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First published online 30 April 2008
doi: 10.1242/dev.018200

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Activation of Eklf expression during hematopoiesis by Gata2 and Smad5 prior to erythroid commitment

Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, Box 1020, 1 Gustave Levy Place, New York, NY 10029, USA.

View larger version (46K): [in this window] [in a new window]   Fig. 1. EKLF expression is initiated prior to erythroid commitment in the mouse fetal liver. (A) Schematic of the Peklf-GFP transgene after site-specific, uni-directional integration into the Ainv18 homing site, yielding the Peklf-GFP ES cell clone. (B-D) Flow cytometric analysis of GFP (B) and CD71/Ter119 expression (C) in fetal liver cells derived from E13.5 Peklf-GFP embryos, as quantified in Table 1. GFP+/Ter119- cells selected for colony assays are marked in D. (E-H) Erythroid (CFU-E and BFU-E), megakaryocyte, granulocyte and macrophage progenitor-derived colonies from GFP+/Ter119- cells were quantified and compared with those derived from GFP-/Ter119- cells. Cells were morphologically assessed by cytospin and May-Grünwald/Giemsa staining. (E) Erythroid cells; (F) megakaryocyte; (G) granulocytes; (H) macrophages. Scale bars: 5 µm. (I) Enrichment is defined as a colony number greater than 10% of total colonies (red vertical line) because 10% of Ter119- sorted fetal liver cells at E13.5 are GFP+. The arithmetic mean of three independent experiments ±s.d. is shown.

View larger version (22K): [in this window] [in a new window]   Fig. 2. EKLF expression is initiated prior to erythroid commitment during EB differentiation. (A) GFP expression in relation to hematopoietic marker gene expression between days 2 and 8 of mouse EB differentiation. Levels of GFP, Eklf, Gata1, Gata2 and globin βH1 (bh1) mRNA in samples harvested at the indicated day after EB differentiation were monitored by qRT-PCR and normalized to 18S rRNA. Values are presented relative to the maximum expression level per gene, which was set to 1. (B) Flow cytometric analysis of GFP expression at day 5.5 of differentiation in EBs derived from Peklf-GFP ES cells. (C) Percentages of progenitor-derived colonies from sorted GFP+ EB cells in one representative experiment.

View larger version (63K): [in this window] [in a new window]   Fig. 3. All three cis-regulatory elements of the Eklf locus contain highly conserved Smad binding motifs adjacent to Gata sites. (A) Alignment of five mammalian Eklf genomic loci encompassing 17 kb (30 kb global alignment length) surrounding the Eklf transcription unit in between the two nearest neighboring genes, Gcdh and Dnase2a. Each sequence is represented as a black line (breaks demarcate gaps in the alignment), repeats or low complexity DNA are represented as blue bars, untranslated regions (UTR) as light tan bars and exons as brown bars. The degree of sequence conservation between species is expressed as an alignment score (y-axis) per nucleotide position (x-axis; global length). Peaks of conservation above 0.6 (red line) within non-coding regions are shaded in orange. The 950 bp region upstream of Eklf exon 1, exon 1 itself, and intron 1 are highlighted in light yellow. (B) Enlarged view of the aligned mouse Eklf sequence from -950 bp to the beginning of exon 2. The three conserved Eklf cis-regulatory elements are indicated (upstream enhancer, proximal promoter, intronic enhancer) together with blocks of four or more nucleotides of perfect homology (green bars), erythroid hypersensitive sites (EHS1 and EHS2, dark yellow bars) and previously known Gata and Cp1 sites (small red bars). Arrows indicate positions of newly identified Smad binding motifs (SBM) and WGATAR motifs. (C) Detail of seven-species alignment of all three Eklf cis-regulatory regions (upstream enhancer, proximal promoter, intronic enhancer) at the single nucleotide level. Newly identified Smad binding motifs (SBM1 through SBM10) are highlighted (blue), as are unassigned conserved blocks of perfect homology (green), WGATAR motifs (red), E-box motifs (yellow), Cp1 (pink) and putative Sp1 (brown) sites. The EHS1/GEG region within the upstream enhancer is underlined.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Gata factor and Smad binding motifs regulate Eklf expression in vivo. (A) Schematic of the Peklf-GFP reporter construct and its four derivatives, which were targeted to the Ainv18 ES cell line, that carry two GATA motif mutations (2xG/A) in the upstream enhancer (Peklf-2xG/A-GFP), or one GATA motif mutation in the proximal promoter (Peklf-G/A-GFP), two Smad binding motif deletions (2xSBM) in the upstream enhancer (Peklf-2xSBM-GFP), or an insertion of Eklf intron 1 (Peklf-intron-GFP). Positions of Eklf cis-regulatory elements and translation start codons (ATG) are indicated and color-coded as in Fig. 3. (B-E) qRT-PCR analysis of GFP transgene expression in the four derivative Ainv18 ES cell reporter clones between days 4 and 7 or 8 of EB differentiation. Expression levels were normalized to 18S rRNA or Gapdh. For Eklf and GFP, normalized levels are presented relative to a value of 1 on EB day 4, whereas for Gata2 (G2) and globin βH1 (bh1) the maximum expression level per gene is set to 1. Arithmetic mean ±s.d.; *, 0.01<P<0.05, n=3; **, 0.001<P<0.01, n=3. (Left) Endogenous Eklf (red) and GFP (green) expression from the Peklf-GFP clone, differentiated in parallel in each experiment, is shown for cross-comparison. (Right) Endogenous Eklf (red) and GFP (green) expression from each mutant clone (Peklf-2xG/A-GFP, Peklf-G/A-GFP, Peklf-2xSBM-GFP, Peklf-intron-GFP) is shown along with that of Gata2 (black) and globin βH1 (orange). Within each panel, high levels in Gata2 expression are indicative of a progenitor population in differentiating EBs prior to day 5.5, after which the onset of globin βH1 expression is indicative of erythroid commitment (expression patterns as in Fig. 2A).

View larger version (19K): [in this window] [in a new window]   Fig. 5. Gata2 to Gata1 switch in occupancy at Eklf cis-regulatory regions during G1E-ER-Gata1 and EB differentiation. Quantitative ChIP analysis of Gata2 and Gata1 binding to cis-regulatory regions of the Eklf or Gata1 locus as indicated. Occupancy values recorded at a non-conserved GATA motif in the Eklf 3'UTR serve as baseline signal. (A) G1E-ER-Gata1 cells before (white) and after (black) 14 hours of estradiol treatment. (B) Differentiating EBs before (day 4, white) or after (day 6, black) erythroid commitment. Normal rabbit immunoglobulin (IgG) ChIP serves as negative control. Arithmetic mean of three independent experiments ±s.d. is shown.

View larger version (27K): [in this window] [in a new window]   Fig. 6. Smad5 knockdown in mouse EBs results in reduced Eklf and Gata1 expression. (A) Schematic of the GFP-Intron-miR transgene after site-specific, uni-directional integration into the Ainv18 homing site, including the location of the miR-30 backbone and shRNA insertions into the GFP intron. (B) Flow cytometric analysis of GFP expression in the Smad5 shRNA-1 Ainv18 ES cell clone in doxycycline treated (24 hours, +dox) or untreated (-dox) EBs harvested at day 5 of differentiation. (C) Analysis of Smad5, Eklf and Gata1 expression by qRT-PCR in the Smad5 shRNA-1 clone. Untreated (-dox) or doxycycline-treated (24 hours; +dox) cells were harvested at day 5 of EB differentiation. To enrich for transgene-expressing populations, cells from untreated EBs were FACS sorted for GFP-negative cells (-dox/GFP-), whereas cells from dox-treated EBs were FACS sorted for GFP expression (+dox/GFP+) prior to being monitored for RNA expression. Expression levels were normalized to Gapdh and untreated samples were set to 1. (D) Analysis of Smad1, Smad5, Eklf, Gata1 and Gata2 expression by qRT-PCR in the shRNA-1 clone in cells harvested from EBs at day 5 of differentiation without FACS sorting. RNA was isolated from untreated (-dox) or doxycycline-treated (24 hours, +dox) cells as indicated. Expression levels were normalized to Gapdh and each untreated sample was set to 1. Arithmetic mean ±s.d. (E) Western blot analysis of Gata2, Eklf, Gata1 and GFP protein expression in shRNA-1, shRNA-2 and shRNA-control Ainv18 ES cell clones harvested at day 6 of EB differentiation. Lysates were prepared from unsorted EBs that had been treated with (+dox) or without (-dox) doxycycline for 48 hours. Hsp90 expression levels were used as a loading control. Molecular mass (kDa) markers for each blot are shown to the left.

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