Mi2β is required for γ-globin gene silencing: temporal assembly of a GATA-1-FOG-1-Mi2 repressor complex in β-YAC transgenic mice

Abstract

Activation of γ-globin gene expression in adults is known to be therapeutic for sickle cell disease. Thus, it follows that the converse, alleviation of repression, would be equally effective, since the net result would be the same: an increase in fetal hemoglobin. A GATA-1-FOG-1-Mi2 repressor complex was recently demonstrated to be recruited to the -566 GATA motif of the (A)γ-globin gene. We show that Mi2β is essential for γ-globin gene silencing using Mi2β conditional knockout β-YAC transgenic mice. In addition, increased expression of (A)γ-globin was detected in adult blood from β-YAC transgenic mice containing a T>G HPFH point mutation at the -566 GATA silencer site. ChIP experiments demonstrated that GATA-1 is recruited to this silencer at day E16, followed by recruitment of FOG-1 and Mi2 at day E17 in wild-type β-YAC transgenic mice. Recruitment of the GATA-1-mediated repressor complex was disrupted by the -566 HPFH mutation at developmental stages when it normally binds. Our data suggest that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that either a trans-acting Mi2β knockout deletion mutation or the cis-acting -566 (A)γ-globin HPFH point mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis.

Figure 1. Expression of murine Mi2β and human and murine β-like globins in erythroid-specific conditional knockout Mi2β β-YAC transgenic mice.

Expression of murine Mi2β, human fetal γ-globin, human adult β-globin and murine adult β^maj-globin was analyzed by real-time qRT-PCR in adult blood samples. A) Murine Mi2β; B) Human γ-globin; C) Human β-globin; D) Murine β^maj-globin. Numbers represent the average of 6 animals (* indicates P<0.05).

Figure 2. Flow cytometry and cytospin analysis of blood from adult erythroid-specific Mi2β conditional knockout β-YAC transgenic mice.

A rabbit polyclonal anti-Mi2 antibody+secondary antibody (panels A–F) or an anti-human hemoglobin F FITC-conjugated antibody (panels G–L) was used to determine the percentage of cells expressing of Mi2 or HbF, respectively. Cytospins were prepared using an anti-human hemoglobin F FITC-conjugated antibody (panels M–P). Antibody employed, followed by sample: A) IgG control, wild-type β-YAC; B) Mi2, wild-type β-YAC; C–F) Mi2, floxed Mi2β μ'LCR β pr-Cre β-YAC 1, 2, 3 and 4, respectively; G) HbF, wild-type β-YAC; H) HbF, −117 Greek HPFH β-YAC; I–L) Mi2 floxed Mi2β μ'LCR β pr-Cre β-YAC 1, 2, 3 and 4, respectively. Cytospins: M) wild-type β-YAC; N) −117 Greek HPFH β-YAC; O–P) floxed Mi2β μ'LCR β pr-Cre β-YAC 1 and 2. Top panels, visible light images of cytospin cells; bottom panels, immunofluorescent images of same fields.

Figure 3. Murine Mi2β protein and human fetal γ-globin mRNA expression in CID-dependent bone marrow cells (BMCs) derived from Mi2β conditional knockout β-YAC transgenic mice.

mRNA and protein levels from in CID-dependent wild-type β-YAC and floxed Mi2β μ'LCR β pr-Cre β-YAC BMCs were analyzed by Western blotting and real-time qRT-PCR, respectively. A) Mi2 Western blotting using an anti-Mi2 antibody; B) γ-globin mRNA.

Figure 4. Temporal recruitment of the GATA-1-FOG1-Mi2 complex to the −566 ^Aγ-globin GATA motif in wild-type β-YAC.

ChIP analysis of the −566 ^Aγ-globin GATA site in fetal liver samples from post-conception days E12–E18 wild-type β-YAC transgenic conceptuses. The relative occupancy of the −566 region of the ^Aγ-globin gene (GenBank coordinates 38772–38937 from accession file GI455025) by GATA-1, FOG-1, and Mi2 proteins (grey bars) is shown in comparison to IgG control samples (black bars). ChIP was carried out using isotype-matched immunoglobulin (IgG), GATA-1, FOG-1 and Mi2 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) as described in Materials and Methods. The results are the average of at least two experiments and each experiment was performed in duplicate (* indicates P<0.05 and ** P<0.01). A) GATA-1 protein occupancy; B) FOG-1 protein occupancy; C) Mi2 protein occupancy.

Figure 5. GATA-2 binding at the −566 GATA site of the ^Aγ-globin gene in wild-type β-YAC.

A) GATA-2 protein occupancy at the −566 ^Aγ-globin GATA site by ChIP analysis. B) GATA-2 protein occupancy at the murine Gata-2 gene −2.8 Kb binding site by ChIP analysis. Binding of GATA-2 at the murine Gata-2 locus was used as a positive control for GATA-2 occupancy. ChIP was performed as described in the legend to Figure 4; labeling is also as illustrated in Figure 4.

Figure 6. Human β-like globin expression in adult −566 ^Aγ-globin HPFH β-YAC transgenic mice.

(A and B) Expression of the fetal γ- and adult β-globin was analyzed by real-time qRT-PCR and the data was normalized to mouse α-globin or Gapdh gene expression. (A) Human fetal γ-globin mRNA expression; B) Adult β-globin mRNA expression. Lines are indicated at the bottom of the plot. Results are the average of triplicate experiments from three animals of lines 20. * indicates P<0.05. (C–F) Flow cytometry analysis was performed using a mouse monoclonal anti-γ-globin antibody to determine the percentage of F cells. C) Wild-type β-YAC; D) −117 Greek HPFH β-YAC; E) −566 ^Aγ-globin HPFH β-YAC transgenic line 20 (1), F) −566 ^Aγ-globin HPFH β-YAC transgenic line 20 (2). Flow cytometry controls are identical to those shown in Figure 2A, 2B and 2G since the experiments were performed together. (G–I) Cytospins were prepared using an anti-human hemoglobin F FITC-conjugated antibody. G) Wild-type β-YAC; H) −117 Greek HPFH β-YAC; I) −566 ^Aγ-globin HPFH β-YAC transgenic line 20. Top panels, visible light images of cytospin cells; bottom panels, immunofluorescent images of same fields.

Figure 7. Disruption of the γ-globin silencing in the −566 ^Aγ-globin HPFH β-YAC transgenic mice.

ChIP analysis of the −566 ^Aγ-globin GATA site and determination of human fetal γ-globin mRNA expression in fetal liver samples from post-conception days E16 and E18–566 ^Aγ-globin HPFH β-YAC line 20 transgenic mice. A) Relative occupancy of the −566 region by GATA-1, FOG-1, and Mi2 proteins (gray bars) is shown in comparison to the IgG control samples (black bars). ChIP was performed as described in the legend to Figure 4. B) Human fetal γ-globin mRNA expression.

Figure 8. Temporal repression model.

A) GATA-2 occupies the −566 ^Aγ-globin GATA gene silencer at day E12. B) Loss of transcriptional activators. The loss of GATA-2 and other transcriptional activator occupancy at the proximal promoter of the ^Aγ-globin gene at days E13 to E15 is the initial step in the silencing cascade. C) GATA-1 occupies the −566 ^Aγ-globin GATA gene silencer at day E16. A post-translational modification of GATA-1 or other determinants might play a role in this occupancy, dictating silencing versus activation by GATA-1. D) Formation of the GATA-1 repressor complex. GATA-1 recruits FOG-1 and Mi2β (and presumably other NuRD components) to the −566 ^Aγ-globin GATA silencer motif. Ikaros also may be recruited to this silencer region at a nearby Ikaros binding site where it interacts with GATA-1 and Mi2β . E) Disruption of the −566 GATA site γ-globin gene repressor. γ-globin gene expression is reactivated in adult definitive erythropoiesis by preventing the recruitment of theGATA-1-FOG-1-Mi2 complex, either by the presence of the −566 ^Aγ-globin HPFH mutation or by knocking down the Mi2 subunit of the repressor complex.