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1 Laboratory of Genetics (B-3), and 2 Developmental Biology Laboratory, Graduate School of Frontier Biosciences and Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
3 Solution-Oriented Research for Science and Technology, Japan Science and Technology Corporation, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
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Abstract |
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N/N). The mDomN/N mice died on embryonic day 11.5 (E11.5), and displayed an anemic appearance and slight deformity of the neural tube. DNA microarray and quantitative RT-PCR analyses revealed that all of the embryonic globin genes and a globin chaperone gene were poorly expressed in the mDomN/N embryo and yolk sac on E8.5, indicating that primitive erythropoiesis was impaired. A hematopoietic colony assay indicated that the hematopoietic activity of the yolk sac was significantly blocked in the mutant mice. We also found that the expression of a limited set of Hox genes, including Hoxa7, Hoxa9 and Hoxb9, was drastically enhanced in the mDomN/N yolk sacs. These results suggest that p400/mDomino plays a critical role in embryonic hematopoiesis by regulating the expression of developmentally essential genes such as those in the Hox gene cluster. |
Introduction |
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p400/mammalian Domino (p400/mDomino, Gene symbol: Ep400) is an Swr1-class DNA-dependent ATPase that was identified as a component of a novel adenovirus E1A-binding complex (Fuchs et al. 2001), and as an interaction partner for a myeloid-specific transcription factor, MZF-2 A (Ogawa et al. 2003a,b). The human and mouse p400/mDomino proteins contain a bipartite SWI2/SNF2-type ATPase domain, a SANT (SWI3, ADA2, NCoR and TFIIIB) domain, an HSA (helicase- and SANT-associated) domain and a glutamine-rich domain, and are highly homologous to the Drosophila Domino A protein and distantly related to the C. elegans ssl-1 and yeast Swr1 proteins (Fuchs et al. 2001; Ruhf et al. 2001; Ogawa et al. 2003b; Ceol & Horvitz 2004; Kobor et al. 2004). The p400/mDomino-containing protein complex consists of more than ten subunits, including the Tip60 histone acetyltransferase and a PI3K family protein kinase TRRAP, and closely resembles the Drosophila dTip60 complex, which contains Domino A, dTip60 and dTra1 (Fuchs et al. 2001; Cai et al. 2005; Jin et al. 2005). Interestingly, this metazoan Tip60-Domino complex seems to correspond to a homologous merge of the yeast SWR1 complex and the NuA4 histone acetyltransferase complex (Doyon & Cote 2004; Cai et al. 2005; Jin et al. 2005). Another mammalian Swr1-class ATPase, SRCAP, is also known to form a multisubunit complex, which shares some components with the Tip60-mDomino complex but does not contain Tip60, suggesting a functional difference between p400/mDomino and SRCAP (Cai et al. 2005; Eissenberg et al. 2005; Jin et al. 2005; Ruhl et al. 2006).
Recent genetic and biochemical studies have demonstrated that the yeast and Drosophila SWR1-class complexes execute a unique remodeling function, called histone variant exchange. The Saccharomyces cerevisiae SWR1 complex associates with Htz1 protein, a histone variant categorized as H2A.Z, and is essential for the deposition of Htz1 into chromatin via its ATP-dependent catalytic activity, thereby replacing histone H2A with Htz1 in nucleosomal arrays (Krogan et al. 2003, 2004; Kobor et al. 2004; Mizuguchi et al. 2004). On the other hand, the Drosophila dTip60-Domino complex possesses activities to acetylate the phosphorylated form of the H2Av histone variant, and to subsequently exchange it with unmodified H2Av in nucleosomal arrays (Kusch et al. 2004). Thus, these SWR1-class remodelers are responsible for the regulated deposition of selective histone H2A variants into nucleosomes, which probably plays essential roles in the epigenetic regulation of gene expression as well as in DNA repair (Jin et al. 2005; Sarma & Reinberg 2005; van Attikum & Gasser 2005). Functional studies using mammalian cultured cell lines have shown that p400/mDomino is involved in the expression of genes that are regulated by transcription factors such as c-Myc, MZF-2 A and E2F (Frank et al. 2003; Ogawa et al. 2003b; Taubert et al. 2004), and plays significant roles in p53/p21-dependent cellular senescence, in E1A-mediated transformation, and in E1A- or DNA damage-induced apoptosis (Fuchs et al. 2001; Chan et al. 2005; Samuelson et al. 2005; Tyteca et al. 2006). Together, these findings suggest that p400/mDomino functions in various biological processes through its role in chromatin remodeling activities. However, its physiological roles in mammalian development and organogenesis remain unknown.
Here, we report that the targeted mutation of p400/mDomino in mice caused early embryonic lethality with profound defects in yolk sac hematopoiesis. The expressions of embryonic globin genes and a globin chaperone gene were markedly reduced in the mDomino-mutated embryos, whereas the expressions of a set of Hox genes were drastically increased, suggesting a critical role for p400/mDomino in the epigenetic regulation of developmentally controlled genes. Intriguingly, Drosophila Domino is essential for the development of fly blood cells, hemocytes, and domino mutations show a genetic interaction with Polycomb group (PcG) and Trithorax group (TrxG) genes, the epigenetic regulators for homeotic gene expression (Braun et al. 1997, 1998; Ruhf et al. 2001). These findings may indicate that the mammalian and Drosophila Tip60-mDomino complexes play conserved roles in the development of analogous tissues, via their chromatin-remodeling activities.
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Results |
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The targeting constructs for the mouse mDomino gene are shown in Fig. 1A. The 1.56 kb region from the KpnI site in exon 2 to the HindIII site in intron 2 of the p400/mDomino gene was replaced with a neomycin-resistance gene cassette (Neo). Mouse embryonic stem (ES) cell clones containing the mDomino-targeted allele were identified by PCR (not shown) and Southern blot analyses (Fig. 1B), and used to generate gene-targeted mice. Although we had designed the targeting vector with the aim of generating mDomino-null mice, this construct resulted in expression of an N-terminally truncated protein of mDomino, as described below. Thus, we named the mutated allele as mDomN. Mice heterozygous for the mutated allele (mDom+/N) were crossed to generate homozygotes, but the genotypic analysis of 121 pups showed 53 wild-type mice, 68 heterozygotes and no homozygotes (Table 1), suggesting that the homozygous mutation was embryonically lethal. Characterization of the mutant embryos through development showed that the homozygous embryos (mDomN/N) were developmentally arrested around embryonic day 9.5 (E9.5), and no mutants were found viable beyond E11.5 (Table 1). Since mDom+/N mice were phenotypically normal and fertile, the truncated mDomino protein was unlikely to behave as a dominant-negative mutant.
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Since the lethality of mDomN/N mice in utero suggested that mDomino plays a critical role in early embryogenesis, we first examined the expression of mDomino in embryos. Quantitative RT-PCR analysis revealed that the mDomino gene was expressed in both the yolk sac and embryo proper at E8.5 (data not shown), and whole mount in situ hybridization using an anti-sense probe of exon 2 showed ubiquitous expression of the mDomino transcript throughout the E8.5 embryo (Fig. 2A).
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N and mDomN/N embryos at E8.5, and analyzed them for mDomino expression by immunoblotting using antibodies specific for the N- or C-terminal region of mDomino (Ogawa et al. 2003b). As shown in Fig. 2B, a 350-kDa product, the predicted size of mDomino, was detected with the anti-Dom-N and anti-Dom-C antibodies in the extracts from wild-type and mDom+/N MEFs but not in those from mDomN/N MEFs. On the other hand, a smaller product (~300 kDa) was detected by the anti-Dom-C antibody in the mDom+/N and mDomN/N extracts. This product was not recognized by the anti-N antibody, and appeared to be a truncated mDomino protein lacking the large N-terminal region encoded by exon 2. To test this, we analyzed RNAs from the wild-type and mDom-targeted MEFs by RT-PCR using primer sets specific for regions between exon 1 and exon 3, 4 or 5 of the mDomino gene, and found that an aberrant mDomino mRNA produced by splicing between exons 1 and 3, which was undetectable in wild-type MEFs, was significantly expressed in mDomN/N and mDom+/N MEFs (data not shown). Thus, the targeted replacement of exon 2 with the neo cassette happened to result in expression of the aberrant mDomino mRNA, which was likely to be translated from an internal ATG codon in exon 3 to produce the mutant protein lacking the N-terminal 471 amino acids. The apparent molecular mass (~300 kDa) of the small product in Fig. 2B was consistent with the calculated molecular mass (282.9 kDa) of the predicted mutant protein.
Immunofluorescence analysis of these MEFs using the anti-Dom-N and anti-Dom-C antibodies showed nuclear staining of both wild-type and mDomN/N cells, indicating that the truncation of the N-terminal domain did not change the nuclear localization of mDomino protein (Fig. 2C). In the nucleus, mDomino was detected as granular dots that were excluded from nucleoli, as shown by fibrillarin staining, and from the DAPI-dense heterochromatic region (Fig. 2D).
Reduced expression of erythroid-specific genes in mDomN/N embryos
Although the mDomN/N embryos displayed a grossly anemic appearance, enlarged pericardial sacs and growth retardation around E9.5–10.5 (Fig. 3A), the mutant embryos at E8.5 were morphologically indistinguishable from their wild-type littermates, except they displayed a wavy neural tube (Figs 2A and 3B). Hematoxylin–eosin staining of sections of the developing neural tube did not reveal evident histological abnormalities that would explain the slight deformity of the neural tube (data not shown). To characterize the neural tube formation of the mutant embryos, we examined the expression of several genes in E8.5 embryos by whole mount in situ hybridization. However, this analysis revealed that neuroepithelial or somite marker genes, such as Pax6, Lunatic-fringe, Sox2, Patched, Ngn2, Mash1, Fgf8, Necab, Id1 and Id3, were expressed in similar patterns in the wild-type, mDom+/N and mDomN/N embryos (Fig. 3B, and data not shown).
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N/N embryos using DNA microarrays consisting of about 36 000 mouse genes. Comparative analysis of the wild-type and mDomN/N embryos (GEO accession number: GSE5358
[NCBI GEO]
) revealed that the expression of ~40 genes was significantly reduced in the mDomN/N E8.5 embryos (with a normalized signal intensity of wild-type > 5.0, and signal intensity ratio of wild-type to mDomN/N greater than twofold), of which 17 genes showed a > 2.5-fold difference (Table 2). Although seven of the genes were functionally unannotated, the array analysis revealed that the expression of the embryonic globin genes Hba-x, Hbb-y and Hbb-bh1 was much lower (4%–21% of the wild-type levels) in the mDomN/N embryos. The gene expression of Hba-a1, which is expressed in both embryonic and adult erythrocytes in mice (Fantoni et al. 1981), and of an erythrocyte-specific globin chaperone protein, AHSP (
-hemoglobin stabilizing protein) (Kihm et al. 2002), was also reduced in the mDomN/N embryos. These results suggest that genes critical for primitive erythrocytes were impaired in the mDomN/N embryos. A reduced expression of the Zic1, Foxd1 and Rnf110 genes in the mutant embryos was also detected (Table 2). Zic1 and Foxd1 are zinc finger- and forkhead box-transcription factors, respectively, and are implicated in early neurogenesis (Herrera et al. 2004; Grinberg & Millen 2005). Rnf110 (Ring finger protein 110, also known as Mel-18, Zfp144 or Pcgf2) is a member of the mammalian PcG genes, which are homologous to Drosophila Posterior sex combs, and is known to play roles in the regulation of Hox gene expression (Akasaka et al. 2001). Thus, the expression of various genes involved in early embryogenesis appeared to be impaired by the mDomino mutation. We could not identify genes that were significantly up-regulated in the mDomN/N embryos compared with the wild-type embryos by the DNA array analysis.
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To confirm the results of the DNA array analysis, we performed quantitative, real-time RT-PCR using the total RNA from E8.5 embryos. Consistent with the microarray data, the embryonic globin and AHSP genes were abundantly expressed in the wild-type embryos, but their mRNA levels were much lower (< 10% of the wild-type level) in the mDomN/N embryos (Fig. 4A, left). We also examined the expression of these genes in the yolk sac, since the hematopoietic events begin in the extra-embryonic blood islands of the yolk sac at E7.0 and the yolk sac hematopoiesis still dominates at the E8.5 stage (Dzierzak et al. 1998). The mRNA levels of the erythroid-specific genes in the mDomN/N yolk sac at E8.5 were about 15%–50% of the levels in the wild-type yolk sac (Fig. 4A, right). To examine the protein levels of the embryonic globins, we performed an immunoblot analysis of the E8.5 embryo proper and yolk sac using a rabbit anti-mouse embryonic hemoglobin EI (x 2y2) polyclonal antibody, which recognizes both the embryonic x-globin (encoded by Hba-x) and y-globin (encoded by Hbb-y) chains (Miwa et al. 1991), with strong preference for the x-globin chain (our unpublished observation). The immunoblot analysis demonstrated that the protein level of x-globin was dramatically lower than wild-type in the mDomN/N embryo proper (Fig. 4B, left), and significantly lower in the mDomN/N yolk sac, although to a lesser extent (Fig. 4B, right). These results suggest that the mDomino mutation impaired the primitive erythropoiesis that occurs in the yolk sac, which in turn resulted in a lack of blood cells in the embryo proper. The severe reduction of globin levels in the mDomN/N embryo proper in comparison with the yolk sac may be partly due to inefficient supply of erythrocytes through the nascent allantoic vasculature, although the chorio-allantoic fusion seemed to occur at E8.5 (6–8 somite pairs) without apparent morphological abnormality (data not shown).
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N E8.5 embryos generated dozens of hematopoietic colonies, whereas the mDomN/N yolk sac cells yielded only a few colonies (Fig. 4C). DAF staining of the resulting colonies showed that the majority of the colonies arising from the mDom+/+ and +/N yolk sacs consisted mostly of hemoglobin-positive erythroid cells (74% and 66%, respectively). The mDomN/N colonies, although the colony number was reduced, contained the same proportion of erythroid colonies (67%). This result suggests that there is a cell-autonomous defect in the yolk sac hematopoietic precursors in the mDomN/N embryos. The mDomino mutation enhances the expression of Hox genes in the yolk sac
Previous studies suggested that the Drosophila domino gene has a repressive function on homeotic genes via interactions with PcG and TrxG genes (Ruhf et al. 2001). A mammalian homologue of Drosophila trithorax, mixed-lineage leukemia (MLL), is known to positively regulate the expression of a set of Hox genes, including Hoxa7 and a9, which appear to play important roles in MLL-dependent leukemogenesis and hematopoiesis (Hess et al. 1997; Yu et al. 1998; Ayton & Cleary 2003; Ernst et al. 2004; Milne et al. 2005). These observations prompted us to examine whether the mDomino mutation could perturb the expression of these Hox genes in early embryos. When compared with the wild-type yolk sac, the mDomN/N yolk sac displayed a markedly increased expression of the Hoxa7 and Hoxa9 genes (eight- and tenfold, respectively), while the mDomN/N embryo proper showed comparable to or slightly lower expressions of these genes than the wild-type embryo proper (Fig. 5A). The expression levels of other genes in the HoxA locus (Hoxa4, a5, a6 and a10) were comparable between the mDom+/+ and mDomN/N yolk sacs (Fig. 5B) and embryos proper (data not shown). We further examined the expression of paralogous genes in other Hox clusters, and found a drastic increase in Hoxb9 expression in the mutant yolk sacs (Fig. 5B). The expression levels of other paralogous genes (Hoxb6, b7, b8, c8, c9 and d8) were comparable between the wild-type and mutant yolk sacs (Fig. 5B, and data not shown). The expression levels of the ß-actin gene (Fig. 4A) and hematopoietic marker genes Scl, Lmo2 and GATA2 (data not shown) were comparable in the wild-type and mutant yolk sacs. These results indicate that the mDomino mutation resulted in the mis-expression or de-repression of only a certain set of Hox genes in the yolk sac cells.
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Discussion |
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In this report, we generated mutant mice of an SWR1-class ATPase p400/mDomino, which exhibited embryonic lethality around E9.5. The aberrant splicing of the transcript from the targeted gene resulted in the expression of the N-terminally truncated mDomino protein. Although we have not succeeded in determining whether the truncated protein is active in the chromatin-remodeling function, it still contains the entire regions of the HSA, ATPase, SANT and glutamine-rich domains, suggesting that the truncated mDomino is likely to retain at least the DNA-dependent ATPase/helicase activity. Thus, we note that the phenotypes observed here would reflect hypomorphic properties of this mutant but not those of the null mutation. It was unlikely that the N-mDomino behaved as a dominant-negative mutant, since no apparent phenotype was observed in the heterozygous mice.
Although the mDomN/N E8.5 embryos did not show apparent morphological abnormality except for a slight deformity of the neural tube, DNA microarray and quantitative RT-PCR analyses revealed that the expression of embryonic globin genes was reduced in the mDomN/N embryos proper and yolk sacs. Consistent with this observation, the hematopoietic colony-forming activity was severely impaired in the mDomN/N yolk sac. These results indicate that mDomino plays a critical role in primitive hematopoiesis in the yolk sac. It is widely known that mid-gestation lethal phenotypes accompanied with hematopoietic defects shown by gene-knockout mice are often attributed to placental insufficiency. However, since our analyses of the mDom mutant mice were performed at E8.5 (5–12 somite pairs), the decrease in primitive hematopoiesis is likely due to the intrinsic defect in proliferating and/or differentiating ability of the hematopoietic progenitors in the yolk sac, although we cannot exclude a possibility that a functional defect of the nascent chorio-allantoic placenta caused the more severe reduction of mDomN/N erythroid cells in the embryo proper than in the yolk sac. The hematopoietic phenotype of the mDomN/N mice may reflect a functional conservation of homologous genes in mammalian and fly development. The Drosophila domino gene was originally identified as a loss-of-function mutation characterized by the total absence of circulating hemocytes, the blood cells for innate immunity in Drosophila (Braun et al. 1997, 1998), and was later shown to be required for cellular viability and proliferation in developing tissues as well as for oogenesis (Ruhf et al. 2001).
Cued by the genetic interaction between the domino gene and PcG/TrxG genes in Drosophila (Ruhf et al. 2001), we investigated Hox gene expression in the mutant mice, and found that a subset of Hox genes, Hoxa7, Hoxa9 and Hoxb9, were over-expressed in the mDomN/N yolk sacs. Ectopic expression or de-repression of these Hox genes has been observed in mice lacking mammalian PcG genes such as Bmi-1 and Mel-18 (Hanson et al. 1999; Akasaka et al. 2001; Cao et al. 2005), whereas Hox gene silencing has been demonstrated in cells from MLL-deficient mice (Yu et al. 1998; Hanson et al. 1999; Ernst et al. 2004). Recent studies demonstrated that MLL specifically associates with a subset of its target loci, including the Hoxa7 and Hoxa9 genes (Milne et al. 2005). In this context, mDomino is likely to play a role in controlling Hox gene expression in the yolk sac, where mDomino would function cooperatively with PcG gene products and/or antagonistically with MLL. This hypothesis is consistent with the function of Drosophila domino, which has a repressive effect on homeotic gene expression via an enhancing interaction with PcG genes and an opposing interaction with TrxG members (Ruhf et al. 2001). A physical association of p400/mDomino with Enhancer of Polycomb (EPc) in a large nuclear complex (Fuchs et al. 2001; Cai et al. 2005) supports this idea. MLL is a histone methyltransferase specific for histone H3-Lys4, whose methylation is positively related to active gene expression and the maintenance of this expression (Milne et al. 2005). In contrast, two Polycomb repressive complexes (PRCs), PRC2 and PRC1, possess distinct histone-modifying activities for H3-Lys27 methylation and H2A-Lys119 monoubiquitylation, respectively, both of which appear to play essential roles in gene silencing and the maintenance of this silenced state (Cao et al. 2005; Martin & Zhang 2005). Therefore, the p400/mDomino chromatin remodeling complex may play a role in linking the TrxG/PcG-mediated histone modifications with the ATP-dependent histone variant exchange for the alternation and/or maintenance of chromatin organization, although we have no experimental evidence for this hypothesis.
The molecular mechanism by which p400/mDomino regulates embryonic hematopoiesis remains unknown, but accumulating evidence suggests that the strictly regulated expression of Hox cluster genes by MLL and PcG members is essential for mammalian hematopoiesis, and a malfunction of MLL or PcG causes hematopoietic disorders such as leukemia/lymphoma or progenitor reduction. MLL-deficient hematopoietic progenitors exhibit a marked reduction in their ability to proliferate and differentiate, which is rescued by the expression of a range of Hox genes (Hess et al. 1997; Ernst et al. 2004). On the other hand, the Bmi-1 and Rae28 PcG genes are required for the proliferative potential, self-renewal and maintenance of hematopoietic stem cells (van der Lugt et al. 1994; Ohta et al. 2002; Lessard & Sauvageau 2003; Park et al. 2003; Iwama et al. 2004; Kim et al. 2004). Although the increased expression of the limited set of Hox genes cannot be simply correlated with the hematopoietic defects observed in the yolk sac of mDomN/N mice, the dysregulated Hox gene expression may contribute, at least in part, to the impaired yolk sac hematopoiesis.
Our studies have shown that mDomino is ubiquitously expressed in early embryos and adult tissues (Ogawa et al. 2003b), and its mutation seems to affect not only primitive hematopoiesis but also neural tube formation and cardiac development. Although a detailed investigation of mDomN/N mice in later developmental stages is hampered by the embryonic lethality, future studies using mice with null mutation or conditional gene-targeted mice would help to elucidate the physiological roles of mDomino in definitive hematopoiesis and the development of other tissues and organs.
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Experimental procedures |
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To construct the targeting vector, a neomycin-resistance gene (Neo cassette) was inserted between the KpnI site in exon 2 and the HindIII site in intron 2 of the mouse mDomino gene, cloned from a mouse 129/Sv genomic DNA library. The diphtheria toxin A fragment gene (DTA cassette) for negative selection was ligated to the 3' end of the 3' homologous arm (Fig. 1A). Production of mDom+/N mice was performed as described (Ueda et al. 2004). In brief, mouse R1 ES cells were transfected with the targeting vector by electroporation, and G418-resistant clones were screened for homologous recombination by PCR. ES clones carrying the single mDomino-targeted allele were injected into BDF1 blastocysts, which were then implanted into recipient ICR female mice. Chimeric mice with a high ES contribution were crossed to C57BL/6 females to yield mDom+/N mice. Germ-line transmission was identified by coat color and then confirmed by PCR. mDomino heterozygotes were intercrossed to generate homozygous mutants. All mice were housed in a specific pathogen-free facility at Osaka University Medical School and all animal experiments were carried out in accordance with protocols approved by the Osaka University Medical School Animal Care and Use Committee (Osaka, Japan).
Genomic DNA for PCR was prepared from tail snips from infant mice or the yolk sacs from embryos. The genotype of the mDomino locus was determined by polymerase chain reaction (PCR) using a mixture of three specific primers: mDom-2S (5'-AGAATGTCCAACATCAGCTGCAGAGGTCTA-3'), mDom-2AS (5'-ATCATGTTTCCCTGTAGATGTTGCAAGAAG-3') and NeoR (5'-GATTCGCAGCGCATCGCCTTCTATCG-3') as described (Ueda et al. 2004). The amplified bands corresponding to the wild-type and mutated alleles of the mDomino gene were 1.18 and 0.7 kb in length, respectively. Southern hybridization analysis was performed as described (Ueda et al. 2004).
Whole mount in situ hybridization
Mouse embryos were isolated and fixed in 4% paraformaldehyde overnight at 4 °C, dehydrated in a graded ethanol series, and then used for the procedure. Whole mount in situ hybridization of the E8.5 embryos was performed using digoxigenin-labeled probes and color development with NBT (Nitro blue tetrazolium chloride)/BCIP (5-Bromo-4-chloro-3-indolyl phosphate, toluidine salt), as described (Maruhashi et al. 2005). The 993-bp RsaI fragment (235–1227, corresponding to a region in exon 2) of the mDomino cDNA was cloned in pBluescript II SK + and used for preparing the anti-sense probe. The RNA probes were prepared by transcription of the linearized plasmid with T3 or T7 RNA polymerase (Promega), using digoxigenin-11-UTP (Roche). After hybridization and color development, the embryos were observed with a stereomicroscope (Stemi DV4, Zeiss).
Preparation of mouse embryonic fibroblasts (MEFs)
Embryo-derived fibroblasts were prepared from E8.5 embryo proper as described (Xu et al. 1999), with some modifications. Briefly, E8.5 embryos from heterozygous breeding pairs were dissected from the decidual tissue and isolated from the yolk sac, and then transferred to individual wells in a 24-well tissue culture plate. Each embryo was trypsinized, dissected by pipetting and cultured in medium containing high-glucose DMEM (Invitrogen), 30% FCS (Invitrogen) and 50 µM 2-mercaptoethanol in a new tissue culture plate. Confluent cells were passaged in the same medium containing 10% FCS.
Immunoblot and immunofluorescence analyses
For the detection of endogenous mDomino protein, MEFs were lyzed directly in Laemmli's sample loading buffer and heated for 30 min at 85 °C and for 5 min at 95 °C. The samples were separated by electrophoresis on a 7.5% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane filter (Millipore). Immunoblot analysis was carried out using the enhanced chemiluminescence system (Perkin-Elmer) with rabbit anti-Dom-N or anti-Dom-C polyclonal antibody (Ogawa et al. 2003b) and peroxidase-conjugated goat anti-rabbit immunoglobulin (Dako). For the detection of the embryonic globin proteins, each embryo proper or yolk sac from litter-matched embryos at E8.5 was lyzed as described above. The lysates were separated by electrophoresis on a 15%–25% SDS-polyacrylamide gel, and analyzed by immunoblotting using rabbit anti-mouse embryonic hemoglobin EI polyclonal antibody (a kind gift from Dr Tadao Atsumi) (Miwa et al. 1991). As a loading control, the membranes were reblotted with an anti-actin monoclonal antibody (clone C4, MP Biomedicals).
For immunofluorescence staining, cells cultured in four-well slide chambers (#154526, Nalgene Nunc) were fixed with 4% paraformaldehyde for 10 min, permeabilized with phosphate-buffered saline (PBS) containing 0.2% Triton X-100 for 20 min at room temperature, blocked with PBS containing 5% skim milk for 30 min, and then incubated with the affinity-purified anti-Dom-N antibody and mouse anti-fibrillarin monoclonal antibody (Upstate Biotechnology) for 60 min at 37 °C. The samples were then washed with PBS, and incubated with the Alexa Fluor 488-conjugated anti-mouse IgG antibody (Invitrogen) and Cy3-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories), for 30 min at 37 °C. The nuclei were counterstained with DAPI, and cells were observed by fluorescence microscopy.
Gene array analysis
Gene array analysis was performed as a ‘custom order’ by Kurabo with a CodeLink Mouse Whole Genome Bioarray (GE Healthcare Bio-Sciences), which displays probe DNAs for about 36 000 mouse genes and expressed sequence tags. Total RNA was prepared from litter-matched embryos at E8.5 (mDom+/+, n = 42; mDomN/N, n = 45) using the RNeasy extraction kit (QIAGEN). Double-stranded cDNA was then prepared from the RNA and was transcribed in vitro with biotin-labeled UTP as a substrate. The complementary RNA was used as a probe for hybridization of the array, and the hybridized RNA was detected with Cy5-conjugated streptavidin. The arrays were scanned using a Biochip Reader (Applied Precision), and the array image was analyzed with CODELINK SYSTEM Software (GE Healthcare Bio-Sciences).
Real-time reverse transcriptase-PCR (RT-PCR) for quantitative analysis of mRNA
Total RNA was prepared from litter-matched embryos proper (mDom+/+, n = 42; mDomN/N, n = 45) and yolk sacs (mDom+/+, n = 8; mDomN/N, n = 10), as described above. Single-stranded cDNA was synthesized from the total RNA (0.5 µg) using an oligo-(dT) primer and Superscript III (Invitrogen). Real-time RT-PCR analysis was carried out using the LightCyclerFastStart DNA Master SYBR Green I kit (Roche), as described (Iida et al. 2005), under the following conditions: 40 cycles of 15 s at 95 °C, 5 s at 62 °C and 20 s at 72 °C. The primer sets used for the real-time RT-PCR were listed in Supplementary Table S1. The specific amplification of each targeted cDNA was confirmed by melting temperature analysis and by the presence of a single PCR product on agarose gel electrophoresis. In all real-time RT-PCR experiments except for Fig. 5B, cloned cDNA fragments (102–108 copies per reaction) containing the respective target region were amplified in parallel and used as standards, and the sample data were normalized against the standards and expressed as copy numbers of target mRNA per nanogram of total RNA. In the experiments in Fig. 5B, the sample data were normalized against the ß-actin standard curve, and were expressed as relative mRNA levels in arbitrary units, where the mRNA level of ß-actin was defined as 106 units.
Hematopoietic colony assay
E8.5 embryos including the yolk sac from wild-type, mDom+/N and mDomN/N littermates were treated with PBS containing 20% FCS and 0.1% collagenase D (Roche) at 37 °C for 60 min, and passed through 27-gauge needles to obtain single-cell suspensions. The cells were washed with PBS containing 20% FCS and then plated in 1% methylcellulose/Iscove's MDM containing 15% FCS, 10 µg/mL insulin, 200 µg/mL transferrin, 50 ng/mL stem cell factor (SCF), 10 ng/mL IL-3, 10 ng/mL IL-6 and 3 units/mL erythropoietin (MethoCult M3434, Stem Cell Technologies). Colonies containing more than 30 cells were counted after 10 days of culture. Erythroid colonies were stained with DAF (2,7-diaminofluorene) (Wako), as described (Worthington et al. 1987).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: fukunaga{at}genetic.med.osaka-u.ac.jp
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References |
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Received: 8 December 2006
Accepted: 31 January 2007
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