Transcription Factor Gata2 Regulates the Myeloid-Lymphoid Fate Decision in Multipotent Progenitors

Hematopoiesis is a continuous process of differentiation whereby hematopoietic stem cells (HSCs) and multipotent progenitor cells (MPPs) generate diverse cell types encompassing myeloid and lymphoid lineages. This dynamic differentiation is orchestrated by transcription factors that regulate gene regulatory networks (GRNs), promoting expected lineage-specific gene expression while concomitantly repressing alternative lineage genes. A transcription factor Gata2, a zinc finger DNA binding protein, has been identified as a master regulator in HSC emergence and maintenance. We previously revealed that Gata2 is essential for the proper generation of dendritic cells, with its depletion leading to the induction of lymphoid gene expressions (Onodera et al., Blood 2016). Additionally, we reported that Gata2 might possess significant roles at the myeloid-T cell differentiation bifurcation point in the thymus (Sano et al., ASH 2023). Gata2 is also prominently expressed in MPPs where lineage fate decisions are at least partially determined. However, the specific role of Gata2 in MPPs, particularly its contribution to myeloid-lymphoid fate decisions in the bone marrow (BM), remains not fully understood. This study aims to delineate the functionally distinctive roles of Gata2 in MPPs utilizing several types of conditional knockout mouse models, with a specific focus on its impact on hematopoietic progenitor differentiation and lineage commitment.

To reveal the roles of Gata2 in hematopoietic progenitors in vivo, we generated Gata2^fl/fl: Mx1-Cre mice enabling efficient Gata2 deletion in hematopoietic stem and progenitor cells by poly(I:C) administration. Intriguingly, the conditional knockout of Gata2 led to increased common lymphoid progenitors (CLPs; lineage^-Sca-1⁺c-Kit⁺Flt3⁺CD127⁺) and early B cell progenitors in the BM. By contrast, common myeloid progenitors (CMPs; lineage^-Sca-1^-c-Kit⁺CD34⁺CD16/32⁺) were significantly reduced. We further generated HSC-specific knockout mice (Gata2^fl/fl: Fgd5-CreERT2) and B cell-specific knockout mice (Gata2^fl/fl: Mb1-Cre) to assess the effects of Gata2 deletion in HSCs and B cells, respectively. HSC-specific Gata2 deletion resulted in a persistent depletion of HSCs (lineage^-Sca1⁺cKit⁺CD48^-) whereas both CLPs and CMPs remained unaffected overtime. On the other hand, B cell-specific Gata2 deletion did not impair B cell development. Taken together, our findings suggest that the Gata2 deletion in MPPs might skew fate determination towards the B cell lineage rather than the myeloid lineage. To investigate whether Gata2-deficient MPPs predominantly differentiate into B cells, we employed both in vivo lineage tracking following transplantation and in vitro OP9-coculture systems utilizing Gata2-deficient MPPs. Our findings demonstrated enhanced B cell output from Gata2-deficient MPPs in vivo, while Gata2-deficient MPP2s, MPP3s, and MPP4s exhibited increased B cell production at the expense of myeloid cell production in vitro. Comprehensive transcriptional analysis (RNA-seq) of MPP2s, MPP3s, and MPP4s with or without Gata2, revealed that gene expression changes by Gata2 depletion were most pronounced in MPP4s. GSEA (Gene Set Enrichment Analysis) on MPP4s revealed that myeloid progenitor cell related gene sets were significantly enriched in control MPP4s compared to Gata2-deficient MPP4s, suggesting that Gata2 in MPP4s biases their trajectory towards myeloid lineage at the transcriptomic levels. These findings collectively suggest that Gata2 promotes myeloid gene expression, which could suppress lymphoid differentiation reciprocally. Consistent with this notion, we demonstrated that Gata2 expression is sharply declined during B cell commitment, including in CLPs, whereas it is maintained up to the CMPs stage.

In conclusion, our study elucidates the role of Gata2 in MPPs. Gata2 facilitates the myeloid differentiation from MPPs, while its downregulation appears to be a prerequisite for appropriate B cell commitment from progenitor cells. Further researches are needed to reveal the precise mechanisms by which Gata2 orchestrates the myeloid and lymphoid GRNs and to determine the regulation of Gata2 expression at the myeloid-lymphoid bifurcation point. These studies will provide more comprehensive insights into the molecular control of hematopoietic lineage commitment and associated hematological disorders.

Kato:Nippon Shinyaku: Research Funding; The Chemo-Sero-Therapeutic Research Institute: Research Funding. Onodera:Pfizer: Honoraria, Research Funding; Asahi Kasei Pharma: Honoraria; Janssen Pharmaceutical: Honoraria; Nippon Shinyaku: Honoraria; Abbvie: Honoraria, Research Funding; Novartis pharma: Honoraria; Meiji Seika Pharma: Honoraria; Astellas pharma: Honoraria; BMS: Honoraria; Otsuka: Honoraria, Research Funding; Chugai Pharmaceutical: Honoraria; Celgene: Research Funding. Fukuhara:Eisai: Honoraria; Takeda: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria; Nippon kayaku: Honoraria; Eli Lilly: Honoraria; Gilead: Honoraria; Janssen: Honoraria; Meiji Seika: Honoraria; AstraZeneca: Honoraria; Incyte and Takeda: Research Funding; LOXO Oncology: Research Funding; Kyowa Kirin: Honoraria, Research Funding; Genmab: Honoraria, Research Funding; Chordia Therapeutics: Research Funding; Chugai Pharma: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Novartis: Honoraria; Ono: Honoraria; Soreisia: Honoraria. Onishi:Incyte: Research Funding; Meiji Seika: Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; BMS: Honoraria, Research Funding; JCR: Research Funding; Abbvie: Research Funding; Sumitomo: Research Funding; Shionogi: Research Funding; AsahiKasei: Honoraria; Kyowa Kirin: Honoraria; Chugai: Honoraria; MSD: Honoraria; Amgen: Honoraria; Asteras: Honoraria; Daiichi Sankyo: Honoraria; Meiji Seika: Honoraria; Symbio: Honoraria; Nippon Shinyaku: Honoraria; IQVIA: Honoraria; Sanofi: Honoraria; Kissei: Honoraria. Harigae:Sumitomo Corporation: Research Funding; Kyowa Kirin: Research Funding; AbbVie Inc.: Honoraria; Novartis International AG: Honoraria; CHUGAI PHARMACEUTICAL CO.,LTD.: Honoraria, Research Funding; Sanofi: Honoraria.