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Keywords:

  • placenta trophoblast differentiation;
  • Alkbh1−/−;
  • AlkB gene family

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES
  8. Supporting Information

E. coli AlkB has been intensively studied since 1983, but the in vivo roles of its mammalian homologue Alkbh1 are unknown. We, therefore, created null mice for Alkbh1. Alkbh1 mRNA is expressed at highest levels in the trophoblast lineages of the developing placenta. Alkbh1−/− placentas have decreased expression of differentiated trophoblast markers including Tpbp, Gcm1, and Pl-1, and increased expression of the trophoblast stem cell marker Eomes. Alkbh1 localizes to nuclear euchromatin, and interacts strongly with Mrj, an essential placental gene that mediates gene repression by recruitment of class II histone deacetylases (HDACs). Competition experiments show Alkbh1 and HDAC4 binding to Mrj are mutually exclusive, which causes decreased HDAC activity and increased target gene expression. Our study demonstrates Alkbh1 performs important functions in placental trophoblast lineage differentiation and participates in mechanisms of transcriptional regulation. Developmental Dynamics 237:316–327, 2008. © 2007 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES
  8. Supporting Information

The AlkB gene family has been extensively studied since E. coli AlkB was found to play critical roles in the genomic response to methylating agents (Kataoka et al.,1983; Kataoka and Sekiguchi,1985; Kondo et al.,1986; Chen et al.,1994). The recent discovery that in vitro AlkB is a Fe-oxygen-α-ketoglutarate-dependent dioxygenase capable of demethylating methyl and etheno modified DNA and RNA has dramatically catalyzed our understanding of this gene family's potential biological roles and given substantial insights into potential mechanisms (Duncan et al.,2002; Falnes et al.,2002,2004; Trewick et al.,2002; Begley and Samson,2003; Koivisto et al.,2003,2004; Falnes,2004; Sedgwick,2004; Delaney et al.,2005; Lee et al.,2005; Mishina et al.,2005). There are eight eukaryotic AlkB gene family members. ALKBH1 has 52% amino acid similarity and 23% identity to the E. coli AlkB (see Supplemental Fig. 1A, which can be viewed at www.interscience.wiley.com/jpages/1058-8388/suppmat). There are seven other homologues (ABH2-8) (Suppl. Fig. 1B) (Kurowski et al.,2003). ALKBH1, ABH2, and ABH3 have been preliminarily characterized. In vitro AlkB, ABH2, and ABH3 can repair methylated and ethylated DNA and RNA substrates (Duncan et al.,2002; Falnes,2004; Falnes et al.,2004; Koivisto et al.,2004; Lee et al.,2005). In vivo AlkB and ABH2, but not ABH3, repair 1-methyl adenine and 1-methyl cytosine (Ringvoll J,2006). Paradoxically, despite the fact that among eukaryotic family members ALKBH1 has the highest amino acid identity to AlkB, ALKBH1 has no detectable activity on any DNA/RNA substrate (Duncan et al.,2002; Falnes,2004; Falnes et al.,2004; Koivisto et al.,2004; Lee et al.,2005). These data have led to the suggestion that ALKBH1 may have roles that are different from the other characterized homologues in DNA/RNA repair.

In order to understand the in vivo roles of ALKBH1, we created a conditional knockout mouse model. Alkbh1−/− mice are viable and fertile, but show intra-uterine growth retardation (IUGR) and placental defects. Alkbh1−/− placentas have impaired trophoblast giant cell, spongiotrophoblast, and glycogen cell and labyrinthine trophoblast cell differentiation. The mechanistic roles of Alkbh1 in mammalian cells are unknown. To gain greater insight into Alkbh1 function, we used full-length human Alkbh1 as bait in yeast two-hybrid analysis to search for interacting proteins encoded in a cDNA library made from mouse e12.5 embryo and placenta. The only consistent and reproducible interacting protein that could be isolated was mouse Mrj (Dnajb6). Because Alkbh1 localizes to nuclear euchromatin and Mrj mediates gene repression by recruiting class II HDAC activity, we performed expression profiling and found a bias towards downregulation of genes of differentiated trophoblast subtype in Alkbh1−/− placentas. Alkbh1 competes for binding to Mrj and disrupts its interaction with class II HDACs, thereby relieving HDAC mediated gene repression. Our study demonstrates Alkbh1 performs important functions in placental trophoblast lineage differentiation and participates in mechanisms of epigenetic gene regulation.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES
  8. Supporting Information

Generation of Conditional Alkbh1 Knockout Mice

In order to understand the biological roles of Alkbh1, we created a conditional knockout mouse model (Fig. 1A–C). This model deletes exon 3, resulting in a frame shift and premature stop codon in exon 4 (Fig. 1D) before the evolutionarily conserved domains in the protein (Fig. 1A and Suppl. Fig. 1A). The mRNA is expressed but lacks exon 3 (Fig. 1C). This would result in a protein of 11 kD if stable versus 42 kD in the wild type. To confirm the null allele in Alkbh1−/− mice, we generated polyclonal antibodies against the recombinant holoprotein. In Western analyses, anti-Alkbh1 antisera reacts against the expected 42-kD protein in wild-type (Wt) mouse embryonic fibroblasts (MEFs), but does not detect any protein of any size in Alkbh1−/− MEFs (Fig. 1E). Therefore, our model is a null allele.

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Figure 1. Generation of Alkbh1-null mice. A: Gene-targeting strategy. The Alkbh1 locus (WT), the homologous recombination targeting construct (HR vector), and the recombinant locus (HR) are shown. The HR 5′ flanking sequence. 5′ flanking plus exon 2 and exon 3, and the HR 3′ flanking sequence from intron 3 are shown. Exon 3 is shown as a black box, and the BamH1 sites used for targeting validation are shown. The neomycin resistance cassette and LoxP sites are indicated. The sequence after Cre-mediated deletion is shown (HR). B: The diagnostic BamH1 digestion products for the wild-type and HR Alkbh1 locus that were probed are shown. Black boxes represent the 5′ and 3′ targeting-vector flanking probes. Southern-blot hybridization. Tail DNA was digested with BamHI and hybridized with 5′ or 3′ flanking probes. For 5′ HR, bands of 13.2 and 11.7 kb, corresponding to Wt and targeted alleles, respectively, are observed. For 3′ HR, bands of 13.0 and 6.7 kb, corresponding to Wt and targeted alleles, respectively, are observed. C: RT-PCR analysis. Whole embryo cDNA from Wt, Alkbh1+/−, and Alkbh1−/− mice was amplified with probes in exon 2 and exon 4, encompassing the targeted exon 3. WT cDNA (exon 2, 3, 4) has slower agarose gel mobility than HR cDNA (exon 2, 4). D: DNA sequencing of cDNA showing the junction of exons 2 and 4 in the Alkbh1−/− embryo. E: Western analysis with polyclonal Alkbh1 reactive anti-sera in Alkbh1−/− and Wt embryos showing a 42-kD band specific for Wt. Beta-actin is used as a protein loading control.

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Impaired Trophoblast Lineage Differentiation and Intra-Uterine Growth Retardation in Alkbh1−/− Mice

Alkbh1−/− mice are viable, but birth weights are significantly smaller than their Wt or heterozygote littermates (+/+ 1.4 ± 0.05, +/− 1.4 ± 0.04, −/− 1.1 ± 0.02) (Suppl. Fig. 2A,B). Except for intrauterine growth retardation (IUGR), Alkbh1−/− mice are anatomically normal, reach adulthood, and are fertile (data not shown). Alkbh1flox/flox lines that retain exon 3 (and Alkbh1+/− mice) express Alkbh1, are indistinguishable from Wt mice, and have no IUGR or placentopathy.

To understand the mechanisms of Alkbh1-deficient IUGR, we analyzed Alkbh1−/− embryos and placentas from Alkbh1+/− backcrosses at a series of different developmental stages. Starting at e12.5, the first appreciable gross defect is that Alkbh1−/− placentas lack the red/pink coloration of Wt placentas, and instead have a pale brown/bluish coloration (Fig. 2A). Furthermore, at e12.5 Alkbh1−/− placental weights lag behind those of Wt siblings (Fig. 2B), while Alkbh1−/− embryo weights and crown-rump lengths are generally normal at this stage (data not shown). At e15.5, Alkbh1−/− embryo weights and crown-rump lengths begin substantially to lag behind Wt at e15.5, which continues through parturition (data not shown). To quantify the relative impact of Alkbh1 nullizygosity on placental and fetal growth, we measured embryo and placenta weights at different time points and derived fetal:placental ratios. The fetal:placental ratio is significantly higher in Alkbh1−/− vs. Wt at e12.5, e15.5, and e.18.5 (P = 0.001, Paired t-test) (Fig. 2C).

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Figure 2. Alkbh1−/− placentas and embryos show severe growth defects. A: Alkbh1−/− and Wt placentas and embryos at the e15.5 stage. B: Comparison of wildtype and Alkbh1−/−placental weights during different stages in development. C: Fetal:Placental ratio was calculated for Wt and Alkbh1−/− mice. Fetal:Placental ratio was significantly higher in mutants as compared to wildtype as indicated by the double asterisk (**P = 0.001). The average fetal:placental ratio ± standard error were e12.5 (+/+) 1.20 ± 0.05, (−/−) 1.73 ± 0.02, e15.5 (+/+) 3.57 ± 0.1, (−/−) 4.35 ± 0.22, e18.5 (+/+) 12.45 ± 0.35, (−/−) 14.7 ± 0.38. D: Volume of Placental components at e12.5. The average volume of labyrinthine ± standard error were (+/+) 16.1 ± 1.0, (−/−) 7.9 ± 0.14, and junctional zone (+/+) 12.9 ± 1.16, (−/−) 7.3 ± 0.38. E: Volume of Placental components at e15.5. The average volume of labyrinthine ± standard error were (+/+) 26.9 ± 1.0, (−/−) 20.1 ± 0.44, and junctional zone (+/+) 22.0 ± 0.95, (−/−) 14.6 ± 0.61. F: Time course of Alkbh1 expression in Wt placenta at different developmental stages using TaqMan (Applied Biosystems). Mean expression is depicted relative to peak e9.5 levels (100%) ± S.E.M. All time points use ≥3 placentas and are triplicate data points in ≥2 performed experiments.

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To quantify if the decrease in placental weight is due to changes in specific components of the placenta, either the labyrinthine or junctional zone, we measured the volume of both these layers at e12.5 and e15.5. At e12.5, the volume of the labyrinthine and junctional zone Alkbh1−/− is significantly reduced by approximately 50% (Fig. 2D) and at e15.5 by 30% (Fig. 2E). These data are consistent with a predominant role for Alkbh1 in placental development.

To understand more precisely its developmental roles, we analyzed placental Alkbh1 expression temporally and spatially. From e8.0 onward, the highest placental Alkbh1 mRNA level measured is at e9.5 and subsequently decreases until parturition (Fig. 2F). Therefore, functional and anatomical Alkbh1−/− placental abnormalities are subsequent to the highest measured level of Alkbh1 mRNA expression. In contrast, Alkbh1 Northern analyses of embryos and adult mice show essentially “flat” expression with no identifiable highly over-expressed tissue or cell type (data not shown).

To understand Alkbh1 expression in different placental cell types, we performed in situ hybridization analyses of Wt e8.5 and e10.5 placentas. At e8.5, Alkbh1 is highly expressed in the chorion and the ectoplacental cone (Fig. 3A–C). The chorion forms the labyrinthine of the functional placenta while the ectoplacental cone gives rise to the spongiotrophoblast layer that supports the growth of the labyrinthine (Rossant and Cross,2001). Furthermore, at e10.5 Alkbh1 expression is most highly expressed in multiple trophoblast lineages (spongiotrophoblasts, giant cell trophoblasts, glycogen cells, and labyrinthine trophoblasts) (Fig. 3D-F).

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Figure 3. In situ hybridization of Alkbh1 at e8.5 (A–C) and in e10.5 (D–F) placenta. A,D: Probe hybridization shows dark purple color, Alkbh1 anti-sense probe. B,E: Control Alkbh1 sense probe hybridization. C,F: H&E only. epc, ectoplacental cone; ch, chorion; em, embryo; MD, maternal decidua; Sp, spongiotrophoblast; L, labyrinthine; G, giant cells.

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Histological Analysis of Alkbh1−/− Placenta

Histological analysis of Alkbh1−/− placentas demonstrated a dramatic reduction in three trophoblast cell lineages: spongiotrophoblasts, giant cell trophoblasts, and glycogen cells (Fig. 4A,C). Tpbp is a well-characterized marker of placental spongiotrophoblasts and glycogen cells (Teesalu et al.,1998; Georgiades et al.,2000). We, therefore, analyzed Tpbp expression in Alkbh1−/− placentas. In situ hybridization analyses demonstrated a substantial reduction in the number of cells expressing Tpbp (Fig. 4B). These data are consistent with a specific reduction in functional spongiotrophoblasts and glycogen cells in the Alkbh1−/− placental junctional zone. While the number of cells expressing Tpbp in Alkbh1−/− placentas is reduced, the intensity of the residual cells expressing this marker appears similar to that seen in Wt placentas. Trophoblast giant cells arise through endoreduplication of their DNA without undergoing mitosis and are also characterized by the expression of PRL family gene members (Soares,1991; Ilgren,1983). DAPI staining of placental sections showed dramatic reduction of large giant cell nuclei in the junctional zone. Additionally, we performed in situ hybridization for Pl-1, a trophoblast giant cell–specific marker (Colosi et al.,1987; Yamaguchi et al.,1994). Alkbh1−/− placentas show a significant decrease in Pl-1 staining cells, consistent with the histology showing a dramatic reduction in the number of trophoblast giant cells (Fig. 4C,D). The decreased number of giant cells, spongiotrophoblasts, and glycogen cells was most dramatic in the central junctional zone (Suppl. Fig. 2C), with less notable differences in the peripheral distribution of these cell lineages compared to Wt placenta. In addition to characterizing defects in the junctional zone, we analyzed the labyrinthine trophoblast marker Gcm1. Gcm1 is a mammalian homolog of the Drosophila glial cell missing gene and is expressed in a subset of labyrinthine trophoblast cells (Eugenia Basyuk,1999; Schreiber et al.,2000). We performed in situ hybridization using the Gcm1 probe in Wt and Alkbh1−/− placenta. The expression of Gcm1 is significantly reduced in the Alkbh1−/− placenta (Fig. 4E). Besides decreased volume of labyrinthine and Gcm1 expression, the labyrinthine appears normal with no significant difference in maternal or fetal blood vessel area (Suppl. Fig. 2D).

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Figure 4. Histology of wild type and Alkbh1−/− placentas. A: H&E staining shows a reduction in spongiotrophoblast and glycogen cells at e15.5 (4×). B: Tpbp, a marker of spongiotrophoblasts, staining significantly decreased in the Alkbh1−/− at e15.5 (10×). C: DAPI staining of nuclei at e11.5 shows severe reduction in the number of giant cells (10×). D: PL-1, a marker for giant cells staining, is greatly reduced in Alkbh1−/− at e11.5 (10×). E: Gcm1 staining is reduced in the Alkbh1−/− labyrinthine (10×). F: Eomes staining in e12.5 shows increased staining of the trophoblast stem cell marker in Alkbh1−/− placenta (20×). MD, maternal decidua; Sp, spongiotrophoblast; L, labyrinthine; G, giant cells; Gly, glycogen cells.

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Trophoblast stem cells (TS cells) can differentiate into either labyrinthine trophoblast or a common spongiotrophoblast/giant cell progenitor. Glycogen cells subsequently differentiate from spongiotrophoblasts (Simmons and Cross,2005). The observation that Alkbh1−/− placentas have decreased numbers of all the trophoblast lineages suggested impaired differentiation of the common progenitor. Eomes is a homeobox and T-box containing transcription factor that plays a critical role in TS cells maintenance (Russ et al.,2000). It is a well-characterized marker of TS cells and is also expressed in spongiotrophoblast and labyrinthine trophoblast progenitors. We therefore used in situ hybridization to analyze Eomes in Wt and Alkbh1−/− placentas. In contrast to the substantial reduction in Tpbp, Pl-1, and Gcm1 expression, we found Alkbh1−/− placentas have increased Eomes expression in the junctional zone and labyrinth compared to Wt (Fig. 4F). In summary, Alkbh1 expression is high in all trophoblast lineages examined. Our data are, therefore, most consistent with impaired TS cells differentiation in all the major trophoblast lineages that make up the placenta.

Gene Expression Profiling Reveals Significant Downregulation of Trophoblast Lineage-Specific Genes

To explore the roles of Alkbh1 in trophoblast lineage differentiation, we molecularly profiled Alkbh1−/− placentas at the e11.5 stage. Expression profiling using Affymetrix mouse gene arrays revealed a significant bias towards downregulation of genes of differentiated trophoblast subtype in Alkbh1−/− placenta compared to Wt (Fig. 5A). Because many of the genes whose expression is perturbed have roles in placental development (Simmons and Cross,2005; Sood et al.,2006), we validated selected candidates using real-time quantitative PCR (qPCR) (Fig. 5B). Ascl2 and Hand1 are helix-loop-helix–containing transcription factors. Hand1 is critical for trophoblast giant cell development (Kraut et al.,1998). Ascl2 is required for spongiotrophoblast differentiation (Guillemot et al.,1994,1995; Tanaka et al.,1997), and also suppresses trophoblast giant cell differentiation. Igf2 stimulates proliferation of all placental cell types (Constancia et al.,2002) but is especially critical for glycogen cell trophoblasts (Simmons and Cross,2005). Esx1 is a paired-like homeobox-containing transcription factor expressed in spongiotrophoblasts, giant cell, and labyrinthine trophoblasts (Li et al.,1997) (and most likely their progenitors as well). Esx1 suppresses spongiotrophoblast and glycogen cell differentiation (Li and Behringer,1998). Because we found Igf2, Hand1, and Ascl2 are significantly decreased, and Esx1 expression increased, in Alkbh1−/− placentas, our data are consistent with a causal downstream role for these genes resulting in decreased giant cell, spongiotrophoblast, and glycogen cell differentiation in Alkbh1−/− placentas. Additionally, molecular profiling suggested dysregulation of trophoblast genes previously identified in other placental dysplasia models such as mouse interspecies hybrid-cross placental dysplasia (IHPD). IHPD models are characterized by abnormal numbers of spongiotrophoblast and glycogen cell lineages and abnormal epigenetic regulation of gene expression (Suemizu et al.,2003; Singh et al.,2004). We validated many of these candidates previously found to be dysregulated in IHPD models. We found that expression of Gpc1, Car2, Cd83, Plac8, Dcn, Gatm, Cd81, Osbpl5, and CalCr correlates in a biologically consistent manner with the observed lineage defects in Alkbh1−/− trophoblasts (Fig. 5B and data not shown).

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Figure 5. A: Gene expression profiling of e11.5 Alkbh1−/− vs. Wt placenta showing dysregulated genes. The gene symbol, fold expression compared to wildtype, and P value are shown. B: Relative expression levels in critical developmental genes in Alkbh1−/− and Wt placentas at a stage preceding anatomical changes (see inset). Expression levels are compared using Assay-on-Demand (Applied Biosystems), for Ascl2/Mash2, Hand1, Igf2, Esx1, Cd81, Calcr, Cd83, Plac8, Gpc1, and Car2. The relative expression ratios were normalized to the housekeeping gene Gapdh and are expressed as fold difference. C: Alkbh1 gene expression at e11.5 in Peromyscus inter-hybrid species placental dysgenesis (placental inset at e17.5) using RT-PCR (SYBR Green, Applied Biosystems). The relative expression ratios were normalized to the housekeeping gene Gapdh and are expressed as fold difference. BWxPO expression is set to 1.0 fold. Three placentas each for BWxPO and POxBW were used with triplicate data points ± S.E.M. and were repeated in 2 experiments.

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Peromyscus is a well-characterized murine system of IHPD (Vrana et al.,1998,2000). Peromyscus polionotus (strain=PO) and Peromyscus maniculatus (strain=BW) are closely-related species whose interbreeding significantly disrupts epigenetic placental gene regulation (Vrana et al.,1998,2000). POxBW placentas are characterized by a significant expansion of spongiotrophoblasts and glycogen cells, while bwxpo placentas have dramatically fewer numbers of these lineages. We, therefore, examined Alkbh1 expression in Peromyscus POxBW and bwxpo IHPD placentas. Consistent with a role for Alkbh1 in spongiotrophoblast and glycogen cell development, Alkbh1 expression is significantly increased in POxBW placentomegaly and lessened in bwxpo placentomalacia (Fig. 5C).

Alkbh1 Localizes to Nuclear Euchromatin and Interacts with the Class II HDAC Modulator Mrj

Previous studies did not link Alkbh1 to any known developmental process. To understand the mechanisms that Alkbh1 participates in, we searched for proteins that interact with Alkbh1 using the yeast two-hybrid system. Screening a mouse embryo/placenta e12.5 library, we identified Mrj (Dnajb6), a mouse homologue of E. coli DnaJ, as a consistent and strong Alkbh1-interacting protein. DnaJ is an E. coli co-chaperone that plays critical roles in host genome and phage λ DNA replication by modulating conformation of the replication machinery complex proteins (Saito and Uchida,1978; Yochem et al.,1978; Georgopoulos et al.,1980). Mrj is a member of a subfamily of 5 eukaryotic co-chaperone proteins, which modulate the conformation and activity of multiple proteins (Dai et al.,2005). Mrj specifically interacts with multiple proteins including class II histone deacetylases (HDACs) (Dai et al.,2005), the mSin3-HDAC transcription co-repressor complex member BRMS1 (Hurst et al.,2006), the transcription factor NFATc3 (Dai et al.,2005), poly(Q) repeat–containing proteins such as mutant Huntingtin (Chuang et al.,2002) that associate with transcription factors (Russ et al.,2000; Yohrling et al.,2003), and intermediate filament proteins that associate with the nuclear membrane (Izawa et al.,2000). Mrj is, therefore, thought to play roles in epigenetic gene repression, as well as other processes like keratin turnover (Watson et al.,2007).

Previous studies have demonstrated that Mrj is essential for placental development. Mrj−/− mice are inviable due to failure of chorioallantoic fusion at e8.5, which prevents formation of the labyrinth (Hunter et al.,1999). In situ hybridization experiments demonstrated that Mrj is expressed in e8.5 chorion and the ectoplacental cone, where trophoblast lineage progenitors are located (Hunter et al.,1999). In situ hybridization in Wt placentas at later stages also demonstrated that Mrj is expressed in all trophoblast lineages, with highest expression in trophoblast giant cells (Hunter et al.,1999).

Mrj localizes to the nucleus (as well as peri-nuclear cytoplasm) (Izawa et al.,2000; Dai et al.,2005; Watson et al.,2007). To analyze Alkbh1 cellular localization, we performed immunofluorescence studies with an epitope-tagged Alkbh1. Alkbh1 predominantly localizes to nuclear euchromatin (Fig. 6A–C), but is largely excluded from heterochromatin or nucleoli. Because transcribed genes localize to euchromatin, these data suggest that Alkbh1, like Mrj, might play roles in regulation of gene expression. Furthermore, the observation that both play roles in placental development and that their expression patterns overlap in trophoblast lineages suggested that they might participate in the same trophoblast lineage developmental processes.

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Figure 6. Alkbh1 is a nuclear protein that localizes to euchromatin. A–C: Full-length Alkbh1 with an appended N-terminal Myc-epitope. A: Alkbh1 cell localization is assayed with anti-Myc and a secondary anti-mouse Cy3 immunofluorescence label (red). B: Counterstained with DAPI (blue). C: Merged. D–F: Mrj interacts directly with Alkbh1. D: Expression of full-length Mrj and Mrj truncations appended to an N-terminal express tag. E: Co-immunoprecipitations with HDAC4 (FLAG tagged) and Western for Mrj (anti-Xpress). F: Co-immunoprecipitations with Alkbh1 (Myc tagged) and Western for Mrj (anti-xpress). *Immunoprecipitated Mrj or Mrj truncations. G: Co-immunoprecipitations are performed with Mrj (Xpress tagged) and Western analysis performed for Alkbh1 (anti-Myc). H: Schematic representation of Alkbh1 Mrj-interacting regions. AlkB, conserved domain; HD, HDAC Class II interacting domain; AB, Alkbh1 interacting domain. I: Binding of Alkbh1 and HDAC4 to Mrj is mutually exclusive. 293T cells co-transfected with Xpress-Mrj, FLAG-HDAC4, and increasing amounts of Myc-Alkbh1. Co-Immunopreciptations were performed against Xpress-Mrj, followed by Western blot detection of HDAC4 and Alkbh1, showing decreasing amounts of HDAC4 in the presence of increasing amounts of Alkbh1.

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To map the Alkbh1-Mrj interaction more precisely, we performed co-immunoprecipitation analyses in a series of deletion mutations. Full-length and c-terminus truncated versions of Mrj tagged with N-terminal express tag were transfected into 293T cells. After confirming expression of all constructs (Fig. 6D), immunoprecipitaions with both HDAC4 and Alkbh1 were performed. These experiments demonstrate that the c-terminal end of the Alkbh1 interaction domain on Mrj lies between amino acids 191 and 210, adjacent to the HDAC binding domain, which is between amino acids 146 and 177 (Fig. 6D–F,H) (Dai et al.,2005). In addition, to map the interaction domain of Mrj on Alkbh1 c-terminal truncations of Alkbh with an N-terminal myc tag were made and co-transfected with full-length Mrj. C-terminal deletions of Alkbh1 show that the N-terminus of Alkbh1 is sufficient for the interaction with Mrj (Fig. 6G,H).

Antagonism of Class II HDAC Activity by Alkbh1 Competition for Mrj Binding

Mrj augments repression of target genes by direct interactions with class II HDACs and transcription factors such as NFATc3 (Dai et al.,2005). This repression is mediated by Mrj recruitment of class II HDACs (HDAC4, 5, 7, 9, and 10) to target gene promoters. This recruitment causes promoter Histone 3 deacetylation and is dependent on interactions between class II HDACs and the Mrj c-terminus (Dai et al.,2005).

The best-characterized example of Mrj- class II HDAC interactions is with HDAC4 on NFAT regulated promoters (Dai et al.,2005). Expression of increasing amounts of Mrj in cardiomyocytes inhibits NFATc3 transactivation through a mechanism of class II HDAC recruitment to NFATc3-responsive promoters and a subsequent decrease in promoter acetyl-histone3 (acetyl-H3). Conversely, siRNA-mediated reduction of Mrj increases NFATc3 transactivation and increases acetyl-H3 content at NFATc3 responsive promoters (Dai et al.,2005).

Because Mrj interacts with both Alkbh1 and the class II HDACs, all three proteins could form one complex (similar to Mrj-HDAC-NFATc) (Dai et al.,2005), or mutually exclusive complexes. To distinguish between these possibilities, we performed co-immunoprecipitation experiments in 293T cells co-transfected with epitope tagged HDAC4, Mrj, and increasing amounts of Alkbh1. Consistent with previous results, immunoprecipitation of Mrj co-precipitates associated HDAC4 (Dai et al.,2005) (Fig. 6I). Co-transfection of increasing amounts of Alkbh1 and immunoprecipitation of Mrj caused a decrease in Mrj-HDAC4 interaction and increased Mrj-Alkbh1 interaction (Fig. 6I). Therefore, our data suggest that Mrj forms mutually exclusive complexes with either Alkbh1 or HDAC4. Since the interaction domains of Alkbh1 and HDAC4 on Mrj are in close proximity, it is likely that the binding of one protein sterically hinders binding of the other protein to Mrj at the same time. Hence, functionally, in the absence of Alkbh1, HDAC4 can bind to Mrj and downregulate genes essential for trophoblast differentiation.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES
  8. Supporting Information

A Critical Role for Alkbh1 in Placental Trophoblast Lineage Development

Here, we demonstrate that Alkbh1 plays an important role in placental trophoblast lineage development. In the formation of mouse placenta, the first placental cell fate decision occurs at the 16 cell stage, when the polarized outer cells that give rise to trophectoderm are segregated from the cells destined to become the embryo (Simmons and Cross,2005). Primary giant cells arise from the mural trophectoderm (the trophectoderm not overlying the inner cell mass) and occupy the periphery of the developing placenta. At e7.5–e8.5, there are two distinct regions: the chorion, which gives rise to the labyrinthine, and the ectoplacental cone, which forms the spongiotrophoblast layer.

Alkbh1 is expressed at e8.5–9.5, when progenitor cells in the ectoplacental cone are differentiating into trophoblast giant cells and spongiotrophoblasts (Fig. 2F). Alkbh1 is highly expressed in the chorion and ectoplacental cone and, subsequently, in trophoblast giant cells, spongiotrophoblasts, glycogen cells, and labyrinthine trophoblasts (Fig. 3). Our data are most consistent with an important role for Alkbh1 in the differentiation of trophoblast stem cells into all the major trophoblast lineages: trophoblast giant cells, spongiotrophoblasts, glycogen trophoblasts, and labyrinthine trophoblasts. At the same time, Eomes, a marker of trophoblast stem cells, is increased in Alkbh1−/− placentas (Fig. 4F). Alkbh1−/− placentas have increased expression of the critical developmental transcription factors Ascl2, Hand1, and decreased Esx1 and Gcm1 expression (Figs. 4E and 5B). Because Hand1 promotes trophoblast giant cell differentiation (Kraut et al.,1998), Ascl2 is essential for spongiotrophoblast differentiation (Guillemot et al.,1995; Tanaka et al.,1997), Esx1 suppresses spongiotrophoblast and glycogen cell differentiation, and Gcm1 promotes labyrinthine trophoblast differentiation, our data are consistent with a causal downstream role for these genes in mediating Alkbh1−/− trophoblast lineage defects.

Interspecies hybrid-cross placental dysplasia (IHPD) models are characterized by abnormal epigenetic regulation of gene expression and abnormal development of spongiotrophoblast and glycogen cell lineages. Peromyscus is a well-characterized murine system of IHPD (Vrana et al.,1998,2000). Alkbh1 expression levels correlate in a biologically consistent manner with the observed placental defects in Peromyscus IHPD (Fig. 5C). Furthermore, many of the genes previously found to be dysregulated in IHPD models and embryo nuclear cloning (both of which cause expansion of spongiotrophoblast/glycogen cell lineages) (Zechner et al.,1996; Humpherys et al.,2002; Ogura et al.,2002; Suemizu et al.,2003; Umashankar Singh,2004) are reciprocally regulated in Alkbh1−/− placentas, which is biologically consistent with the observed defects (Fig. 5C and data not shown). In addition, Peromyscus IHPD model of growth retardation shows downregulation of Igf2 and upregulation of Esx1 (Duselis and Vrana,2007; Loschiavo et al.,2007) as seen in the Alkbh1−/− placentas. These data suggest Alkbh1 may play important roles in the mechanisms causing placentopathy in these models, perhaps involving epigenetic gene regulation, and merit further exploration.

Alkbh1 Is a Nuclear Protein That Participates in Epigenetic Regulation of Gene Expression

The predominant nuclear euchromatin cellular localization of Alkbh1 (Fig. 6A), in combination with the expression profiling studies and interaction with Mrj, suggested that Alkbh1 might participate in mechanisms of transcriptional regulation. Mrj interacts with multiple proteins associated with transcriptional repression, including class II histone deacetylases (HDACs) (Dai et al.,2005) and the mSin3-HDAC transcription co-repressor complex member BRMS1 (Hurst et al.,2006). Mrj is a co-chaperone that is part of a subfamily of 5 Mrj-related proteins, as well as the larger family of eukaryotic DnaJ co-chaperones (Dai et al.,2005). Other chaperones/co-chaperones that have been implicated in transcriptional repression include Hsp70, HSC4, and Droj2, which associate with Polycomb protein–containing repressive complexes (Levine et al.,2002; Wang and Brock,2003). These proteins modulate protein conformation, although their substrates and the precise mechanisms through which they enhance gene repression are not well characterized.

Mrj has specifically been shown to recruit class II HDACs to target gene promoters as one mechanism of gene repression. This recruitment causes promoter Histone 3 deacetylation and is dependent on interactions between class II HDACs and the Mrj c-terminus (Dai et al.,2005). The observation that Mrj interacts with both class II HDACs and Alkbh1, and that their binding to Mrj is mutually exclusive (Fig. 6D–F,I), suggests a possible mechanism for Alkbh1 in transcriptional regulation. When Alkbh1 is present, it competes with class II HDACs for Mrj binding. This interaction diminishes class II HDAC recruitment to target promoters, and lessens repression of these targets. When Alkbh1 is absent, Mrj-mediated recruitment of class II HDACs to target genes is increased, resulting in enhanced repression. This repression also involves interaction with sequence-specific transcription factors. The best-characterized Mrj interaction causing class II HDAC recruitment is with NFAT family members (Dai et al.,2005). However, it is highly likely that Mrj interacts with other transcription factors as well.

The Alkbh interaction with Mrj is mediated through the N-terminal 120 amino acids. This Alkbh sequence has low amino acid identity with Abh2, Abh3, and the other homologues, in contrast to the higher similarity in the c-terminus of these proteins (Suppl. Fig. 1A). These data suggest that Abh2, Abh3, and the other AlkB family members are unlikely to interact with Mrj, an observation that would be consistent with the in vitro and in vivo phenotypes that Alkbh plays a very different role from the other AlkB homologues (Duncan et al.,2002; Falnes,2004; Falnes et al.,2004; Koivisto et al.,2004; Lee et al.,2005; Ringvoll,2006).

Alkbh1 and Mrj Roles in Placental Development

Mrj is essential for chorioallantoic fusion, which is required for formation of the labyrinth and labyrinthine trophoblast development. The critical gene targets of Mrj-class II HDAC interactions, or other mechanisms that Mrj participates in, are unknown. While Mrj has not been explicitly tested for expression in the same cells as Eomes, because Mrj is expressed highly in the chorion and is expressed in all trophoblast cell lineages, it is likely that Mrj is expressed in TS cells. Our analyses of Alkbh1−/− placentas suggest that Alkbh1 and Mrj promote the differentiation of distinct trophoblast lineages. Because Alkbh1 functionally antagonizes Mrj-class II HDAC-mediated gene repression, it is tempting to speculate this interaction affects regulation of critical target genes in TS cells. Future experiments will be required to identify target genes of Alkbh1 and Mrj.

Distinct In Vivo Roles for Alkbh1 vs. Abh2 and Abh3

AlkB is a Fe-oxygen-α-ketoglutarate-dependent dioxygenase capable of demethylating methyl and etheno modified DNA and RNA. Mammalian homologues ABH2 and ABH3 have similar in vitro activities. Ringvoll et al. (2006) recently generated mouse models for Abh2 and Abh3. They demonstrated Abh2−/− (but not Abh3−/−) cells are deficient in repair of 1-methyladenine and 3-methylcytosine. They also demonstrated Abh2−/−, Abh3−/− and Abh2−/−/Abh3−/− mice are all viable and have no overt phenotype, although the placenta was not among the tissue examined (Ringvoll et al.,2006). Our data that Alkbh1 deficiency causes a phenotype distinct from Abh2 and Abh3 knockouts is consistent with the in vitro DNA/RNA repair studies (Duncan et al.,2002; Falnes et al.,2002,2004; Trewick et al.,2002; Begley and Samson,2003; Koivisto et al.,2003,2004; Falnes,2004; Sedgwick,2004; Delaney et al.,2005; Lee et al.,2005; Mishina et al.,2005), which demonstrate that Alkbh1 plays a very different role from the other homologues. In their discussion section, Ringvoll et al. (2006) state they have also produced Alkbh1 knockout mice and that they are viable. However, no data are shown or additional comments made. Our study is, therefore, consistent with their observation, and significantly extends beyond what is known about the role of Alkbh1 in mammals.

Because of the 23% amino acid identity between AlkB and ALKBH1 across two billion years of evolutionary history, we hypothesized that Alkbh1 would perform critical functions in eukaryotes. Alkbh1 contains all the critical amino acid residues to act as a Fe-oxygen-α-ketoglutarate-dependent dioxygenase (Kurowski et al.,2003; Yamane et al.,2006).Yet, despite the fact that it has been intensively investigated, ALKBH1 has no known enzymatic function or substrate (Duncan et al.,2002; Falnes et al.,2002,2004; Trewick et al.,2002; Begley and Samson,2003; Koivisto et al.,2003,2004; Falnes,2004; Sedgwick,2004; Delaney et al.,2005; Lee et al.,2005; Mishina et al.,2005). Mrj is the first known protein that associates with Alkbh1 with high affinity. In future experiments, it will be important to analyze whether Alkbh1-Mrj complexes may act on methyl and etheno modified DNA and RNA, or non-nucleic acid substrates.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES
  8. Supporting Information

Generation of an Alkbh1-Null Allele in Embryonic Stem Cells

A genomic fragment of 7.6 kb containing Exon 3 and flanking sequence of Alkbh1 derived from a 129 Sv/Ev phage library was subcloned into the NotI site of pNT loxP, and a genomic fragment of 5.3 kb of the Alkbh1 intron 3 was subcloned into the EcoRI site. This construct places the PGK promoter–neomycin cassette, flanked on both 5′ and 3′ ends by loxP sites, in the opposite transcriptional orientation as Mlh3. The targeting vector (50 μg) was linearized at the single NotI side and electroporated into 2.0 ×107 129 Sv/Ev embryonic stem (ES) cells. The ES cells were selected in neomycin, and resistant colonies isolated as previously described. We screened genomic DNA from individual colonies by Southern analysis using genomic DNA digested with BamHI and a 5′ flanking probe. Homologous recombinant ES cell clones testing positive in this screen were confirmed by Southern analysis using a probe external to the targeting vector from Alkbh1 intron 3. Alkbh1+/− mice were generated by mating with E2A Cre mice (Lipkin et al.,2002). These mice were intercrossed to generate Alkbh1−/− mice. Details for Southern blotting, RT-PCR of Alkbh1 mRNA, and Western analysis of Alkbh1 fibroblast protein are available on request.

Mice were handled according to institutional guidelines for the humane care and use of experimental animals, and with approval for all studies from the appropriate Institutional Animal Care Committees at the University of California, Irvine. Mice were fed ad libitum with standard pet chow and water, and housed under conditions of controlled light (12 h/12 hr light/dark cycle) and temperature (27°C).

Histology, Placental Stereology, and In Situ Hybridization Analyses

Placentas and embryos were fixed in 4% buffered formalin for 1–12 hr. We processed fixed tissues for histology using routine methods and sectioned the paraffin-embedded tissues at 5 μm. In situ hybridization analyses were performed with the 3′untranslated mRNA sequence (sense and anti-sense) of Alkbh1, 4311/Tpbp, PL-1, Gcm1, and Eomes (kind gifts from Dr. G. Leone, Ohio State University) probe as previously described (Li et al.,1997; Lipkin et al.,2002; Wu et al.,2003). Histology of Peromyscus placentas was performed according to Duselis et al. (2005). To calculate the volume of placental components, they were hemidissected, weighed, and immediately fixed. The stereology work was carried out using methods described previously (Coan et al.,2004). Three mice from at least 3 different litters were analyzed for each stage.

RNA Isolation and Microarray Experiments

Total RNA was isolated by using the TRIzol method (Invitrogen) from e11.5 Wt and Alkbh1−/− placenta (experiment was carried out as three independent samples for each genotype), followed by purification, using RNeasy columns (Qiagen, Valencia, CA). Double-stranded cDNA was synthesized from the total RNA, and an in vitro transcription reaction was then performed on biotin-labeled RNA that was made from the cDNA. Labeled RNA was hybridized with MG-U74Av2 chips (Affymetrix, Santa Clara, CA) and washed according to the manufacturer's recommendations. The hybridized probe array was then stained with streptavidin-conjugated phycoerythrin, and each GeneChip was scanned twice in an HP GeneArray confocal laser scanner at 570 nm with a laser resolution of 3 mm by using MAS 5.0 Microarray Suite software (Affymetrix) to produce a .cel file for further data processing. Data was analyzed as previously described (Lin et al.,2004).

Quantitative RT–PCR

Wt and Alkbh1−/− mouse e11.5 placentas and Peromyscus placentas were dissected and total RNA extracted using Trizol. Applied Biosystems TaqMan mouse Assay-on-Demand assays were used to quantify gene expression on an ABI 7900HT. Expression levels were normalized to Gapdh.

Immunoflourescence

For analysis of Alkbh1 intracellular localization, mouse fibroblasts were infected with MSCV-Myc-Alkbh1 or control MSCV-GFP retrovirus. The cells were then fixed in 4% paraformaldehyde and blocked with 5% BSA. Staining was carried out using standard procedures with primary antibody (mouse monoclonal anti-Myc, 1:500, Invitrogen), followed by the secondary antibody (Cy3 conjugated anti-mouse, 1:2,000, Jackson Labs). DAPI was used as a counterstain.

Yeast Two-hybrid Analysis

For screening, the full-length human ALKBH1 cDNA was expressed as a fusion protein with the GAL4 DNA-binding domain (bait). A GAL4 activation domain cDNA library prepared from mouse e12.5 embryo and placenta was introduced into the yeast reporter strain with ALKBH1-GAL4 bait as described previously (Kudryavtseva et al.,2003). Colonies were screened for β-galactosidase expression and (-Leu -His -Ade) selective plates. In protein–protein interaction assays, β-galactosidase units were calculated according to standard methods (Clontech, Matchmaker two-hybrid system).

Generation of Alkbh1-Reactive Antisera

The full-length ALKBH1 protein was expressed in E. coli as a histidine fusion protein, purified to homogeneity and polyclonal reactive sera raised in rabbits as previously described (Lipkin et al.,2002).

Protein Immunoprecipitation

Full-length Alkbh1, or Alkbh1 with the c-terminal 65 or 265 aa deleted, were subcloned into pcDNA6 with an N-terminal Myc-epitope tag. Mrj, or Mrj with the N-terminal 123, 174, 191, and 210 aa, were subcloned into the same vector with an N-terminal Xpress epitope tag (Invitrogen). Briefly, 293 cells were transiently transfected, and co-immunoprecipitations processed as previously described (Lipkin et al.,2000). For competition assays, 293T cells were transiently transfected with Myc-Alkbh1 (0–9 μg), Flag-Hdac4 (provided by Dr. Yang, McGill University, Montreal, Canada), Xpress-Mrj, and GFP vector. Transfected cells were lysed with NP-40 buffer (50 mM Tris-HCL, pH 7.4, 150 mM NaCL, 0.5% NP-40) and co-immunoprecipitation against Xpress-Mrj was carried out.

Peptide Homology Alignment and Evolutionary Relationships

We constructed phylogenetic trees for the ALKB family using algorithms contained within the PHYLIP Phylogeny Inference Package, version 5.5, as previously described (Kurowski et al.,2003). Briefly, we generated a multiple sequence alignment using CLUSTALW with the entire ALKB protein sequence. We used PROTDIST on these nine sequences to calculate a distance matrix according to the Dayhoff PAM probability model. The distances computed represent the expected fraction of amino acid substitutions between each pair of sequences. We used the distance matrix to estimate phylogenies using the Fitch-Margoliash least-square distance method. In this method, the sum of branch lengths between any two species is expected to equal the distances between species found in the calculated matrix. We performed all FITCH runs with global rearrangement and multiple jumbles (reordering of the data set 10,000 times) to evaluate the effect of different input orders on the derived trees and to assure that none of the subtrees have become caught in a region of the tree representing a statistical local minimum. We examined each data set five times in this fashion, producing trees with identical sum-of-squares and average percent standard deviation statistics. We used CONSENSE to compute the consensus tree by majority-rule method.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES
  8. Supporting Information

The Supplementary Material referred to in this article can be viewed at www.interscience.wiley.com/jpages/1058-8388/suppmat

FilenameFormatSizeDescription
dvdy21418-DVDY21418SuppFig1.eps9999KSuppl. Fig. 1.A:The evolutionary relationship ofE. coliAlkB, the human homologue ALKBH1, and other human AlkB homologues. ALKBH1 is 52% similar and 23% identical to theE. coliAlkB. AlkB, ALKBH1, as well as ABH2 and ABH3, the best-characterized mammalian AlkB family members, are in bold.B:CLUSTALW Alignment of ALKBH1 homologues in highly divergent species. *The start of the deleted Alkbh1 exon 3, resulting in a frame-shifted protein.
dvdy21418-DVDY21418SuppFig2.eps21480KSuppl. Fig. 2.A:Four-week-oldWtandAlkbh1 −/− mice.B:Birth weights ofAlkbh1 +/+ , Alkbh1 +/− , andAlkbh1 −/− mice are shown for four litters.C:4311/ Tpbpin situ hybridization of e15.5Alkbh1 −/− andWtplacentas. Blue color indicatesTpbpanti-sense probe hybridization. H+E stained adjacent sections are also shown.D:Areas of maternal and fetal vessels in the placenta ofAlkbh1 −/− and wild-type embryos at E12.5. Placental sections were subjected to hematoxylin-eosin staining for detection of maternal vessels (containing denucleated red blood cells) and fetal vessels (containing nucleated red blood cells). The areas of maternal and fetal vessels were measured by image analysis (NIH image Software). Twelve different fields (magnification, 60×) for each genotype were randomly selected for analysis, and the means ± standard errors of the means of the percentage area of each vessel type in each field were calculated. n.s., not significant atP= 0.05,t -test).

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