An Erg-driven transcriptional program controls B cell lymphopoiesis

ARTICLE https://doi.org/10.1038/s41467-020-16828-y OPEN 1234567890():,; An Erg-driven transcriptional program controls B cell lymphopoiesis Ashley P. Ng 1,2 ✉, Hannah D. Coughlan2,3, Soroor Hediyeh-zadeh 3, Kira Behrens 1, Timothy M. Johanson2,4, Michael Sze Yuan Low2,4,5, Charles C. Bell 6,7, Omer Gilan6,7, Yih-Chih Chan 6,7, Andrew J. Kueh1,2, Thomas Boudier 2,8, Rebecca Feltham9, Anna Gabrielyan9, Ladina DiRago1, Craig D. Hyland 1, Helen Ierino1, Sandra Mifsud1, Elizabeth Viney1, Tracy Willson1, Mark A. Dawson 6,7,10, Rhys S. Allan 2,4, Marco J. Herold1,2, Kelly Rogers2,8, David M. Tarlinton 11, Gordon K. Smyth 2,3, Melissa J. Davis 2,3, Stephen L. Nutt 2,4 & Warren S. Alexander1,2 B lymphoid development is initiated by the differentiation of hematopoietic stem cells into lineage committed progenitors, ultimately generating mature B cells. This highly regulated process generates clonal immunological diversity via recombination of immunoglobulin V, D and J gene segments. While several transcription factors that control B cell development and V(D)J recombination have been defined, how these processes are initiated and coordinated into a precise regulatory network remains poorly understood. Here, we show that the transcription factor ETS Related Gene (Erg) is essential for early B lymphoid differentiation. Erg initiates a transcriptional network involving the B cell lineage defining genes, Ebf1 and Pax5, which directly promotes expression of key genes involved in V(D)J recombination and formation of the B cell receptor. Complementation of Erg deficiency with a productively rearranged immunoglobulin gene rescued B lineage development, demonstrating that Erg is an essential and stage-specific regulator of the gene regulatory network controlling B lymphopoiesis. 1 Blood Cells and Blood Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia. 2 Department of Medical Biology, The University of Melbourne, Parkville, VIC 3010, Australia. 3 Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia. 4 Immunology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia. 5 Monash Haematology, Monash Hospital, Clayton, VIC 3004, Australia. 6 Peter MacCallum Cancer Centre, Parkville, VIC 3000, Australia. 7 Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC 3010, Australia. 8 Advanced Technology and Biology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia. 9 Inflammation Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3010, Australia. 10 Centre for Cancer Research, The University of Melbourne, Parkville, VIC 3010, Australia. 11 Department of Immunology and Pathology, Monash University, Melbourne, VIC 3004, Australia. ✉email: NATURE COMMUNICATIONS | (2020)11:3013 | https://doi.org/10.1038/s41467-020-16828-y | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-16828-y T ranscription factors are critical for controlling the expression of genes that regulate B-cell development. The importance of specific B-lymphoid transcription factors is highlighted by the phenotype of gene knockout models. Failure of B-cell lineage specification from multi-potential progenitors occurs with deletion of Ikzf11 and Spi1 (Pu.1)2, while deletion of Tcf3 (E2A)3 and Foxo14 results in failure of B-cell development from common lymphoid progenitors (CLPs). Developmental arrest later in B lymphopoiesis is observed with deletion of Ebf1 and Pax5 at the pre–proB and proB stages, respectively5,6. This sequential pattern of developmental arrest associated with the loss of gene function, along with ectopic gene complementation studies2, gene expression profiling7 and analysis of transcription factor binding to target genes, support models in which transcription factors are organised into hierarchical gene regulatory networks that specify B-lymphoid lineage fate, commitment and function8. Two transcription factors that have multiple roles during B-cell development are Ebf1, a member of the COE family, and Pax5, a member of the PAX family. While Ebf1 and Pax5 have been shown to bind to gene regulatory elements of a common set of target genes in a co-dependent manner during later stages of B lineage commitment9, both manifest distinct roles during different developmental stages. Ebf1 has been proposed to form a transcriptional network with E2A and Foxo1 in CLPs that appears important in early B-lymphoid fate determination10, while during later stages of B lymphopoiesis, Ebf1 acts as a pioneer transcription factor that regulates chromatin accessibility at a subset of genes co-bound by Pax511 as well as at the Pax5 promoter itself12. Pax5 in contrast, regulates B-cell genomic organisation13 including the Immunoglobulin heavy chain (Igh) locus during V(D)J recombination, co-operating with factors such as CTCF14, as well as transactivating15 and facilitating the activity of the recombinase activating gene (Rag) complex16. It is unclear, however, how these various functions of Ebf1 and Pax5 are co-ordinated during different stages of B-lymphoid development. In particular, it would be important to ensure coordinated Ebf1 and Pax5 co-expression before the pre-BCR checkpoint, such that Ebf1 and Pax5 co-regulated target genes required for V(D)J recombination and pre-B-cell receptor complex formation are optimally expressed9. Here we show that the ETS-related gene (Erg), a member of the ETS family of transcription factors, plays this vital role in B lymphopoiesis. Deletion of Erg from early lymphoid progenitors resulted in developmental arrest at the early pre–proB-cell stage and loss of VH-to-DJH recombination. Gene expression profiling, DNA-binding analysis and complementation studies demonstrated Erg to be a transcriptional regulator that lies at the apex of an Erg-dependent Ebf1 and Pax5 gene regulatory network commencing in pre–proB cells. This co-dependent transcriptional network directly controls expression of the Rag1/Rag2 recombinase activating genes and the Lig4 and Xrcc6 DNA repair genes required for V(D)J recombination, as well as expression of components of the pre-BCR complex such as CD19, Igll1, Vpreb1 and Vpreb2. Taken together, we define an essential Erg-mediated transcription factor network required for regulation of Ebf1 and Pax5 expression that is exquisitely stage specific during early Blymphoid development. Results Erg is required for B-cell development. To build on prior work defining the role of the transcription factor Erg in regulation of hematopoietic stem cells (HSCs)17 and megakaryocyteerythroid specification18, we sought to identify whether Erg played roles in other hematopoietic lineages. Erg expression in 2 adult hematopoiesis was first examined by generating mice carrying the Ergtm1 (...truncated)