C/EBPα determines hematopoietic cell fate in multipotential progenitor cells by inhibiting erythroid differentiation and inducing myeloid differentiation by Hyung Chan Suh, John Gooya, Katie Renn, Alan D. Friedman, Peter F. Johnson, and Jonathan R. Keller Blood Volume 107(11):4308-4316 June 1, 2006 ©2006 by American Society of Hematology

Increased erythroid development in C/EBPα–/– mice. Increased erythroid development in C/EBPα–/– mice. (A) Cytocentrifuge preparations of C/EBPα–/– FL cells were stained with Wright-Giemsa, and differential cell counts were performed on 400 cells from 3 individual mice. The data are reported as the mean number of cells ± standard error (SE). (B) FL cells (2.5 × 104) from C/EBPα+/+, C/EBPα +/–, and C/EBPα–/– embryos at E15.5 were cultured in complete IMDM (cIMDM) containing 1.1% methylcellulose, 25% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM l-glutamine (P/S/G), and cultures were supplemented with murine (m) IL-3 (30 ng/mL), stem cell factor (mSCF, 100 ng/mL), and human erythropoietin (hEpo, 5 U/mL). The mean number of BFU-E colonies ± SE of triplicate plates is shown. (C-D) C/EBPα–/– and C/EBPα+/+ FL cells were transduced with MSCV-GFP retroviral vectors to track donor erythroid reconstitution. Fetal liver cells were resuspended at 2 × 105/mL in cIMDM and cultured with 8 μg/mL polybrene, 100 ng/mL mSCF, 100 ng/mL thrombopoietin (hTpo), 100 ng/mL hFlt-3L, and 50 ng/mL IL-6. A total of 3 retrovirus infections were performed, after which the cells were cultured with the same cytokine mixture. GFP-positive (GFP+) cells were isolated 2 days after the last infection by FACS sorting (FACSAria [BD Biosciences] or MoFlo [Cytomation, Fort Collins, CO]). BM cells were harvested 4 months after transplantation and then analyzed for the expression of the erythroid markers (TER119 and CD71) and myeloid cell markers (Gr-1 and Mac-1). (D) Horizontal bars indicate median values. (E) GFP+ BM cells were purified by FACS and cultured in methylcellulose (7.5 × 104/plate) or soft agar (1 × 105/plate) to determine the number of erythroid or myeloid progenitors. For myeloid progenitors, cells were plated in cIMDM containing 10% FCS, P/S/G, and 0.35% sea plaque agarose in the presence or absence of 50 ng/mL hG-CSF, 100 ng/mL M-CSF, 100 ng/mL mSCF, 20 ng/mL mGM-CSF, 30 ng/mL mIL-3. The mean number of BFU-E and CFU-c colonies ± SE of triplicate plates is shown. Hyung Chan Suh et al. Blood 2006;107:4308-4316 ©2006 by American Society of Hematology

C/EBPα promotes myeloid development versus erythroid development in vivo. C/EBPα promotes myeloid development versus erythroid development in vivo. Femoral BMCs were prepared from 8-week-old inbred female C57Bl/6 mice that had been treated with 5-fluorouracil (5-FU) 4 days earlier to enrich for hematopoietic stem cells, and then infected with retrovirus containing MSCV-GFP or MSCV-C/EBPα-GFP. GFP+ BM cells (1 × 106) were transplanted into lethally irradiated mice with host bone marrow cells (2 × 105) to promote radioprotection. Four months after transplantation, the presence of donor-derived (GFP+) hematopoietic cells was determined by flow cytometry. Granulocytes and macrophages (Gr-1 and Mac-1) and erythroid cells (CD71 and TER119) in peripheral blood are presented in panels A-B, while myeloid (Mac-1) and erythroid (CD71 and TER119) cells in bone marrow are presented in panels C-D. These flow cytometry data were collected from 3 different transplantation experiments. (B, D) Horizontal bars indicate median values. Hyung Chan Suh et al. Blood 2006;107:4308-4316 ©2006 by American Society of Hematology

C/EBPα inhibits erythroid differentiation of MEL cells. C/EBPα inhibits erythroid differentiation of MEL cells. (A) MOCK and C/EBPα/hER-transduced MEL clones were incubated in the presence of HMBA (3 mM) and β-estradiol (1 μM) for 96 hours. Red cell pellets indicate terminal differentiation of MEL cells. C/EBPα/hER and α-actin protein expression of each clone was confirmed by Western blot analysis as described in “Materials and methods.” (B) Hemoglobinization of MEL, MOCK, and αER-E4 cells was evaluated at the indicated times after treatment with (i) HMBA or (ii) HMBA + β-estradiol by benzidine staining. The mean benzidine positivity of each cell group ± SE of triplicate experiments is shown. (C) β-Globin mRNA levels were measured in HMBA-treated MOCK and αER-E4 cells by Northern blot analysis as described in “Materials and methods” using 447-bp fragment of mouse β-globin cDNA. Control lanes show 18S and 28S ribosomal RNA. Hyung Chan Suh et al. Blood 2006;107:4308-4316 ©2006 by American Society of Hematology

C/EBPα switches lineage potential of MEL and EML cells toward myeloid development. C/EBPα switches lineage potential of MEL and EML cells toward myeloid development. (A) MEL cells were transduced by retrovirus containing MSCV-C/EBPα-GFP or MSCV-GFP as described in “Materials and methods.” After sorting GFP+ cells, MEL cells were cultured in vitro for an additional 3 days, after which cell morphology was determined by Wright-Giemsa staining, and cell lineage by naphthol AS-D chloroacetate esterase and MPO staining (× 1000). Arrows indicate stained myeloid granules. (B) RT-PCR was performed using total RNA harvested from the cultured MEL cells 3 days after transduction with retrovirus containing MSCV-C/EBPα-GFP or MSCV-GFP. (C) The SCF-dependent EML cell line (gift of Dr Schickwann Tsai) was maintained in IMDM supplemented with 20% horse serum, 8% conditioned medium from BHK/MKL cells containing mSCF, and P/S/G. EML cells were cultured with mSCF (100 ng/mL) and hEpo (40 U/mL) 24 hours prior to transduction with retrovirus containing pMSCV-C/EBPα-GFP or pMSCV-GFP. EML cells were transduced with the retroviral vectors that express MSCV-C/EBPα-GFP or MSCV-GFP and then cultured for 3 days with mSCF (100 ng/mL) and hEpo (40 U/mL) to promote erythroid development. The expression of (GFP+) granulocytes and monocytes (Gr-1 and Mac-1) and erythroid cells (CD71 and TER119) was determined by flow cytometry. Hyung Chan Suh et al. Blood 2006;107:4308-4316 ©2006 by American Society of Hematology

C/EBPα induces myeloid differentiation of primary MEPs C/EBPα induces myeloid differentiation of primary MEPs. (A) Lineage-negative (Linneg) cell population was stained with the following cocktail of directly conjugated monoclonal antibodies: PE anti-cKit, FITC anti-CD34, APC anti–Sca-1, PE-Cy7 anti-FcRII/III (... C/EBPα induces myeloid differentiation of primary MEPs. (A) Lineage-negative (Linneg) cell population was stained with the following cocktail of directly conjugated monoclonal antibodies: PE anti-cKit, FITC anti-CD34, APC anti–Sca-1, PE-Cy7 anti-FcRII/III (2.4G2), and PE-Cy5 anti–IL-7Rα. These cells were then sorted by high-speed sorter (MoFlo; Cytomation) for the IL-7Rα–/c-Kit+/Sca-1–/CD34–/FcRII/IIIlo cell population identified as MEPs. Normal MEPs were transduced with retrovirus containing pMSCV-C/EBPα-GFP or pMSCV-GFP. GFP+ MEPs were isolated and cultured for 3 days in cIMDM containing mSCF (100 ng/mL), hEpo (10 U/mL), and hTpo (100 ng/mL). Lineage differentiation was analyzed by flow cytometry using Gr-1 and TER119 antibodies and differential cell counts of Wright-Giemsa–stained cytocentrifuge preparations. Arrow indicates segmented neutrophils. (B) GFP+ MEPs (5 × 104/plate) were also cultured in 1.1% methyl-cellulose containing mSCF (100 ng/mL), hEpo (5 U/mL) to detect erythroid progenitors as described in “Materials and methods.” The mean number of erythroid colonies ± SE of triplicate plates is shown. Hyung Chan Suh et al. Blood 2006;107:4308-4316 ©2006 by American Society of Hematology

Regulation of gene expression in MEL cells by C/EBPα. Regulation of gene expression in MEL cells by C/EBPα. (A) MEL cells and αER-E4 clone cells were treated with 3 mM HMBA and 1.0 μM β-estradiol. Total cell lysates were prepared from treated cells at the indicated times and 30 μg was analyzed for GATA-1, PU.1, and α-actin expression by Western blot analysis as described in “Materials and methods.” (B) Total RNA was also obtained from MEL cell clones 10 hours after treatment with HMBA and β-estradiol. Expression of the indicated genes was evaluated by RT-PCR using the indicated cycle numbers (24, 26, 28, and 30 cycles for β-globin, or 26, 28, 30, and 32 cycles for all other genes). The line graph represents densitometry scan of each band on an agarose gel. Hyung Chan Suh et al. Blood 2006;107:4308-4316 ©2006 by American Society of Hematology

Regulation of EpoR and Id1 expression by C/EBPα during erythroid differentiation. Regulation of EpoR and Id1 expression by C/EBPα during erythroid differentiation. (A) MEL cells, MOCK cells, and αER-E4 cells were cultured in 3 mM HMBA and/or 1.0 μM β-estradiol as indicated. Id1 protein expression was determined 24 hours after treatment by Western blot analysis as described in “Materials and methods.” (B) MEL cells were transduced with control (pMSCV-GFP) or Id1-expressing (pMSCV-Id1-GFP) retroviral vectors. Stable cell lines were cloned and Id1 expression was determined by Western blot analysis. Asterisks indicate the cell lines used for induction of erythroid differentiation. (C) Cloned MEL cells were seeded at 4 × 105/mL in cDMEM containing 3 mM hexamethylene bisacetamide (HMBA) and incubated at 37°C. Erythroid differentiation of Id1-expressing cell lines was determined by benzidine staining 4 days after HMBA treatment (3 mM). (D) Total RNA was obtained from MOCK and αER-E4 cell clones at the indicated times after treatment with HMBA (3 mM) and β-estradiol (1 μM). EpoR mRNA expression in MOCK and αER-E4 cells was analyzed by Northern blot analysis before and after treatment with HMBA and β-estradiol. The EpoR probe was an XhoI 1450-bp fragment of the murine EpoR cDNA (gift of Dr Sandy Ruscetti, NCI-Fredrick. (E) Total cellular extracts were prepared from C/EBPα–/–, C/EBPα+/–, C/EBPα+/+ FL cells, and EpoR expression was determined by Western blot analysis of 30 μg protein. (F) L cells were transfected with the pGL3, 2xC/EBPα-Luc, mEpoR-Luc, alone or with increasing amounts (20, 50, 100 ng) of C/EBPα expression vector by liposomal method (FuGENE 6). Cell extracts were assayed after 48 hours of incubation with complete DMEM media. The histograms represent means ± SE of the luciferase activity from triplicates normalized for transfection efficiency using renilla luciferase. **P < .05 t test. Hyung Chan Suh et al. Blood 2006;107:4308-4316 ©2006 by American Society of Hematology