A recurrent network involving the transcription factors PU.1 and Gfi1 orchestrates innate and adaptive immune cell fates

Abstract

The transcription factor PU.1, encoded by the Sfpi1 gene, functions in a graded manner to regulate macrophage versus B cell generation; its higher concentration favors the macrophage fate. We demonstrated that Gfi1 reciprocally promoted B cell fate choice at the expense of myeloid progeny. Gfi1(-/-) multipotential progenitors (MPPs) were unable to constrain the expression of PU.1 because Gfi1 functioned to repress the Sfpi1 gene by displacing PU.1 from positive autoregulatory elements. Attenuating a transcriptional module composed of PU.1 and Egr suppressed the B lineage developmental defects of Gfi1(-/-) MPPs. Finally Ikaros, a transcription factor required for B cell development, promoted Gfi1 and antagonized PU.1 expression in MPPs. Our results reveal that a core transcriptional regulatory network used for directing cell fate choice in the innate immune system has been co-opted by Ikaros to orchestrate B lymphocyte generation. These findings have important implications for the evolution of the adaptive immune system.

Figure 1. Gfi-1 regulates B versus myeloid cell fate choice

(A) MPPs (Lin⁻Sca-1⁺c-Kit^hi) were isolated from the bone marrow of WT or Gfi-1^−/−animals. Cells were directly plated on OP9 stroma and analyzed by flow cytometry at day 12. Data are representative of at least three independent experiments. (B) WT MPPs were transduced with a control vector (MIGR1) or one expressing Gfi-1 (MIGR1-Gfi-1). GFP⁺ transductants were plated on OP9 stroma and examined for the generation of myeloid and B-lineage progeny after 7 days. Data are representative of at least three independent experiments.

Figure 2. Gfi-1^−/− MPPs express increased levels of PU.1 and its target genes

(A) Semi-quantitative RT-PCR analysis of cDNA (3X serial dilution) from WT or Gfi-1^−/− MPPs. Transcript levels were normalized to Hprt. (B) qPCR analysis of PU.1, c-fms and Flt3 transcripts in WT or Gfi-1^−/− MPPs. (C) Histograms represent c-fms or FcγRII/III expression on the surface of WT (gray shaded area) or Gfi-1^−/− (black line) MPPs. (D) and (E) WT or Gfi-1^−/− MPPs were plated in methylcellulose containing erythropoietin, stem cell factor, IL-3, IL-6, and Flt3 ligand. Colony numbers were scored 8 days after plating and analyzed for the generation of CFU-Mix (erythroid/granulocyte/macrophage), CFU-M (macrophage) or CFU-GM (granulocyte/macrophage) by Wright staining of cytospins. Data is from two independent experiments.

Figure 3. PU.1 heterozygosity promotes B cell development in Gfi-1^−/− mice

(A) Bone marrow cells from WT, Gfi-1^−/− and Gfi-1^−/−PU.1^+/− animals were analyzed by flow cytometry for B-lineage (B220⁺CD19⁺) and myeloid (Mac1⁺) precursors. (B) Absolute numbers of B-lineage (B220⁺CD19⁺ or B220⁺CD43⁺) cells in the bone marrow of WT (n=5), Gfi-1^−/− (n=5), and Gfi-1^−/−PU.1^+/− (n=7) animals. (C) Quantitative analysis of B-lineage potential in MPPs from WT (solid line), Gfi-1^−/− (long dashed line), or Gfi-1^−/−PU.1^+/− (short dashed line) mice. MPPs were plated on OP9 stroma in limiting dilution as described in Fig. 1. Cultures were analyzed for wells containing CD19⁺ cells at day 12. Data are representative of two independent experiments.

Figure 4. Attenuation of PU.1 activity promotes B cell fate specification

(A) shRNA-mediated knockdown of PU.1 in MPPs. WT MPPs were transduced with a MSCV-shLuciferase or MSCV-shPU.1 vector. GFP⁺ transductants were analyzed for the expression of PU.1 and c-fms transcripts. Data are representative of two independent experiments. (B) WT MPPs transduced with MSCV-shLuciferase (gray shaded area) or MSCV-shPU.1 (black line) were plated on OP9 stroma and analyzed for the presence of c-Kit⁺ precursors (gated on Mac1⁺ cells) 7 days post-sort. Data are representative of two independent experiments. (C) Gfi-1^−/− MPPs were transduced with MSCV-shLuciferase or MSCV-shPU.1 and analyzed by flow cytometry for the generation of Mac1⁺ and CD19⁺ precursors after 10 days.

Figure 5. Egr activity in MPPs antagonizes B cell development

(A) WT MPPs transduced with a control vector (MIGR1) or one expressing a dominant-negative version of Egr-2 (MIGR1-ΔEgr) were plated on OP9 stroma and analyzed for Mac1⁺ and CD19⁺ precursors after 7 days. (B) GFP⁺ transductants plated in limiting dilution were analyzed for the generation of CD19⁺ progeny by flow cytometry 7 days post-sort. Data are representative of at least three independent experiments. (C) Total bone marrow cells from WT, Egr1^−/−, Gfi-1^−/− and Egr1^−/−Gfi-1^−/− animals were analyzed for B-lineage (B220⁺CD19⁺) and myeloid (Mac1⁺) cells by flow cytometry. Data is representative of three mice for each genotype.

Figure 6. Gfi-1 targets the PU.1 locus

(A) Schematic structure of the murine PU.1 locus. Sequence alignment between the mouse and human orthologs showing putative PU.1 (blue) and Gfi-1 (red) binding sites in the PU.1 promoter and URE. (B) PU.1 and Gfi-1 compete for overlapping sites within the PU.1 URE. Binding reactions containing either PU.1 and/or Gfi-1 in vitro translation extracts with radiolabelled oligonucleotides were analyzed by EMSA. Unlabelled PU.1 (λB) and Gfi-1 binding site oligonucleotides were used as competitor DNAs. Arrows indicate migration positions of PU.1 or Gfi-1 protein-DNA complexes. (C) ChIP analysis of PU.1 (blue bars) and Gfi-1 (red bars) to the URE in unstimulated and OHT-treated PUER cells. Fold enrichment was assessed by qPCR after normalization to α-actin. Data are representative of three experiments. (D) ChIP-on-chip analysis of PU.1 target promoters in OHT-treated PUER cells. Putative Gfi-1 binding sites were identified in PU.1 target promoters (see Experimental Procedures). The histogram displays the distribution of the various configurations of overlapping presumptive PU.1 and Gfi-1 binding sites in the PU.1 ChIP sequences (blue bars). A randomized set of DNA sequences (see Experimental Procedures) was used to generate a control distribution (red bars). The Gfi-1 binding site matrix is displayed below the frequency distributions. The configurations of the overlapping presumptive PU.1 and Gfi-1 binding sites are tabulated based on the distance (bp) from the center of the Gfi-1 core AATC to the center of the PU.1 core GGAA. PU.1 core sequences that reside either upstream or downstream of the Gfi-1 core sequence are denoted by negative or positive coordinates, respectively.

Figure 7. Ikaros promotes Gfi-1 expression and represses PU.1

(A) RT-PCR analysis of the indicated transcripts in WT and Ik^−/− MPPs. (B) Flow cytometric analysis of c-fms and FcγRII/III expression on WT (gray shaded area) and Ik^−/− (black line) MPPs. Data are representative of three independent experiments. (C) Expression of the Gfi-1-GFP knock-in allele in MPPs from Gfi-1^+/− (gray shaded area) or Ik^−/−Gfi-1^+/− (black line) mice by flow cytometry. Data are representative of two independent experiments. (D) A proposed gene regulatory network that dictates myeloid versus B cell fate choice in the context of a MPP. Arrows represent gene activation and barred lines represent gene repression. Hatched lines represent regulatory connections whose molecular basis remains to be elucidated. The shaded region encompasses a core gene regulatory network that is used in a recurring manner to orchestrate innate as well as adaptive immune cell fates.