Severe Congenital Neutropenia-Associated Mutations Reveal Stage-Specific Gfi1-Dependent Checkpoints in Myeloid Development

Growth factor independent-1 (GFI1) is a zinc-finger transcription factor that is critically required for both murine and human granulopoiesis. Mutations in GFI1 zinc fingers (e.g. N382S in zinc finger 5) have previously been found in humans with severe congenital neutropenia (SCN). We noted that clinical resequencing of neutropenic patients identified a number of novel GFI1 sequence changes of unknown significance. To clarify their impact, we used viral vectors to force expression of 13 GFI1 polymorphisms/mutations in primary-murine progenitors followed by colony-forming-unit assays for differentiation. While these assays infer new potential clinical significance to some genetic alterations, we focused on the sixth zinc finger of GFI1. While Gfi1 zinc-finger 6 was previously found to be dispensable for DNA binding and repression of a high affinity GFI1 DNA-binding-site reporter, the biological impact of expressing GFI1 zinc finger 6 mutants suggests a functional role for this domain in granulopoiesis. Therefore, we generated mice with patient-derived SCN-associated mutations (zinc finger 5; N382S, zinc finger 6; K403R, and R412X) in the murine Gfi1 locus. The resulting defects in murine neutrophil production are proportional to the severity of SCN in patients with the same GFI1 mutation (ranging from mild to profound neutropenia); with neonates displaying more profound defects. Neonatal and adult Gfi1^N382S/- and Gfi1^R412X/- mice are neutropenic, but Gfi1^K403R/- mice have normal steady-state neutrophil levels. While Gfi1^-/- HSC are profoundly abnormal, SCN-associated mutant HSC are phenotypically normal; dissociating Gfi1 control of hematopoietic stem cell biology from granulopoiesis. Similar to Gfi1^-/-, the Gfi1^N382S/- and Gfi1^R412X/- mice accumulate cells within the "granulocyte monocyte progenitor" (GMP) gate as well as those that express Sca1 (known as emergency GMP; eGMP); however, unlike Gfi1^-/- mice, the serum levels of known cytokine inducers of eGMP are normal in SCN-associated mutants. Moreover, isolated progenitors from all three mutants form significantly more monocytic colonies at the expense of granulocytic colonies in methylcellulose assays. Together, these data suggest that the mutations induce a cell-intrinsic defect.

We recently utilized single-cell RNA-Seq (scRNA-Seq) to identify a gene regulatory network (GRN) centered on the Gfi1 and Irf8 transcription factors that underlies the granulocytic versus monocytic lineage decision of myeloid progenitors at steady state. To pinpoint the molecular deregulation underlying Gfi1-mutant progenitors, we performed scRNA-Seq. Notably, Gfi1^R412X/- induced deregulation of gene expression which increases in amplitude and complexity as myeloid differentiation proceeds; coincident with induction of Gfi1 expression. ChIP-Seq analysis reveals that most of the deregulated genes are normally bound by Gfi1. Using murine fluorescent-protein knock-in reporter alleles for Gfi1 and Irf8, we demonstrate the stage-specific regulation of the granulocyte-monocyte lineage decision, and how SCN mutations deregulate the Gfi1-Irf8 network. Finally, we developed an alternative flow-cytometric strategy that highlights individual Gfi1-dependent checkpoints in myeloid differentiation (identified by comparative analysis of Gfi1^-/- versus knock-in mutants). We find that the accumulated Gfi1^N382S/- GMP are unspecified (similar to Gfi1^-/-); however, Gfi1^R412X/- GMP exhibit only a modest reduction in the granulocytic specified population and instead display a profound block at granulocytic commitment. The latter effects can be partially explained by the protein-destabilizing effect of mutations in zinc finger 6; suggesting that there are dose-dependent roles for Gfi1 in granulopoietic specification and commitment. In summary, Gfi1 knock-in mice provide a mouse model of human SCN, and both flow-cytometric and scRNA-Seq dissection of hematopoiesis reveal myelopoietic-stage-specific deregulation to both block granulopoiesis and deregulate monocytic subsets.