Fig 1.
Generation of B6 Mice with Floxed Cebpa +37 kb Enhancer Alleles.
A) Diagram of a wild-type genomic allele in the vicinity of the +37 kb Cebpa enhancer (B6 WT), the knockin (KI) vector, and a targeted genomic allele (B6 KI). The conserved 439 bp enhancer is flanked by a frt-PGK-Neo-frt cassette and two loxP sites. Cre-mediated deletion of the DNA between the loxP sites will remove the enhancer, 214 bp of DNA 5’ to the enhancer, and 35 bp of DNA 3’ to the enhancer, 688 bp in total. Positions of SpeI sites, fragment sizes expected after SpeI digestion, and locations of the 5’ and 3’ probes used for Southern blotting are also shown. B) Southern blots obtained after SpeI digestion of genomic DNA isolated from parental B6 BL-1 ESC (B6) or from three targeted lines (KI ESC lines) using the 5’ or 3’ probes. Locations and sizes of fragments derived from the WT and KI alleles are indicated. C) PCR of tail snip DNA, using loxP5 primers spanning the 5’ loxP site, from homozygous Enh(f/f), WT (+/+), and heterozygous (f/+) mice obtained after breeding. Locations of bands obtained from the KI and WT alleles are indicated.
Fig 2.
Effect of in vitro Enhancer Deletion on Cebpa Expression and Myelopoiesis.
A) Mononuclear marrow cells from WT or Enh(f/f) mice were placed in IMDM/FBS with IL-3, IL-6 and SCF for 24 hr, transduced with pBabePuro (Puro) or pBabePuro-Cre (Cre) for 48 hr, puromycin selected for an additional 48 hr, and finally lineage-depleted. Genomic DNA was then subjected to PCR using the loxP5 or EnhΔ primer pairs followed by agarose gel electrophoresis and visualization by ethidium bromide staining. B) Total cellular RNAs were analyzed for Cebpa and large ribosomal subunit mS16 mRNA expression. Cebpa RNA expression, normalized using mS16 expression and set to 100 for WT marrow transduced with Cre, is shown (left, mean and SD from 3 determinations). Total cellular proteins isolated from the same groups of Lin- cells were subjected to Western blotting for C/EBPα and β-actin; locations of the p42 and p30 C/EBPα alternative translation variants are indicated (right). C) Lin- cells were placed in liquid culture with IMDM/FBS, IL-3, IL-6, and SCF and analyzed for surface CD11b and Gr-1 expression on day 4 (D4; mean and SD from three determinations). D) The morphology of Puro- or Cre-transduced Enh(f/f) cells from these cultures was assessed on D4 by Wright’s Giemsa staining; g—granulocyte; m—monocyte; b—blast. E) Lin- cells were cultured similarly in methylcellulose at 1E3 cell/mL, and myeloid CFUs were enumerated 7–8 days later (Gen1). CFU cells were then collected, washed with PBS, replated at 1E3 cells/mL, and analyzed similarly each 7 days (Gen 2 to Gen 12). In addition, a proportion of Gen5 cells were evaluated for their ability to proliferate in liquid culture in IMDM/FBS with IL-6/SCF or IL-3.
Fig 3.
Effect of in vivo Enhancer Deletion on Cebpa Hematopoietic Expression.
A) Representative FACS analyses of marrow stem/progenitor subsets. GMP, CMP, and MEP were analyzed within the Lin-Sca-1-c-kit+ (LK) subset, CLP were analyzed within the Lin-Sca-1loc-kitlo (LSloKlo) subset, and MPP, ST-HSC, LT-HSC, or LSK/SLAM cells were enumerated within the Lin-Sca-1+c-kit+ (LSK) subset. B) Total cellular RNAs from the LSK, CMP, GMP, MEP, CLP, LSK/SLAM, granulocyte, or monocyte marrow subsets isolated from Enh(f/f) or Enh(f/f);Mx1-Cre mice that had been subjected to pIpC injections and allowed to recover for 4 wks were analyzed for relative Cebpa mRNA expression, normalized to mS16 mRNA expression (mean and SD from three determinations). C) Relative Cebpa mRNA expression was analyzed similarly from marrow mononuclear cells isolated from Enh(f/f) mice versus mice lacking both PGK-Neo cassettes (ff;ΔNeo) or Enh(f/f);Vav-Cre mice and from wild-type (++) versus Enh(f+);CMV-Cre (Δ+) mice (n = 3).
Fig 4.
Effect of Germline Enhancer Deletion on Cebpa Expression in Non-Hematopoietic Tissues.
A) Total RNAs from marrow mononuclear cells, brown fat (bFat), white fat (wFat), liver, lung, small intestine (GI), skeletal muscle, or kidney from wild-type (++) and Enh(f/f);CMV-Cre (ΔΔ) mice were analyzed similarly for relative Cebpa expression (n = 3). B) Total embryo DNA from a litter obtained at E16.5 from a cross between Enh(f/+);CMV-Cre (Δ+) mice was subjected to PCR using the loxP5, EnhΔ, and Cre primer pairs, followed by agarose gel electrophoresis and ethidium bromide staining. Enhancer and Cre genotypes are indicated. C) Tail DNA from a litter of 4 wk pups obtained from a cross between a WT and Δ+ mouse, both lacking CMV-Cre, was analyzed similarly (left). DNA from a mouse harboring Mx1-Cre served as a positive control for the Cre PCR (Cre+). DNAs from adult tissues of one of the Δ+ pups were also analyzed using loxP5 and EnhΔ5 PCR (right). * - p<0.05, ** - p<0.01, *** - p<0.001.
Fig 5.
Effect of in vivo Enhancer Deletion on Hematopoiesis.
A) Peripheral blood counts from 14–16 wk old Enh(f/f) and Enh(f/f);Mx1-Cre mice obtained 4 wks after completion of pIpC injections. B) Total marrow cellularity from Enh(f/f) versus Enh(f/f);Mx1-Cre mice exposed 4 wks earlier to pIpC. C) Marrow CD11b+Gr-1+ granulocytes, CD11b+Gr-1- monocytes, Ter119+ erythroid cells, B220+ B cells, CD3+ T cells, or Lin- cells lacking these markers from these same mice. D) Marrow granulocytes, monocytes, erythroid, B and T cells from 4 wk old Enh(f/f) and littermate Enh(f/f);Vav-Cre mice. E) Marrow granulocytes, monocytes, erythroid, B and T cells from 5 wk old Enh(f/f) (++), Enh(f/+);CMV-Cre (Δ+), and Enh(f/f);CMV-Cre (ΔΔ) mice. F) E16.5 FL cells obtained from embryos of indicated genotypes derived from Enh(f/+);CMV-Cre (Δ+) parents were subjected to FACS analysis for these same hematopoietic subsets. G) Marrow stem/progenitor subsets, as determined by FACS analysis, from the same mice evaluated in B and C. H) Myeloid, BFU-E, and B lymphoid CFUs were in enumerated after culture at 1E4 cells/mL in methylcellulose in the presence of IL-3/IL-6/SCF or at 2E5 cells/mL in EPO or IL-7, respectively (mean and SD from three determinations). I) Morphology of marrow obtained from Enh(f/f) or Enh(f/f);Mx1-Cre mice 4 wks after pIpC injections and from Enh(f/f);Vav-Cre or Enh(f/f);CMV-Cre mice.
Fig 6.
Effect of in vivo Enhancer Deletion on Selected Transcription Factor, Myeloid Cytokine Receptor, or Bcl2 Expression.
A) Total cellular RNAs from the GMP, CMP, or LSK marrow subsets isolated from Enh(f/f) or Enh(f/f);Mx1-Cre mice that had been subjected to pIpC injections and allowed to recover for 4 wks were analyzed for relative expression of the indicated mRNAs (mean and SD from three determinations). Relative expression of each RNA is compared in GMP versus CMP versus LSK cells in Enh(f/f) marrow, with the average value in GMP or CMP set to 1.0 (top panel). Expression in Enh(f/f);Mx1-Cre divided by expression in Enh(f/f) marrow is shown for each RNA in each subset analyzed (bottom panel). Dashed red line is the 1.0 level for both graphs. GR—Gcsfr, MR—Mcsfr, GMRa—Gmcsfrα. B) Myeloid CFUs obtained from Enh(f/f) or Enh(f/f);Mx1-Cre mice exposed 4 wks earlier to pIpC were in enumerated after culture at 2E4 cells/mL in methylcellulose in the presence of M-CSF, G-CSF, or GM-CSF (mean and SD from six determinations).
Fig 7.
Effect of in vivo Enhancer Deletion on Progenitor Cell Proliferation and Survival.
A) Enh(f/f) or Enh(f/f);Mx1-Cre mice exposed 4 wks earlier to pIpC received a BrdU injection 3 hr prior to marrow harvest, followed by staining for surface markers to allow gating on the LSK, CMP, GMP, and MEP subsets and for intracellular incorporation of BrdU and 7AAD into DNA after fixation, permeabilization, and DNase exposure. G0/G1 (BrdU-7AAD-) and S phase (BrdU+) cells were then enumerated (mean and SD from three determinations). B) Marrow from Enh(f/f) or Enh(f/f);Mx1-Cre mice exposed 4 wks earlier to pIpC were stained for LSK, CMP, GMP, and MEP and for intracellular Ki67 and 7AAD. G0 (Ki67-7AAD-) cells were enumerated (mean and SD from three determinations). C) Marrow from a similar group of mice was stained for the same progenitors, for surface Annexin-V, and for permeability to 7AAD in the absence of fixation. Non-viable Annexin-V+7AAD+ (A+7+) and early apoptotic Annexin-V+7AAD- (A+7-) cells were then enumerated (mean and SD from three determinations).
Fig 8.
Effect of in vivo Enhancer Deletion on Functional Long-Term Hematopoietic Stem Cells.
A) Diagram of competitive transplantation assay. 2E5 CD45.2+ nucleated marrow cells from Enh(f/f);Mx1-Cre mice exposed 4 wks earlier to pIpC were mixed with equal numbers of CD45.1+ WT marrow cells and transplanted into lethally irradiated WT recipients. At 19 wks, peripheral blood (pB) and bone marrow (BM) cells were analyzed, and 1E6 marrow cells were transplanted into secondary transplant (2°TP) recipients, 1 recipient/donor. Those surviving were then analyzed 16 wks later. B) Percent of CD45.1+ or CD45.2+ cells amongst total marrow nucleated cells, and the percent of CD11b+Gr-1+ granulocytes, CD11b+Gr-1- monocytes, Ter119+ erythroblasts, B220+ B lymphoid cells, CD3+ T lymphoid cells, Lin-, Lin-Sca-1-c-kit+ (LK), or Lin-Sca-1+c-kit+ (LSK) cells within the CD45.1+ or CD45.2+ subsets. C) Percent of CD45.1+ or CD45.2+ cells amongst nucleated peripheral blood cells in primary transplant recipients (mean and SD; n = 7). D) Percent of CD45.1+ or CD45.2+ cells amongst nucleated peripheral blood cells in secondary transplant recipients (mean and SD; n = 3).
Fig 9.
Model for the Role of C/EBPα During Hematopoiesis.
In this model, HSC give rise to lymphoid-myeloid progenitors (LMP) and to MEP, MEP generate megakaryocyte (Meg) and erythroid (E) progenitors, and GMP give rise to granulocytic progenitors (G) in the presence of high-level C/EBPα and to monocytic progenitors (Mo) in the presence of low-level C/EBPα. CLP give rise to T and B cell progenitors in the absence and to bipotent B/Mo progenitors in the presence of C/EBPα. The latter diverge to B cell or monocytic progenitors, again correlated with and potentially guided by loss or retention of C/EBPα.