GATA-2 Regulates Dendritic Cell Differentiation

(Background) Dendritic cells (DCs) are critical regulators of the immune response, but their differentiation mechanism remains unclear. Heterozygous germline GATA-2 mutations in humans cause MonoMAC syndrome, characterized by monocytopenia and predisposition to myelodysplasia/acute myeloid leukemia. In this syndrome, DC count decreases profoundly, with an increased susceptibility to viral infections, impaired phagocytosis, and decreased cytokine production. In the present study, we analyzed the role of GATA-2 in DC differentiation and the underlying molecular mechanisms.

(Method) Gata2 haploinsufficient mice (Gata2^+/−: Tsai et al. Nature 1994) and tamoxifen-inducible Gata2-knockout mice (Gata2^flox/flox/ER-Cre: Charles et al. Molecular Endocrinology 2006) were used. To generate conditional Gata2 knockouts in vivo, Gata2^flox/flox/ER-Cre mice were intraperitoneally injected with 1-μg tamoxifen on days 1–3 and 8–10 and evaluated on days 20–22. Isolation of splenic DCs and bone marrow (BM) precursors, including LSK (Lin^–Sca1⁺Kit⁺ cell), CMP (common myeloid-restricted progenitor), GMP (granulocyte-macrophage progenitor), CLP (common lymphoid-restricted progenitor), and CDP (common dendritic cell precursor), were separated with both MACS (Miltenyi Biotech) and BD FACSAria II (BD Biosciences). For the in vitro analysis of Gata2-knockout, BM cells were cultured with CD45.1⁺BM feeder cells from SJL mice (The Jackson Laboratory) with FLT3L (200 ng/mL) and 4-hydroxytamoxifen (Sigma). For transcription profiling, SurePrint G3 mouse GE microarray (Agilent) was used, and the data was subsequently analyzed with ImmGen database (http://www.immgen.org). Promoter assay was conducted with Dual Luciferase Reporter Assay system (Promega). Quantitative chromatin immunoprecipitation (ChIP) analysis was performed using CMP fraction and erythroid–myeloid–lymphoid (EML) hematopoietic precursor cell line (ATCC) with antibodies to GATA-2 (sc-9008, Santa Cruz Biotechnology).

(Results) Quantitative RT-PCR analysis showed abundant Gata2 expression in LSK and CMP fractions, with detectable expression in GMP, CLP, and CDP fractions and in vitro differentiated DCs. Although the DC count did not change in Gata2 haploinsufficient mice, it significantly and profoundly decreased in Gata2 conditional knockout mice. To examine the role of GATA-2 during DC differentiation, we knocked out Gata2 during in vitro DC differentiation, starting from LSK, CMP, GMP, CLP, and CDP fractions obtained from Gata2^flox/flox/ER-Cre mice. Gata2 knockout significantly decreased CD11c⁺DC counts from LSK, CMP, and CDP fractions, while those from CLP and GMP were unaffected, implying the importance of GATA-2 during DC differentiation in the pathway from LSK to CDP via CMP, not via CLP nor GMP.

To elucidate the underlying molecular mechanisms, we performed expression profiling with control and Gata2-knockout DC progenitors from CMP of Gata2^flox/flox/ER-Cre mice. Gata2 knockout caused >5-fold upregulation and downregulation of 67 and 63 genes, respectively. Although genes critical for the DC differentiation, e.g., Spi1, Ikzf1, and Gfi1, were not detected among the GATA-2-regulated gene ensemble, we found significant enrichment of myeloid-related and T lymphocyte-related genes among the downregulated and upregulated gene ensembles, respectively. We focused on Gata3 upregulation (7.33-fold) as a potential key mechanism contributing to Gata2 knockout-related impaired DC differentiation.  Quantitative ChIP analysis with both CMP fraction and EML cell line demonstrated obvious GATA-2 chromatin occupancy at the consensus GATA-binding motif within Gata3 +190 kb, which was conserved with human. Furthermore, addition of Gata3 +190 kb region to the Gata3 promoter (~0.5 kb) significantly decreased luciferase activity, which was significantly recovered by the deletion of GATA sequence within Gata3+190 kb, in EML cells.

(Conclusion) GATA-2 seems to play an important role for cell fate specification toward myeloid versus T lymphocytes, and thus contributing to the DC differentiation. Our data offer a better understanding of the pathophysiology of MonoMAC syndrome.