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Normal hematopoiesis

Gata2 −77 enhancer regulates adult hematopoietic stem cell survival

Leukemia volume 35, pages 901–905 (2021)Cite this article

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GATA2 is a master transcription factor that governs hematopoietic stem cell (HSC) generation in the mammalian embryo and confers multi-lineage differentiation of hematopoietic stem and progenitor cells (HSPCs) [1,2,3,4]. Previously, we demonstrated that Gata2 expression in fetal liver myeloid progenitors (MPs) is controlled by its upstream −77 enhancer [5, 6]. Although −77−/− fetal liver HSCs exhibited normal or greater than normal long-term repopulating activity, MP differentiation potential was impaired, thus causing anemia and embryonic lethality. However, whether the −77 enhancer has essential functions to regulate adult hematopoiesis has not been established.

To determine whether the −77 enhancer regulates HSPCs by conferring Gata2 expression, its only known activity thus far, Gata2 mRNA levels were quantified in WT, −77+/−, and Gata2+/− LSK and MP cell populations using quantitative reverse-transcriptase PCR (qRT-PCR). Consistent with the prior report [8], Gata2 expression was reduced in Gata2+/− LSKs and MPs. Surprisingly, Gata2 expression in −77+/− LSKs and MP populations was also lower than that of WT cells (Supplementary Fig. S3). We analyzed transcript levels of genes localized adjacent to Gata2, Eefsec, Rab7, and Rpn1. These genes are known not to be regulated by −77 in fetal liver cells [5]. The mRNA levels of these genes in −77+/− and Gata2+/− LSK and MPs were indistinguishable from those in WT cells (Supplementary Fig. S3). Furthermore, we sorted WT and −77+/− HSCs and MPPs and quantified mRNA levels of HSC stemness genes and apoptosis genes using qRT-PCR (Supplementary Fig. S4). We found that c-Kit, Myb, Mpl, and Gata2 were upregulated in −77+/− HSCs (Supplementary Fig. S4a), whereas c-Kit, Myb, and Mpl were upregulated and Gata2 was downregulated in −77+/− MPPs (Supplementary Fig. S4b). For the apoptosis genes, genes promoting cell survival were upregulated and genes promoting apoptosis were downregulated in −77+/− HSCs and MPPs. Our data suggest that the qualitatively different phenotypes of −77+/− and Gata2+/− HSCs is associated with the opposite regulation of Gata2 expression in HSCs. This may reflect downregulation of Gata2 expression in some cells of the −77+/− mice vs. in all cells of the Gata2+/− mutants. Alternatively, this phenotypic difference may reflect a previously unrecognized −77 enhancer function in HSCs.

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Fig. 1: Increased HSC survival in Gata2 −77+/− mice.
Fig. 2: Gata2 -77+/− bone marrow cells exhibit increased long-term reconstitution activity in primary recipients.

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Acknowledgements

We are grateful to Dr. Stuart Orkin for generously providing Gata2+/− mice. We thank the University of Wisconsin Carbone Comprehensive Cancer Center (UWCCC) for use of its Shared Services (Flow Cytometry Laboratory, Genome Editing and Animal Models Shared Resource, and Experimental Pathology Laboratory). The Flow Cytometry Laboratory was supported by NIH Shared Instrument Grants 1S10RR025483-01 (BD FACS AriaII BSL-2 Cell Sorter) and 1S100OD018202-01 (BD LSR Fortessa). This work was supported by NIH grants CA152108 to J.Z and DK69634 to E.H.B and NIH/NCI P30 CA014520-UW-Comprehensive Cancer Center (UWCCC) Support.

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Authors and Affiliations

  1. McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI, USA

    Xiaona You, Yuan-I Chang, Guangyao Kong, Yun Zhou & Jing Zhang

  2. University of Wisconsin–Madison Blood Research Program, Madison, WI, USA

    Xiaona You, Yun Zhou, Kirby D. Johnson, Charu Mehta, Emery H. Bresnick & Jing Zhang

  3. Institute of Physiology, National Yang-Ming University, Taipei City, Taiwan

    Yuan-I Chang

  4. National Local Joint Engineering Research Center of Biodiagnostics and Biotherapy, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, P. R. China

    Guangyao Kong

  5. Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI, USA

    Erik A. Ranheim

  6. Carbone Cancer Center, Department of Cell and Regenerative Biology, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA

    Kirby D. Johnson, Charu Mehta & Emery H. Bresnick

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  1. Xiaona You
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Contributions

The contributions of individual authors are listed below: XY for experimental design and execution, as well as manuscript preparation; YIC and GK for experimental execution; EAR for histopathology analysis and manuscript preparation; YZ, KDJ, and CM for technical or material support; EHB and JZ for experimental design and manuscript preparation.

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Correspondence to Jing Zhang.

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You, X., Chang, YI., Kong, G. et al. Gata2 −77 enhancer regulates adult hematopoietic stem cell survival. Leukemia 35, 901–905 (2021). https://doi.org/10.1038/s41375-020-0942-6

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