defects under lymphoid condition with LPS. The switch/sugar nonfermenting (SWI/SNF) family of chromatin remodeling complexes have been implicated in normal hematopoiesis. The ARID2 protein is a component of the polybromo-associated BAF (PBAF), one of the two main SWI/SNF complexes. In the current study, we used a conditional Arid2 knockout mouse model to determine its role in normal hematopoiesis. We found that the loss of Arid2 has no discernable effects on steady-state hematopoiesis, with the exception of a modest effect on erythropoiesis. On bone marrow transplantation, however, the loss of Arid2 affects HSC differentiation in a cell-autonomous manner, resulting in significant decreases in the ability to reconstitute the lymphoid lineage. Gene expression analysis of Arid2 knockout cells revealed enrichment of myeloid-biased multipotent progenitor (MPP) cell signatures, while the lymphoid-biased MPPs are enriched in the wild type, consistent with the observed phenotype. Moreover, Arid2 knockout cells revealed enrichment of inflammatory pathways with upregulation of TLR receptors, as well as downstream signaling cascade genes. Furthermore, under lymphocyte-biased growth conditions in vitro, Arid2 null bone marrow cells have significantly impaired proliferation, which decreased further on lipopolysaccharide stimulation. Overall, these data suggest that the loss of Arid2 impairs HSC differentiation ability, and this effect may be mediated through upregulation of inflammatory pathways. The switch/sugar nonfermenting (SWI/SNF) family of chromatin remodeling complexes have been implicated in normal hematopoiesis. The ARID2 protein is a component of the polybromo-associated BAF (PBAF), one of the two main SWI/SNF complexes. In the current study, we used a conditional Arid2 knockout mouse model to determine its role in normal hematopoiesis. We found that the loss of Arid2 has no discernable effects on steady-state hematopoiesis, with the exception of a modest effect on erythropoiesis. On bone marrow transplantation, however, the loss of Arid2 affects HSC differentiation in a cell-autonomous manner, resulting in significant decreases in the ability to reconstitute the lymphoid lineage. Gene expression analysis of Arid2 knockout cells revealed enrichment of myeloid-biased multipotent progenitor (MPP) cell signatures, while the lymphoid-biased MPPs are enriched in the wild type, consistent with the observed phenotype. Moreover, Arid2 knockout cells revealed enrichment of inflammatory pathways with upregulation of TLR receptors, as well as downstream signaling cascade genes. Furthermore, under lymphocyte-biased growth conditions in vitro, Arid2 null bone marrow cells have significantly impaired proliferation, which decreased further on lipopolysaccharide stimulation. Overall, these data suggest that the loss of Arid2 impairs HSC differentiation ability, and this effect may be mediated through upregulation of inflammatory pathways. Adult hematopoiesis originates from the hematopoietic stem cells (HSCs), which reside in the bone marrow. HSCs are multipotent, adult stem cells with the hallmark ability to self-renew and differentiate into all blood lineages. Epigenetic regulators have been reported to play important roles in HSC function, and their dysregulation leads to hematological malignancies [1Lessard JA Crabtree GR Chromatin regulatory mechanisms in pluripotency.Annu Rev Cell Dev Biol. 2010; 26: 503-532Crossref PubMed Scopus (135) Google Scholar]. These includes the switch/sugar nonfermenting (SWI/SNF) family of chromatin remodeling complexes [2Wilson BG Roberts CWM SWI/SNF nucleosome remodellers and cancer.Nat Rev Cancer. 2011; 11: 481-492Crossref PubMed Scopus (821) Google Scholar, 3Shain AH Pollack JR The spectrum of SWI/SNF mutations, ubiquitous in human cancers.PLoS One. 2013; 8: e55119Crossref PubMed Scopus (327) Google Scholar, 4Buscarlet M Krasteva V Ho L et al.Essential role of BRG, the ATPase subunit of BAF chromatin remodeling complexes, in leukemia maintenance.Blood. 2014; 123: 1720-1728Crossref PubMed Scopus (69) Google Scholar]. In humans, the SWI/SNF family of chromatin remodelers consists of two main protein complexes, BRG1-associated factor (BAF) and Polybromo-associated BAF (PBAF). These protein complexes are evolutionary conserved from Saccharomyces cerevisiae and Drosophila melanogaster to humans [5Neigeborn L Carlson M Genes affecting the regulation of SUC2 gene expression by glucose repression in Saccharomyces cerevisiae.Genetics. 1984; 108: 845-858Crossref PubMed Google Scholar,6Tang L Nogales E Ciferri C Structure and function of SWI/SNF chromatin remodeling complexes and mechanistic implications for transcription.Prog Biophys Mol Biol. 2010; 102: 122-128Crossref PubMed Scopus (129) Google Scholar]. The human orthologous complexes share a conserved core set of nine proteins, including the catalytic ATPases and Brahma-related gene 1 (BRG1) or Brahma (BRM) and are distinguished by unique AT-rich interactive domain-containing (ARID) proteins. ARID1A and ARID1B (BAF250A and BAF250B, respectively) are mutually exclusive to BAF, while ARID2 (BAF200) is unique to PBAF. Protein Polybromo 1 (PBRM1/BAF180) and bromodomain containing 7 (BRD7) additionally distinguish PBAF from BAF [7Imbalzano AN Kwon H Green MR Kingston RE Facilitated binding of TATA-binding protein to nucleosomal DNA.Nature. 1994; 370: 481-485Crossref PubMed Scopus (512) Google Scholar, 8Kwon H Imbalzano AN Khavari PA Kingston RE Green MR Nucleosome disruption and enhancement of activator binding by a human SW1/SNF complex.Nature. 1994; 370: 477-481Crossref PubMed Scopus (622) Google Scholar, 9Phelan ML Sif S Narlikar GJ Kingston RE Reconstitution of a core chromatin remodeling complex from SWI/SNF subunits.Mol Cell. 1999; 3: 247-253Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar, 10Wang W Côté J Xue Y et al.Purification and biochemical heterogeneity of the mammalian SWI–SNF complex.EMBO J. 1996; 15: 5370-5382Crossref PubMed Scopus (651) Google Scholar, 11Wang X Nagl NG Wilsker D et al.Two related ARID family proteins are alternative subunits of human SWI/SNF complexes.Biochem J. 2004; 383: 319-325Crossref PubMed Scopus (124) Google Scholar, 12Kaeser MD Aslanian A Dong MQ Yates JR Emerson BM BRD7, a novel PBAF-specific SWI/SNF subunit, is required for target gene activation and repression in embryonic stem cells.J Biol Chem. 2008; 283: 32254-32263Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 13Yan Z Cui K Murray DM et al.PBAF chromatin-remodeling complex requires a novel specificity subunit, BAF200, to regulate expression of selective interferon-responsive genes.Genes Dev. 2005; 19: 1662-1667Crossref PubMed Scopus (176) Google Scholar]. SWI/SNF complexes bind to nucleosomes and use ATP to disrupt nucleosome–DNA binding interactions. This disruption alters a nucleosome position by sliding or ejecting the nucleosome from chromatin, affecting biological processes using chromatin as template, including that of gene transcription [2Wilson BG Roberts CWM SWI/SNF nucleosome remodellers and cancer.Nat Rev Cancer. 2011; 11: 481-492Crossref PubMed Scopus (821) Google Scholar,14Saha A Wittmeyer J Cairns BR Chromatin remodelling: the industrial revolution of DNA around histones.Nat Rev Mol Cell Biol. 2006; 7: 437-447Crossref PubMed Scopus (425) Google Scholar,15Lorch Y Maier-Davis B Kornberg RD Mechanism of chromatin remodeling.Proc Natl Acad Sci USA. 2010; 107: 3458-3462Crossref PubMed Scopus (78) Google Scholar]. SWI/SNF subunits have been reported to play important roles in normal hematopoiesis, including that of BAF53, BAF45A, and BAF180 in HSC function [16Krasteva V Buscarlet M Diaz-Tellez A Bernard MA Crabtree GR Lessard JA The BAF53a subunit of SWI/SNF-like BAF complexes is essential for hemopoietic stem cell function.Blood. 2012; 120: 4720-4732Crossref PubMed Scopus (71) Google Scholar, 17Krasteva V Crabtree GR Lessard JA The BAF45a/PHF10 subunit of SWI/SNF-like chromatin remodeling complexes is essential for hematopoietic stem cell maintenance.Exp Hematol. 2017; 48: 58-71Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 18Lee H Dai F Zhuang L et al.BAF180 regulates cellular senescence and hematopoietic stem cell homeostasis through p21.Oncotarget. 2016; 7: 19134-19146Crossref PubMed Scopus (32) Google Scholar]; BRG1 in erythroid development and myeloid differentiation [19Bultman SJ Gebuhr TC Magnuson T A Brg1 mutation that uncouples ATPase activity from chromatin remodeling reveals an essential role for SWI/SNF-related complexes in β-globin expression and erythroid development.Genes Dev. 2005; 19: 2849-2861Crossref PubMed Scopus (125) Google Scholar,20Vradii D Wagner S Doan DN et al.Brg1, the ATPase subunit of the SWI/SNF chromatin remodeling complex, is required for myeloid differentiation to granulocytes.J Cell Physiol. 2006; 206: 112-118Crossref PubMed Scopus (41) Google Scholar]; SMARCD2 in granulopoiesis [21Priam P Krasteva V Rousseau P et al.SMARCD2 subunit of SWI/SNF chromatin-remodeling complexes mediates granulopoiesis through a CEBPε dependent mechanism.Nat Genet. 2017; 49: 753-764Crossref PubMed Scopus (33) Google Scholar]; and BRG1, BAF57, and BAF155 in lymphopoiesis [22Chi TH Wan M Zhao K et al.Reciprocal regulation of CD4/CD8 expression by SWI/SNF-like BAF complexes.Nature. 2002; 418: 195-199Crossref PubMed Scopus (205) Google Scholar]. In this study, we characterized the hematopoietic phenotype of the loss of Arid2, one of the proteins that distinguishes the PBAF from the BAF complex. Whole-body homozygous loss of Arid2 is embryonic lethal at E12.5–E14.5 because of congenital heart defects [23He L Tian X Zhang H et al.BAF200 is required for heart morphogenesis and coronary artery development.PLoS One. 2014; 9e109493Crossref PubMed Scopus (20) Google Scholar]. Arid2 has been found to have tissue-specific functions by modulating specific gene expression important in osteoblast differentiation [24Xu F Flowers S Moran E Essential role of ARID2 protein-containing SWI/SNF complex in tissue-specific gene expression.J Biol Chem. 2012; 287: 5033Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar]. Additionally, ARID2 alters cell cycle entry of hematoma cells. ARID2 knockdown in HepG2 cells leads to accelerated G1-to-S transition [25Duan Y Tian L Gao Q et al.Chromatin remodeling gene ARID2 targets cyclin D1 and cyclin E1 to suppress hepatoma cell progression.Oncotarget. 2016; 7: 45863-45875Crossref PubMed Scopus (20) Google Scholar]. Using a conditional Arid2 knockout mouse model, we showed that loss of Arid2 resulted in impairment of lymphoid lineage regeneration and correlated with transcriptional upregulation of inflammatory pathways. A full description of mouse experiments, bone marrow transplantation, flow cytometry and sorting, [3H]thymidine incorporation, lipopolysaccharide (LPS) stimulation and immunoblotting, colony formation assay, RNA sequencing, quantitative reverse transcription polymerase chain reaction (RT-qPCR), data analysis, statistical methods, list of primers (Supplementary Table E1), flow cytometry antibodies (Supplemental Table E2, and immunoblotting antibodies (Supplementary Table E3) are included in the Supplementary materials (online only, available at www.exphem.org). We examined expression of Arid2 mRNA level using a published database, which indicated that Arid2 is expressed relatively evenly across all hematopoietic lineages (Supplementary Figure E1, online only, available at www.exphem.org) [26Lara-Astiaso D Weiner A Lorenzo-Vivas E et al.Immunogenetics: chromatin state dynamics during blood formation.Science. 2014; 345: 943-949Crossref PubMed Scopus (479) Google Scholar]. To understand the role of Arid2 during normal hematopoiesis, we obtained a transgenic Arid2 knockout-first allele mouse (derived on the C57/BL6 background) from The European Mouse Mutant Archive (EMMA) in which exon 4 of ARID2 (Arid2tm1a/+) is flanked by LoxP sites [27INFRAFRONTIER Consortium. INFRAFRONTIER—providing mutant mouse resources as research tools for the international scientific community.Nucleic Acids Res. 2015; 43: D1171-D1175Crossref PubMed Scopus (22) Google Scholar,28Raess M de Castro AA Gailus-Durner V Fessele S Hrabě de Angelis M INFRAFRONTIER ConsortiumINFRAFRONTIER: a European resource for studying the functional basis of human disease.Mamm Genome. 2016; 27: 445-450Crossref PubMed Scopus Google Scholar]. These with which the to the gene flanked by The to by the or the of Arid2 and exon 4 in a and of an The during embryonic development of hematopoietic and cells P S H et transgenic a for in hematopoietic and 2002; PubMed Scopus Google Scholar]. are with normal and we expression is in hematopoietic and bone marrow cells by of T P D J of the implications for of normal and Cell 2016; 7: Full Text Full Text PDF PubMed Scopus Google Scholar]. in bone marrow is by by and that in the is by bone marrow is on of Arid2 To the effect of loss of Arid2 on steady-state hematopoiesis, we blood and BM from and for blood cell and The BM of these further for in hematopoietic stem and progenitor cells in Arid2 null revealed a in blood cell to with decreased to and to a is significantly decreased to in the while no in cell lineages in the and BM Supplementary Figure E2, online only, available at www.exphem.org). no significant in the or cell of including and all and and and downstream including that of and in the BM Arid2 and in the model of Arid2 in the model consistent with the model, with the of a significant in T cells in the to and to and a significant in to and to cells in the BM Overall, loss of Arid2 to with decreases in lymphoid and progenitor during steady-state hematopoiesis in adult To the function of Arid2 null we and transplantation, to by or BM into C57/BL6 and and by flow cytometry analysis of In the as by the of of Arid2 null in the and a modest to at significant in Arid2 null and Supplementary Figure online only, available at www.exphem.org). no significant in the BM or in and myeloid cell for a in to in the BM Additionally, decreases in to and to in the BM BM from into into In the and significantly decreased in Arid2 null with and the further in the to at with the to at Supplementary Figure In the a significant or a to in in the to and to and a consistent significant in to to We significant in or cell lineages in the in the or In the a significant in myeloid to in the as well as a significant in to in the BM Furthermore, a significant in to in the BM To further the effect of the loss of Arid2 on HSC function, we of Arid2 null BM from or at a with BM from and into in Figure Arid2 null cells have decreased as by the in in the with the of Supplementary Figure a significant in and in the and with a in in the The in the at the a which because of a in the a in to and a significant in cells to while the and progenitor exhibit significant these data suggest that the loss of Arid2 in a decreased ability of the with to a ability to differentiate into lymphoid lineage. To that impaired HSC function in Arid2 null BM is to an we and using the expression mouse model T P D J of the implications for of normal and Cell 2016; 7: Full Text Full Text PDF PubMed Scopus Google Scholar]. Arid2 by 4 for to We the of and in the and BM at the in the impaired HSC function in the model with the to the from in the model, we observed significant decreases in Arid2 null and in the to the Supplementary Figure online only, available at www.exphem.org). 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These data suggest that the effect of the loss of Arid2 on the ability of HSPCs is to in or cell cycle To these data suggest that the loss of Arid2 impairs the function of Arid2 null HSCs have decreased differentiation with while HSC is relatively This decreased differentiation is in decreased with lymphoid and decreased ability to differentiate into lymphoid lineages. The effects on the ability of the HSPCs are to impairment in the of these as by the observed in the and To understand the Arid2 function in we analysis by in cells from or Gene expression analysis revealed that of are expressed knockout using an of and the expressed are and are Supplementary Table online only, available at www.exphem.org). of Arid2 in the mRNA for exon 4 RNA Supplementary Figure online only, available at www.exphem.org). The expression of Arid2 the and the conditional is that mRNA (Supplementary Figure We gene set enrichment analysis of the expression data using gene D et of multipotent blood in normal and Cell. 2015; Full Text Full Text PDF PubMed Scopus Google and found enrichment of the myeloid-biased MPP in while the lymphoid-biased MPP enriched in the cells Moreover, using the revealed enrichment inflammatory pathways in including and signaling pathways and the signaling cascade mediated by and which to factor B activation of the including are in while the is (Supplementary and online only, available at www.exphem.org). Additionally, we used to transcription factor in the significant expressed and found enrichment of and the enriched C de B D S an for the of regulatory and Acids Res. 2012; Scopus Google H Z D S enrichment analysis in mouse and Acids Res. 2015; 43: PubMed Scopus Google Scholar]. Furthermore, is an enrichment of target and significant upregulation of in the cells target genes. Scholar]. factor for and as well as an enrichment and upregulation of in the cells Supplementary Figure de et is required for the of multipotent to lymphoid PubMed Scopus Google Scholar]. This is consistent with the erythroid and lymphoid lineage impairment we as have that a expression level impairs erythroid differentiation S D The transcription factor Cell Biol. PubMed Scopus Google as well as and development H of B and development by expression of PubMed Scopus Google AH expression of in hematopoietic T cell development at the cell 2002; Full Text Full Text PDF PubMed Scopus Google Scholar]. we dysregulation of inflammatory signaling the of Arid2 null cell To this we and signaling as well as cell proliferation in the of and stimulation and Arid2 null cells. We examined the level of and stimulation in cells. We significant in (Supplementary Figure online only, available at and the for because of the level of at the in the of LPS. [3H]thymidine revealed that Arid2 null cells have significantly decreased proliferation with their in the of further in cell proliferation of Arid2 null cells with the of of has no effect on the cells Moreover, this effect of is lymphoid as we in myeloid colony formation Arid2 null and cells (Supplementary Figure that these inflammatory in HSC a of hematopoietic stem cell in and 2017; PubMed Scopus Google Scholar, A Y Y L hematopoietic stem cell and lymphoid progenitor cell-autonomous in a model of 2015; PubMed Scopus Google Scholar, H K et signaling in hematopoietic stem cells proliferation Cell. 2017; Full Text Full Text PDF PubMed Scopus Google Scholar, Y S et on hematopoietic progenitor cells 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, T et to a TLR hematopoietic stem cells.J 2011; PubMed Scopus Google and myeloid at the of development C S et stem cells myeloid differentiation at the of Cell Biol. 2016; PubMed Scopus Google these are consistent with observed phenotype of impaired lymphoid ability in the analysis of data in Arid2 null pathways from the in or pathways are in transcription factor significantly enriched transcription factor with enrichment by the expressed in cells C de B D S an for the of regulatory and Acids Res. 2012; Scopus Google H Z D S enrichment analysis in mouse and Acids Res. 2015; 43: PubMed Scopus Google Scholar]. of transcription factor to by in and cells. enrichment of target and target genes. Scholar]. of expressed target in or BM cells from and in or for and with [3H]thymidine for 4 by Figure SWI/SNF have roles in hematopoiesis and function as in malignancies [2Wilson BG Roberts CWM SWI/SNF nucleosome remodellers and cancer.Nat Rev Cancer. 2011; 11: 481-492Crossref PubMed Scopus (821) Google Scholar, 3Shain AH Pollack JR The spectrum of SWI/SNF mutations, ubiquitous in human cancers.PLoS One. 2013; 8: e55119Crossref PubMed Scopus (327) Google Scholar, 4Buscarlet M Krasteva V Ho L et al.Essential role of BRG, the ATPase subunit of BAF chromatin remodeling complexes, in leukemia maintenance.Blood. 2014; 123: 1720-1728Crossref PubMed Scopus (69) Google Scholar]. we used mouse to understand the role of Arid2, a unique subunit of in normal hematopoiesis and found that the loss of Arid2 in and a in B and T cells in adult On hematopoietic examined by transplantation, Arid2 null BM cell-autonomous as by decreased in Supplementary Figure as well as a in in Supplementary Figure Moreover, we decreases in in and The revealed of myeloid-biased in the and in in the BM these data suggest HSC differentiation is impaired on the loss of The differentiation impairment is largely to a in progenitor with in of observed in experiments, are largely the and and Supplementary and the and suggest that the observed Arid2 null and are to an of Arid2 null cells and this study, et L Wan X P et chromatin remodeling subunit normal hematopoiesis and 11: PubMed Scopus Google published their of the hematopoietic phenotype using Arid2 knockout mouse data with their in that observed in steady-state hematopoiesis and decreased differentiation ability of Arid2 null HSPCs in BM during in to their we HSC in the model at Additionally, HSC by and showed at to the relatively as by in ARID2 null and cells. et HSC in their published We RNA to understand the the impairment of Arid2 null RNA analysis of the expressed Arid2 null HSPCs and that in et L Wan X P et chromatin remodeling subunit normal hematopoiesis and 11: PubMed Scopus Google by We to at a the of the analysis and because et use as is required for a statistical analysis of gene RNA data enrichment of the myeloid-biased MPP in Arid2 knockout of lymphoid-biased MPP This is consistent with the observed in the with lymphoid and the lymphoid cells in the we inflammatory including TLR as the in Arid2 knockout cells. Furthermore, we showed that stimulation by a impairs Arid2 null cell proliferation under of inflammatory pathways a for observed lymphoid as activation of TLR pathways has been to to a phenotype a of hematopoietic stem cell in and 2017; PubMed Scopus Google Scholar, A Y Y L hematopoietic stem cell and lymphoid progenitor cell-autonomous in a model of 2015; PubMed Scopus Google Scholar, H K et signaling in hematopoietic stem cells proliferation Cell. 2017; Full Text Full Text PDF PubMed Scopus Google Scholar, Y S et on hematopoietic progenitor cells 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, T et to a TLR hematopoietic stem cells.J 2011; PubMed Scopus Google Scholar, C S et stem cells myeloid differentiation at the of Cell Biol. 2016; PubMed Scopus Google as we observed in Arid2 knockout model, the impaired regeneration ability of HSPCs the lymphoid lineage. In upregulation of may to the phenotype we are required to the functional of the inflammatory pathways and expression in the Arid2 knockout as well as Arid2 regulates pathways through transcriptional The no The for with cell This by the in by from the and the of with with

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