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. 2013 Jan 14;210(1):71–84. doi: 10.1084/jem.20120993

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© 2013 Guiu et al.

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Figure 2. — Occupancy of the Gata2 promoter by HES-1 and Notch/RBPJ. (A) Relative expression of the indicated genes from E10.5 AGM after 3 d of 4-OH-tamoxifen treatments in explant cultures by qRT-PCR. Fold increase relative to the wild-type control after β2-Microglobulin and GAPDH normalization. Spearman test was used to assess the significance between number of wild-type alleles and gene expression indicated on the top of the figure. Correlation between number of wild-type Hes alleles and Gata2 expression was significant (P ≤ 0.001 and ρ = 0.985) but not for c-Myb (P = 0.126 and ρ = 0.985) and Runx1 expression (P = 0.072 and ρ = 0.591). The mRNA levels of Runx1, Gata2, and c-Myb are significantly up-regulated in cells with three to four deleted compared with zero to one deleted hes alleles (Student’s t test, P < 0.05). Bars represent the mean ± SD fold increase determination from two independent experiments. (B) Representative in situ hybridization of Gata2 in the wild-type (n = 2) or Hes mutant embryos (n = 2, each genotype). Images show transversal section of E10.5 embryos in the AGM region. Arrowheads indicate the structures (Ao, aorta; IAHC, intra-aortic hematopoietic cluster; m, mesonephros; nt, neural tube). Details of IAHC in the right panels correspond to the boxed areas in the left panels. Bars: (left) 200 µm; (right) 20 µm. (C) Graphical representation of the mouse Gata2 promoter showing the putative RBPJ- or HES-binding consensus found by bioinformatic analysis. Asterisks indicate the conserved sites in the human Gata2 locus (IS, specific promoter; IG, general promoter). (D) Sequence and location of the human and mouse conserved RBPJ- and HES-binding sites relative to IS and IG promoters. (E) ChIP with the anti–HES-1, anti-ICN1, anti-RBPJ, and anti-IgG antibodies from myeloid 32D cells and analysis of the Gata2 locus by qPCR. One representative of two experiments is shown. (F and G) ChIP with the anti–HES-1, anti-ICN1, anti-RBPJ, and anti-IgG antibodies from E10.5 AGM cells and analysis of the Gata2 locus by qPCR. One representative from three experiments is shown. (E–G) Error bars represent SD from qPCR values.