Friend of GATA (FOG) Interacts with the Nucleosome Remodeling and Deacetylase Complex (NuRD) to Support Primitive Erythropoiesis in Xenopus laevis

Friend of GATA (FOG) plays many diverse roles in adult and embryonic hematopoiesis, however the mechanisms by which it functions and the roles of potential interaction partners are not completely understood. Previous work has shown that overexpression of FOG in Xenopus laevis causes loss of blood suggesting that in contrast to its role in mammals, FOG might normally function to repress erythropoiesis in this species. Using loss-of-function analysis, we demonstrate that FOG is essential to support primitive red blood cell (RBC) development in Xenopus. Moreover, we show that it is specifically required to prevent excess apoptosis of circulating primitive RBCs and that in the absence of FOG, the pro-apoptotic gene Bim-1 is strongly upregulated. To identify domains of FOG that are essential for blood development and, conversely, to begin to understand the mechanism by which overexpressed FOG represses primitive erythropoiesis, we asked whether FOG mutants that are unable to interact with known co-factors retain their ability to rescue blood formation in FOG morphants and whether they repress erythropoiesis when overexpressed in wild type embryos. We find that interaction of FOG with the Nucleosome Remodeling and Deacetylase complex (NuRD), but not with C-terminal Binding Protein, is essential for normal primitive RBC development. In contrast, overexpression of all mutant and wild type constructs causes a comparable repression of primitive erythropoiesis. Together, our data suggest that a requirement for FOG and its interaction with NuRD during primitive erythropoiesis are conserved in Xenopus and that loss of blood upon FOG overexpression is due to a dominant-interfering effect

Loss of circulating RBCs in FOG morphants is due to increased apoptosis.

<p>(A) TUNEL staining of circulating blood cells. White arrows indicate cells that are both propidium iodide and TUNEL positive. (B) Scatter plot showing the distribution of numbers of propidium iodide (PI) and TUNEL positive cells per high-power field in blood from uninjected control and FOG MO injected embryos. Red bars indicate the mean (<i>n = 3</i>). (C) Graph of the number of TUNEL positive cells as a percentage of total (propidium iodide positive) cells in uninjected control and FOG MO injected embryos. (D) qPCR measuring expression of <i>Bim1</i>, <i>globin</i> and <i>GATA-1</i> in FOG MO injected embryos at the tailbud stage. Expression is normalized to expression of <i>ODC</i> and reported as percent of uninjected control <i>(n = 3). Error bars reflect S.D. for both Northern and qPCR analyses. Paired t test results are as follows:</i> *, <i>p</i>≤<i>0.05.</i></p

Overexpression of both wild type and mutant forms of xFOG inhibit primitive erythropoiesis.

<p>(A) Northern blot of <i>globin</i> expression in tailbud stage embryos in which wildtype xFOG RNA was injected into the VMZ of eight-cell embryos (<i>n = 8</i>). (B) Schematic of MYC epitope tagged wild type and mutant xFOG isoforms. The MYC tag is in blue, the NuRD binding domain in red, the CtBP binding domain in yellow and the zinc fingers are in grey. (C) Northern blot of <i>globin</i> expression in tailbud stage embryos that overexpress mutant forms of xFOG RNA injected into the VMZ of eight-cell embryos (<i>n</i>≥<i>4</i>). Levels of <i>globin</i> expression are normalized to expression of <i>ODC</i> and reported as a percentage of control. <i>Error bars reflect S.D. Paired t test results are as follows: ***, p</i>≤<i>0.0005;</i> *, <i>p</i>≤<i>0.05.</i> ΔN = xFOGΔNuRD, ΔC = xFOGΔCtBP, ΔNΔC = xFOGΔNuRD/ΔCtBP, 4ZM = xFOG4ZM.</p

FOG is required for primitive erythropoiesis in <i>Xenopus laevis</i>.

<p>(A) Alignment of sequence surrounding the translation start site of the two <i>Xenopus FOG</i> alleles. Sequences to which FOG MOs bind are indicated by the black bars and the ATG start codon is in red. (B) Western blot of lysates from embryos injected with MYC epitope tagged wild type xFOG or xFOGr RNA, which harbors silent mutations that prevent MO binding, with either FOG MO or Control MO. Note that FOG MOs inhibit the translation of xFOG-MYC RNA but do not affect translation of xFOGr-MYC RNA. Actin is shown as a loading control. (<i>n = 3</i>) (C) Schematic of experimental design. MO and/or RNA is targeted to the ventral marginal zone (VMZ) of an eight-cell embryo. Injected embryos are cultured to the tailbud stage and assayed for <i>globin</i> expression by whole mount <i>in situ</i> hybridization or Northern analysis. (D) <i>In situ</i> hybridization analysis of <i>globin</i> expression (purple stain) in embryos injected with increasing doses of FOG MO (<i>n = 4</i>). Arrowheads indicate the anterior-most aspect of the VBI, derived from dorsal cells not targeted by injections. (E) Northern analysis of <i>globin</i> expression in embryos injected with increasing doses of FOG MO (<i>n</i>≥<i>3</i>). (F) Northern analysis of <i>globin</i> expression in embryos injected with FOG MO (40 ng), xFOGr RNA (200 pg), or both (<i>n</i>≥<i>4</i>). Levels of <i>globin</i> expression are normalized to expression of the housekeeping gene <i>ODC</i> and reported as a percentage of control in the graphs in panel E and F. <i>Error bars reflect S.D. Paired t test results are as follows: **, p</i>≤<i>0.005;</i> ***, <i>p</i>≤<i>0.0005.</i></p