Title: NSD1 is essential for early post-implantation development and has a catalytically active SET domain
Abstract: Article16 June 2003free access NSD1 is essential for early post-implantation development and has a catalytically active SET domain Geetha Vani Rayasam Geetha Vani Rayasam Laboratory of Receptor Biology and Gene Expression, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Olivia Wendling Olivia Wendling Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 10142, 67404 Illkirch, Cedex, France Search for more papers by this author Pierre-Olivier Angrand Pierre-Olivier Angrand Cellzome AG, Meyerhofstr. 1, D-69117 Heidelberg, Germany Search for more papers by this author Manuel Mark Manuel Mark Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 10142, 67404 Illkirch, Cedex, France Search for more papers by this author Karen Niederreither Karen Niederreither Departments of Medicine and Molecular and Cellular Biology, Center for Cardiovascular Development, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030 USA Search for more papers by this author Luyan Song Luyan Song Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 10142, 67404 Illkirch, Cedex, France Search for more papers by this author Thierry Lerouge Thierry Lerouge Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 10142, 67404 Illkirch, Cedex, France Search for more papers by this author Gordon L. Hager Gordon L. Hager Laboratory of Receptor Biology and Gene Expression, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Pierre Chambon Corresponding Author Pierre Chambon Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 10142, 67404 Illkirch, Cedex, France Search for more papers by this author Régine Losson Régine Losson Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 10142, 67404 Illkirch, Cedex, France Search for more papers by this author Geetha Vani Rayasam Geetha Vani Rayasam Laboratory of Receptor Biology and Gene Expression, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Olivia Wendling Olivia Wendling Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 10142, 67404 Illkirch, Cedex, France Search for more papers by this author Pierre-Olivier Angrand Pierre-Olivier Angrand Cellzome AG, Meyerhofstr. 1, D-69117 Heidelberg, Germany Search for more papers by this author Manuel Mark Manuel Mark Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 10142, 67404 Illkirch, Cedex, France Search for more papers by this author Karen Niederreither Karen Niederreither Departments of Medicine and Molecular and Cellular Biology, Center for Cardiovascular Development, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030 USA Search for more papers by this author Luyan Song Luyan Song Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 10142, 67404 Illkirch, Cedex, France Search for more papers by this author Thierry Lerouge Thierry Lerouge Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 10142, 67404 Illkirch, Cedex, France Search for more papers by this author Gordon L. Hager Gordon L. Hager Laboratory of Receptor Biology and Gene Expression, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Pierre Chambon Corresponding Author Pierre Chambon Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 10142, 67404 Illkirch, Cedex, France Search for more papers by this author Régine Losson Régine Losson Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 10142, 67404 Illkirch, Cedex, France Search for more papers by this author Author Information Geetha Vani Rayasam1, Olivia Wendling2, Pierre-Olivier Angrand3, Manuel Mark2, Karen Niederreither4, Luyan Song2, Thierry Lerouge2, Gordon L. Hager1, Pierre Chambon 2 and Régine Losson2 1Laboratory of Receptor Biology and Gene Expression, National Institutes of Health, Bethesda, MD, 20892 USA 2Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 10142, 67404 Illkirch, Cedex, France 3Cellzome AG, Meyerhofstr. 1, D-69117 Heidelberg, Germany 4Departments of Medicine and Molecular and Cellular Biology, Center for Cardiovascular Development, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030 USA ‡P.Chambon and R.Losson contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:3153-3163https://doi.org/10.1093/emboj/cdg288 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The nuclear receptor-binding SET domain-containing protein (NSD1) belongs to an emerging family of proteins, which have all been implicated in human malignancy. To gain insight into the biological functions of NSD1, we have generated NSD1-deficient mice by gene disruption. Homozygous mutant NSD1 embryos, which initiate mesoderm formation, display a high incidence of apoptosis and fail to complete gastrulation, indicating that NSD1 is a developmental regulatory protein that exerts function(s) essential for early post-implantation development. We have also examined the enzymatic potential of NSD1 and found that its SET domain possesses intrinsic histone methyltransferase activity with specificity for Lys36 of histone H3 (H3-K36) and Lys20 of histone H4 (H4-K20). Introduction Nuclear receptors are members of a superfamily of sequence-specific transcription factors that play diverses roles in the control of cell growth and differentiation, development and homeostasis by stimulating or repressing target gene expression (reviewed in Ciana et al., 2002). Upon binding to their cognate DNA response elements, nuclear receptors modulate transcription through the recruitment of various co-regulatory proteins, called co-repressors and co-activators (reviewed by Robyr et al., 2000; Rosenfeld and Glass, 2001). Co-repressors that are recruited in the absence of ligand, such as N-CoR and SMRT, are part of multiple histone deacetylase complexes, which stabilize chromatin structure and repress transcription. Co-activators that are recruited in a ligand-dependent fashion, such as CBP, p/CAF and members of the p160 family, possess or can recruit histone acetyltransferase and histone methyltransferase (HMTase) activities that are capable of chromatin remodeling/modification, whereas other ligand-recruited complexes such as the TRAP–DRIP–ARC–SMCC complex appear to act more directly on the basal transcriptional machinery (Rosenfeld and Glass, 2001; and references therein). In a screen to isolate co-regulators for retinoic acid receptor, a novel nuclear receptor-binding SET domain-containing protein (NSD1) was identified (Huang et al., 1998) and subsequently shown to belong to an emerging family of proteins that includes NSD2 [also known as MMSET (Chesi et al., 1998) and WHSC1 (Stec et al., 1998)] and NSD3 (Angrand et al., 2001). The domain structure that characterizes these proteins contains a SET domain (Tschiersch et al., 1994), a PWWP domain (Stec et al., 2000) and multiple PHD fingers (Aasland et al., 1995). In addition to these conserved domains, which are present in members of the Trithorax gene family and other chromatin modulators acting positively and/or negatively on transcription, NSD1 contains two distinct nuclear receptor-interacting domains (NIDs) that bind the apo- and holo- forms of the ligand-binding domain of different subsets of nuclear receptors, with characteristics of both co-activators and co-repressors (Huang et al., 1998). Supporting the notion that NSD1 may act as a bifunctional transcriptional cofactor playing a dual role in transcription, NSD1 was also reported to possess distinct activation and repression domains (Huang et al., 1998). The SET domain is an evolutionarily conserved sequence motif of 130–150 amino acids, which initially was identified in the Drosophila position effect variegation (PEV) suppressor Su(var)3-9, the Polycomb group protein Enhancer of zeste [E(z)] and the trithorax group protein Trithorax (TRX) (Tschiersch et al., 1994), and was later found in a variety of chromatin-associated proteins from yeast to mammals (reviewed in Jenuwein, 2001; Schneider et al., 2002). A growing number of SET domain proteins recently have been shown to harbor HMTase activity towards specific lysine residues along the N-terminal tail of histones (reviewed in Jenuwein, 2001; Kouzarides, 2002; Schneider et al., 2002). Among these enzymes, the mammalian homologs of Drosophila Su(var)3-9, SUV39H1 (Rea et al., 2000) and Suv39h2 (O'Carroll et al., 2000), its Schizosacharomyces pombe homolog Clr4 (Rea et al., 2000), the human G9a protein (Tachibana et al., 2002) and the mouse ESET/SETDB1 protein (Yang et al., 2002) specifically methylate histone H3 on Lys9 (and Lys27 in the case of G9a) and require the SET domain and two adjacent cysteine-rich regions [the pre-SET (also called SAC; Huang et al., 1998) and post-SET domains] for enzymatic activity (Rea et al., 2000). Importantly, it has been shown that H3 Lys9 (H3-K9) methylation by SUV39H1 creates a high-affinity binding site for the heterochromatin protein HP1 and thus contributes to heterochromatin-mediated silencing (Jenuwein, 2001; Kouzarides, 2002; and references therein). Recent biochemical purification of HMTase activities from HeLa cells has identified SET7 (also called Set9) as a novel, mammalian SET domain-containing protein that specifically methylates H3 on Lys4 (Wang et al., 2001; Nishioka et al., 2002a). In contrast to H3-K9 methylation, H3-K4 methylation by SET7/Set9 has been shown to activate transcription by inhibiting the association of the NuRD deacetylase complex with the H3 tail and precluding H3-K9 methylation by Suv39h1 (Wang et al., 2001; Nishioka et al., 2002a). In budding yeast, a H3-K4-specific methyltransferase has also been identified (Set1) that is able to catalyze both the di- and tri-methylated state of K4 (Santos-Rosa et al., 2002); interestingly, only the tri-methyl state of K4 was linked to activation of transcription, indicating that not only the site of methylation but also the methyl status of the site are important determinants for gene activity (Santos-Rosa et al., 2002). Recently, SET domain-containing proteins that methylate Lys20 of H4 (H4-K20) have also been described in Drosophila and mammals (Fang et al., 2002; Nishioka et al., 2002b), that are associated with silent chromatin. In an attempt to understand the structure and molecular basis of catalysis of the SET domain HMTases, the three-dimensional structures of diverse SET domain proteins with different substrate specificities have been determined (Marmorstein, 2003; and references therein). Comparison of these three-dimensional structures revealed a common two-domain architecture, consisting of a conserved antiparallel β-barrel structure and a structurally variable insert, with the cofactor-binding site and the catalytic center constructed on an unusual but conserved knot-like substructure. On the basis of sequence similarity within the SET domain, NSD1 has been defined as a SET family member of the Ash1 subclass (also called SET2; Huang et al., 1998; Schneider et al., 2002). This subfamily of SET domain proteins contains, in addition to NSD1 and the related proteins NSD2 and NSD3, the Drosophila trithorax group protein Ash1 (Beisel et al., 2002; and references therein) and the Saccharomyces cerevisiae protein Set2 (Strahl et al., 2002). The SET domain in this subclass is flanked by pre- and post-SET domains and is centrally located (Kouzarides, 2002). Ash1 recently has been demonstrated to be a multicatalytic HMTase that activates transcription by methylating Lys4 and Lys9 in H3, and Lys20 in H4 (Beisel et al., 2002). Set2, which is a nucleosomal H3-K36-selective methyltransferase (Strahl et al., 2002), methylates at the coding and promoter regions of target genes and functions through specific association with the elongating form of RNA polymerase II that is hyperphosphorylated (Xiao et al., 2003), indicating that methylation mediated by Set2 may be involved in regulating transcription elongation. Members of the NSD family have all been implicated in human malignancy, which suggests a key role in controlling cell growth and differentiation for this subgroup of SET domain proteins. The human NSD1 gene, which is located at the chromosomal locus 5q35, has been isolated recently in the context of a fusion transcript with the nucleoporin gene (NUP98) in a recurrent translocation, t(5;11)(q35;p15.5), specifically associated with de novo childhood acute myeloid leukemia (AML; Jaju et al., 2001). More recently, NSD1 has also been implicated in Sotos syndrome, a rare growth disorder also known as cerebral gigantism (Kurotaki et al., 2002,. Through a search for genes located in the Wolf–Hirschhorn syndrome (WHS) critical region, the human NSD2 gene was localized on 4p16.3 (WHSC1; Stec et al., 1998) and was found to be disrupted by t(4;14) translocations causing lymphoid multiple myeloma (MMSET; Chesi et al., 1998). Using fluorescence in situ hybridization, NSD3 was mapped to 8p12 and was shown to be amplified in several tumor-derived cell lines and primary breast carcinomas (Angrand et al., 2001). Recently, NSD3 has also been identified as a translocation partner of NUP98 in AML (Rosati et al., 2002). We demonstrate here that NSD1 is a developmental regulatory protein that exerts cellular function(s) essential for early post-implantation mouse development. We also provide biochemical evidence that the NSD1 SET domain functions as an HMTase to methylate H3-K36 and H4-K20 in vitro. We discuss the implications of these data for mechanistic models of NSD1 function in mammals. Results Targeted disruption of the mouse NSD1 gene Using two genomic clones that contain a portion of the NSD1 gene (see Materials and methods), we generated a targeting vector, pNSD1(LNL:L), in which a neomycin resistance selection cassette (Neo) flanked by two loxP sites was introduced into intron 1 and a loxP site was inserted into intron 2 (Figure 1A; see Materials and methods). This targeting vector was designed with the expectation that upon homologous recombination and subsequent Cre recombinase-mediated excision, exon 2 of NSD1 together with the Neo cassette would be deleted, thereby causing a frameshift mutation with a premature termination codon in exon 3 (Figure 1B). The putative product of this deleted gene would correspond to a truncated NSD1 protein, lacking the NIDs and all conserved domains (Figure 1B). Figure 1.Targeted disruption of NSD1 using the Cre-loxP strategy. (A) Diagram showing a partial map of the genomic locus surrounding the NSD1 exon 2 (E2), the targeting construct, and the targeted allele before (L3) and after (L2 and L−) Cre-mediated excision of the neomycin resistance selection marker (Neo). The 5′ and 3′ probes used for Southern blot analyses and the fragment sizes detected with the 5′ probe upon XbaI digestion and with the 3′ probe upon HindIII digestion are indicated. Relevant restriction sites: X, XbaI; K, KpnI; E, EcoRI; H, HindIII; S, SmaI; A, AflII; Sp, SpeI. (B) Schematic representation of wild-type (WT) and mutant NSD1 proteins. The structural and functional domains are indicated. The putative product of the deleted NSD1 gene corresponds to a C-terminally truncated protein consisting of the first 310 amino acids of NSD1. (C) Southern blot analysis of DNAs derived from wild-type (P1) and targeted (BT157 and BT259) ES cells. Genomic DNA was digested with HindIII, blotted and hybridized with the 3′ probe. (D) Southern blot analysis of ES cell subclones using the 5′ probe, after Cre-mediated excision in BT259 NSD1L3/+ ES cells. Download figure Download PowerPoint The targeting vector pNSD1(LNL:L) was electroporated into 129/Sv P1 embryonic stem (ES) cells, and 280 G418-resistant clones were screened for homologous recombination by Southern blot analysis using an ‘outside’ probe corresponding to a sequence 3′ of the recombination site (3′ probe; see Figure 1A). Two positive clones, BT157 and BT259, were obtained (Figure 1C), which were confirmed to carry a single-copy integration at the NSD1 locus as revealed by hybridization with a Neo probe (data not shown). One of these targeted cell lines (BT259) was transiently transfected with a Cre-encoding expression plasmid (PIC-Cre) to test whether a Cre-mediated excision of the targeted allele (L3) could be achieved. Clones harboring either a partially excised L2 allele without the loxP-flanked Neo cassette (BT259-69; Figure 1D) or a completely excised L− allele lacking the DNA sequences between the three loxP sites (BT259.81) were identified by Southern blotting (Figure 1D). The two independent NSD1L3/+ ES cell lines, BT157 and BT259, were injected into C57BL/6 blastocysts to produce chimeric mice, and both contributed to the germline. Mice heterozygous for the targeted NSD1 gene (NSD1L3/+) were crossed with cytomegalovirus-Cre transgenic mice (CMV-Cretg/0) expressing the Cre recombinase in the germline (Dupé et al., 1997). Tail DNA of the offspring was analyzed by genomic PCR to detect Cre-mediated excision (Figure 2A). Animals in which PCR assays detected an excised L− allele were crossed with wild-type C57BL/6 mice to produce Cre-negative NSD1L−/+ mice, hereafter referred to as NSD1−/+. Figure 2.Morphology of wild-type and NSD1−/− mutant embryos at E7.5 and E8.0. (A) PCR strategy for amplification of wild-type (WT) and excised (L−) NSD1 alleles. DNA samples were subjected to PCR amplification using a mixture of three primers (see Materials and methods). PCR amplification of the wild-type NSD1 allele by sense and antisense primers ZB197 and AAW199 produces a 550 bp DNA fragment (B, upper band), while PCR amplification of the excised allele by sense and antisense primers ZB197 and ZB200 produces a 350 bp DNA fragment (B, lower band). (B) Representative genotypic analysis of E7.5 embryos from an NSD1+/− intercross. (C) Dissected wild-type (left) and NSD1−/− mutant (right) littermates at E7.5 and E8.0. Abbreviations: ee, epiblast; ep, ectoplacental cone; h, head folds. Download figure Download PowerPoint NSD1 is essential for early post-implantation development Mice heterozygous for the NSD1 mutation were viable and fertile. Genotype analysis of progeny from heterozygote intercrosses revealed that 37% were wild type, 63% heterozygous and none homozygous (Table I), indicating that the NSD1 mutation is recessive embryonic lethal. Table 1. Genotype analysis of NSD1+/− intercross progeny Stage Genotype Resorption Total +/+ +/− −/− Newborn 43 73 0 – 116 E10.5 10 16 0 6 32 E9.5 4 5 5a – 14 E7.5 3 11 3a – 17 E6.5 2 8 4a – 14 aEmbryos were either severely growth retarded or were being resorbed. To determine the time of embryonic lethality, embryos from heterozygote intercrosses were collected at various stages of gestation and genotyped (Table I). No homozygous mutant embryos were recovered at, or after, E10.5 (Table I and data not shown), and the percentage of resorptions at this time was unusually high (19%; Table I). At E9.5, a set of degenerating embryos that consisted primarily of extraembryonic tissues was identified and genotyped as NSD1−/− mutants (Table I). At E8.0 and E7.5, all of the morphologically abnormal mutants recovered were also genotyped as mutants (Figure 2B and Table I); these homozygous mutant embryos were severely growth retarded when compared with their NSD1+/+ and NSD1+/− littermates (Figure 2C) and did not contain any structures that resemble those of control embryos (see Figure 2C legend). Mutant embryos generated from the two independently targeted ES cell clones showed identical phenotypes. These results indicate that the NSD1 gene is absolutely required for early post-implantation development in mice. Disruption of the NSD1 gene leads to increased apoptosis and mesodermal defects To analyze the NSD1 mutant phenotype further, we examined histological sections of embryos from heterozygote intercrosses, collected in utero from E6.5 to E8.0 (Figure 3). At the E6.5 egg cylinder stage, normal embryos displayed a well-organized structure, in which two layers of ectodermal and endodermal cells enclose the proamniotic cavity (pa, Figure 3A). A characteristic groove (yellow arrowheads in Figure 3A) separates the epiblast (ee) from the extraembryonic portion of the primitive ectoderm (ex, Figure 3A), the latter being capped by the ectoplacental cone (ep) which invades the maternal decidua. The visceral endodermal layer is composed of two cell subpopulations, the proximal cuboidal cells surrounding the extraembryonic ectoderm (ce) and the distal squamous cells surrounding the epiblast (se, Figure 3A). All abnormal presumptive NSD1−/− E6.5 embryos (n = 4), identified by their small size and absence of the typical egg cylinder shape, displayed the same histological phenotype (Figure 3B). The two cell types of the visceral endoderm and the ectoplacental cone were readily identified (ce, se and ep, Figure 3B). However, the ectoderm (e, Figure 3B) did not exhibit its characteristic groove and always showed an abnormal large gap (see asterisk in Figure 3B). Numerous dying cells marked by pyknotic nuclei were detected on each side of this gap and also within the proamniotic cavity (black arrowhead in Figure 3B, and data not shown). Figure 3.Histological sections of normal (A, C and E) and presumptive NSD1−/− (B, D and F) embryos at E6.5 (A and B), E7.5 (C and D) and E8.0 (E and F). In (A) and (B), deciduas were kept intact. In (C–F), embryos were dissected out of the decidua. Yellow arrowheads in (A) and (D) show the boundary between embryonic and extraembryonic regions. Asterisks in (B) and (D) indicate gaps in the mutant epiblast, and black arrowheads point to pyknotic cells in the proamniotic cavity. Abbreviations: ac, amniotic cavity; al, allantois; am, amnion; ce, cuboidal visceral endoderm; e, ectoderm; ee, epiblast; ep, ectoplacental cone; etc, ectoplacental cavity; ex, extraembryonic ectoderm; exc, exocoelom; h, head folds; m, mesoderm; n, neurectoderm; pa, proamniotic cavity; se, squamous visceral endoderm. Scale bar: 20 μm (A and B); 70 μm (C–F). Download figure Download PowerPoint At E7.5 and E8.0, normal embryos have formed mesoderm (m) and neurectoderm (n, Figure 3C and E), as a consequence of gastrulation. Their anterior and posterior extremities are defined by the headfolds (h) and allantois (al) (Figure 3C and E), respectively; their proamniotic cavity has been partitioned by amniotic folds into amniotic (ac), exocolomic (exc) and ectoplacental (etc) cavities (Figure 3C and E). Abnormal presumptive NSD1−/− E7.5 embryos (n = 4) displayed a groove separating the embryonic and extraembryonic ectoderm (yellow arrowheads in Figure 3D). Few cells detaching from the ectoderm and resembling normal mesodermal cells (m, Figure 3D) were present between the ectoderm and the visceral endoderm. However, the headfolds and the allantois could not be identified, and the proamniotic cavity (pa, Figure 3D) remained undivided. At E8.0, abnormal presumptive NSD1−/− embryos (n = 4) had developed slightly further (Figure 3F) as they now displayed amniotic folds and three distinct cavities resembling those normally derived from the proamniotic cavity (ac, exc and etc, Figure 3F). Ectodermal interruptions and the presence of many pyknotic nuclei in the ectoderm and the proamniotic or amniotic cavity were hallmarks of the presumptive NSD1−/− embryos at both E7.5 and E8.0 (asterisk and arrow in Figure 3D, and data not shown), suggesting that abnormal cell death occurs by apoptosis in the absence of NSD1. To confirm this hypothesis, TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling) assays were performed to detect the fragmented DNA characteristic of apoptotic cells. At E7.5, numerous TUNEL-positive cells (7.25 ± 2.70%) were seen in the four presumptive mutant embryos, whereas <1% (0.57 ± 0.20%) of cells were positive in the littermate controls (compare Figure 4A and B). Altogether, these data indicate that NSD1−/− mutant embryos exhibit a retarded and abnormal gastrulation process, including a marked increase in apoptosis. Figure 4.TUNEL and in situ hybridization analyses of E7.5 normal and mutant embryos. Sections of E7.5 normal embryos (A, C, E, G, I and K) and their presumptive mutant littermates (B, D, F, H, J and L) were subjected to TUNEL reaction (A and B) and were hybridized with Brachyury (C and D), Shh (E and F), Twist (G and H), Hoxa1 (I and J), and Hoxb1 (K and L) antisense probes. In (A) and (B), brown-stained nuclei indicate end incorporation in DNA (arrowheads). Abbreviations: a, axial mesendoderm; al, allantois; h, head folds; m, mesoderm; no, node; s, primitive streak. Scale bar: 60 μm. Download figure Download PowerPoint To characterize gastrulation defects further, presumptive NSD1−/− embryos were analyzed at E7.5 by in situ hybridization (ISH) using markers of mesodermal structures [Brachyury (T), Sonic hedgehog (Shh) and Twist] as well as positional markers of the anteroposterior body axis (Hoxa1 and Hoxb1). Expression of T is detectable in the early mesoderm as it ingresses through the primitive streak (s), in the node (no) and in the axial mesendoderm (a, Figure 4C) (Kispert and Herrmann, 1994). Shh is expressed in the node and the axial mesendoderm (Figure 4E; Echelard et al., 1993). Expression of Twist is observed in the paraxial and lateral mesoderm at a later stage than that of T (m, Figure 4G) (Stoetzel et al., 1995). Hoxa1 and Hoxb1 are expressed along the anteroposterior body axis with anterior boundaries that lie within the hindbrain (Figure 4I and K; Murphy and Hill, 1991). In the three presumptive NSD1−/− embryos analyzed, T was detected in an irregular cluster of embryonic cells (s, Figure 4D). No expression of the two other mesodermal markers (Shh and Twist) and of the two axial markers (Hoxa1 and Hoxb1) was observed in these embryos (Figure 4F, H, J and L). Thus, the primitive streak of mutant embryos has a markedly disorganized aspect, does not form a node and fails to produce mesendoderm and embryonic mesoderm. Moreover, the anteroposterior axis is not specified in these mutant embryos. NSD1 expression during mouse development The expression pattern of NSD1 was examined by ISH at various developmental stages (Figure 5). At the early post-implantation E5.5 stage, NSD1 expression was detected in the developing embryo (em) as well as in the outer region of the uterine decidua (de, Figure 5A and B). In sections of gastrulation stage embryos (E7.5), we found NSD1 uniformly expressed throughout the embryo, in both embryonic and extraembryonic tissues (Figure 5C and D). This ubiquitous expression profile persisted until E14.5 (see E9.5 in Figure 5E and F, and data not shown). After this time, differential expression was seen, with the highest levels of NSD1 expression in proliferative cell populations. Enriched NSD1 levels were detected at E16.5 in the telencephalic region of the brain (br), spinal cord (sc), intestinal crypt cells (in), tooth buds (tb), thymus (th) and salivary glands (sg) (Figure 5G–J). NSD1 expression was also observed in the region of ossification of the developing bones (bo) and in the periosteum (pe), while it was absent in chondrocytes (ca, Figure 5K and L). Taken together, these results are consistent with the gastrulation defects displayed by the NSD1-deficient embryos and also suggest a critical role for NSD1 during post-gastrulation development (see Discussion). Figure 5.In situ hybridization analysis of NSD1 transcript distribution at various stages of mouse development. Brightfield and darkfield views of the histological sections are shown side by side (left and right panel, respectively), revealing the signal grain as white dots. (A and B) E5.5 embryo sectioned in utero. Insert panels show magnification of the embryo. (C and D) Ubiquitous expression of NSD1 in the ectoplacental cone (ep), extraembryonic (ex) and embryonic (em) germ layers of an E7.5 embryo. (E and F) Ubiquitous NSD1 expression in an E9.5 embryo. ba, branchial arches; br, brain; sc, spinal cord. (G and H) Enhanced expression of NSD1 in the brain (br), intestine (in), spinal cord (sc), thymus (th) and tooth buds (tb) of an E16.5 fetus. (I and J) Detail of the E16.5 heart (ht), thymus (th) and salivary gland area (sg). (K and L) Section through the ossification center of the femur. bo, bone tissue; ca, cartilage (chondrocytes); pe, periosteum. Download figure Download PowerPoint The SET domain of NSD1 methylates H3-K36 and H4-K20 To investigate whether the SET domain of NSD1 exhibits HMTase activity, we performed an in vitro methylation assay using native histones as substrates. Recombinant GST–NSD1 (amino acids 1700–1987), GST–SUV39H1 (amino acids 82–412) and GST were incubated with a mixture of native calf thymus histones and S-adenosyl-[methyl-3H]L-methionine as a methyl donor. Reaction products were separated by SDS–PAGE