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, 115 (11), 2156-66

FOG1 Requires NuRD to Promote Hematopoiesis and Maintain Lineage Fidelity Within the Megakaryocytic-Erythroid Compartment

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FOG1 Requires NuRD to Promote Hematopoiesis and Maintain Lineage Fidelity Within the Megakaryocytic-Erythroid Compartment

Gregory D Gregory et al. Blood.

Abstract

Nuclear factors regulate the development of complex tissues by promoting the formation of one cell lineage over another. The cofactor FOG1 interacts with transcription factors GATA1 and GATA2 to control erythroid and megakaryocyte (MK) differentiation. In contrast, FOG1 antagonizes the ability of GATA factors to promote mast cell (MC) development. Normal FOG1 function in late-stage erythroid cells and MK requires interaction with the chromatin remodeling complex NuRD. Here, we report that mice in which the FOG1/NuRD interaction is disrupted (Fog(ki/ki)) produce MK-erythroid progenitors that give rise to significantly fewer and less mature MK and erythroid colonies in vitro while retaining multilineage capacity, capable of generating MCs and other myeloid lineage cells. Gene expression profiling of Fog(ki/ki) MK-erythroid progenitors revealed inappropriate expression of several MC-specific genes. Strikingly, aberrant MC gene expression persisted in mature Fog(ki/ki) MK and erythroid progeny. Using a GATA1-dependent committed erythroid cell line, select MC genes were found to be occupied by NuRD, suggesting a direct mechanism of repression. Together, these observations suggest that a simple heritable silencing mechanism is insufficient to permanently repress MC genes. Instead, the continuous presence of GATA1, FOG1, and NuRD is required to maintain lineage fidelity throughout MK-erythroid ontogeny.

Figures

Figure 1
Figure 1
Expansion of early progenitors in Fogki/ki BM. Total BM from WT and Fogki/ki mice at 5 to 7 weeks of age. (A) LSK cells from BM were identified via flow cytometry. LK myeloid progenitors were divided into CMP, GMP, and MEP using CD34 and FcγR II/III. (B) The proportions of LSK, CMP, GMP, and MEP were calculated for the BM (n = 10). Errors bars represent SEM. *P < .05, **P < .01 by Student t test.
Figure 2
Figure 2
Erythroid defects in Fogki/ki CMP and MEP. (A) CMP, GMP, and MEP were cultured in the presence of EPO and SCF and scored at day 14. As control, WT and Fogki/ki GMP produced similar G and M progeny. Results are the average of 3 independent experiments. (B) BFU-E from CMP and MEP were categorized into large (> 50 clusters), medium (16-50 clusters), and small (5-15 clusters); n = 3. (C) Benzidine staining of representative large WT and Fogki/k BFU-E derived from MEP. Original magnification ×10.
Figure 3
Figure 3
Defective megakaryopoiesis in Fogki/ki CMP and MEP. (A) CMP, GMP, and MEP were cultured with TPO, IL-3, and IL-6 and scored at day 8. CFU-MKs were identified as AchE+ colonies with 3 or more MKs per colony. Other cells were identified by counterstain with Harris hematoxylin. Bars represent numbers of colonies per plated progenitors (n = 3). (B) Representative CFU-MKs derived from WT and Fogki/ki MEP. (C) Representative CFU-MK/G/M (left) and CFU-G/M (right) from Fogki/ki MEP.
Figure 4
Figure 4
Myeloid potential of Fogki/ki MEP. (A) CMP, GMP, and MEP were cultured in the presence of IL-3, IL-6, SCF, and EPO and scored at day 14 (n = 3). (B) Flow cytometric analyses of individual colonies with antibodies against Gr1 (granulocytes), Mac1 (macrophages), and Kit/FcϵR1α (MC). (C) MGG and Toluidine blue stains of representative MC containing colonies from CFU-MC/G/M and CFU-MC. MCs (mc), monocytes (mo), neutrophils (n), and eosinophils (e). Original magnification ×40.
Figure 5
Figure 5
Increased MC gene expression in Fogki/ki MEP as determined by gene arrays. Genes were grouped according to their expression in stem cell, stem-myeloid, myeloid-MC, erythroid, MK, and lymphoid compartments. Data are shown as fold change (± SEM) from WT and are the average of 3 independent samples analyzed by microarray (6 arrays total). Other indicates relevant hematopoietic transcription factors.
Figure 6
Figure 6
Elevated expression of MC-specific genes in Fogki/ki MEP, committed erythroid cells, and MKs. (A-D) mRNA levels of indicated MC genes as determined by quantitative RT-PCR in freshly sorted MEP (A), CD71+Ter119+ basophilic erythroblasts from BM (B) or spleen (C), and cultured fetal liver–derived MK (D). Data were normalized to actin (A) or gapdh (B-D). Numbers above bars indicate the fold change from WT cells. nd indicates not determined. Errors bars represent SEM; n = 3.
Figure 7
Figure 7
GATA factors and FOG1 regulate MC genes in erythroid cells. (A) mRNA levels of indicated genes as determined by quantitative RT-PCR of G1E cells, and G1E-ER4 and G1E(V205M)-ER cells treated with estradiol for 20.5 hours. Data were normalized to actin and plotted as fold change compared with uninduced G1E cells; n = 3-5. *P < .001; **P < .05; ns indicates not significant. (B) Overexpression of GATA2 in G1E-ER4 cells (G1E-ER4 + GATA2) stimulates MC gene expression, whereas estradiol-activated GATA1-ER (+est.) represses them; n = 6. *P < .05 comparing G1E-ER4 + GATA2 ± estradiol treatment. (C-G) ChIP for GATA2 (C-D), GATA1 (E-F), and FOG1 (G) at FcerIb and Cpa3 in G1E cells (C,E,G) or BMMC (D,F). Control regions included: −7 kb upstream of the FcerIb promoter (UR), an intronic region 3 kb downstream of the Cpa3 promoter (5′TR), the 5′transcribed region of Cd4 (Cd4 5′TR), and a region −224.9 kb upstream of the Kit gene (UR). Positive control, the GATA1-activated Eraf erythroid gene. n = 3-8 independent ChIP experiments per primer set. (H-I) ChIP against MTA2 (H) and RbAp46 (I) in G1E cells and G1E-ER4 (+est.); n = 4-6. The promoter of GATA1-activated Hbb-b1 served as a positive control. *P < .01, #P < .05 by Student t test compared with IgG controls (C-I). (J) ChIP for acetylated histone H3 (acH3) in G1E cells, and G1E-ER4 and G1E(V205M)-ER cells treated with estradiol for 20.5 hours; n = 4-6. *P < .01, #P < .05 by Student t test. Error bars represent SEM.

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