Beta-1,4-galactosyltransferase-3 deficiency suppresses the growth of immunogenic tumors in mice.

Background: Beta-1,4-galactosyltransferase-3 (B4GALT3) belongs to the family of beta-1,4-galactosyltransferases (B4GALTs) and is responsible for the transfer of UDP-galactose to terminal N-acetylglucosamine. B4GALT3 is differentially expressed in tumors and adjacent normal tissues, and is correlated with clinical prognosis in several cancers, including neuroblastoma, cervical cancer, and bladder cancer. However, the exact role of B4GALT3 in the tumor immune microenvironment (TIME) remains unclear. Here, we aimed to elucidate the function of B4GALT3 in the TIME. Methods: To study the functions of B4GALT3 in cancer immunity, either weakly or strongly immunogenic tumor cells were subcutaneously transplanted into wild-type (WT) and B4galt3 knockout (KO) mice. Bone marrow transplantation and CD8+ T cell depletion experiments were conducted to elucidate the role of immune cells in suppressing tumor growth in B4galt3 KO mice. The cell types and gene expression in the tumor region and infiltrating CD8+ T cells were analyzed using flow cytometry and RNA sequencing. N-glycosylated proteins from WT and B4galt3 KO mice were compared using the liquid chromatography tandem mass spectrometry (LC-MS/MS)-based glycoproteomic approach. Results: B4galt3 KO mice exhibited suppressed growth of strongly immunogenic tumors with a notable increase in CD8+ T cell infiltration within tumors. Notably, B4galt3 deficiency led to changes in N-glycan modification of several proteins, including integrin alpha L (ITGAL), involved in T cell activity and proliferation. In vitro experiments suggested that B4galt3 KO CD8+ T cells were more susceptible to activation and displayed increased downstream phosphorylation of FAK linked to ITGAL. Conclusion: Our study demonstrates that B4galt3 deficiency can potentially boost anti-tumor immune responses, largely through enhancing the influx of CD8+ T cells. B4GALT3 might be suppressing cancer immunity by synthesizing the glycan structure of molecules on the CD8+ T cell surface, as evidenced by the changes in the glycan structure of ITGAL in immune cells. Importantly, B4galt3 KO mice showed no adverse effects on growth, development, or reproduction, underscoring the potential of B4GALT3 as a promising and safe therapeutic target for cancer treatment.

A degron system targeting endogenous PD-1 inhibits the growth of tumor cells in mice.

Recently, targeted protein degradation systems have been developed using the ubiquitin-proteasome system. Here, we established Programmed cell death-1 (PD-1) knockdown mice as a model system for subjecting endogenous mouse proteins to the small molecule-assisted shutoff (SMASh) degron system. SMASh degron-tagged PD-1-mCherry in Jurkat cells and CD3+ splenocytes were degraded by the NS3/4A protease inhibitors, asunaprevir (ASV) or grazoprevir (GRV). Growth of MC-38 colon adenocarcinoma cells injected in Pdcd1-mCherry-SMASh homozygous knock-in (KI) mice was repressed by ASV or GRV. Moreover, growth of MC-38 cells was suppressed in wild-type mice transplanted with KI bone marrow cells after GRV treatment. This is the first study to use a degron tag targeting an endogenous mouse protein in vivo. Our experimental system using the SMASh degron may be employed for treating diseases and characterizing the cellular functions of essential proteins.

Digenic inheritance of mutations in EPHA2 and SLC26A4 in Pendred syndrome.

Enlarged vestibular aqueduct (EVA) is one of the most commonly identified inner ear malformations in hearing loss patients including Pendred syndrome. While biallelic mutations of the SLC26A4 gene, encoding pendrin, causes non-syndromic hearing loss with EVA or Pendred syndrome, a considerable number of patients appear to carry mono-allelic mutation. This suggests faulty pendrin regulatory machinery results in hearing loss. Here we identify EPHA2 as another causative gene of Pendred syndrome with SLC26A4. EphA2 forms a protein complex with pendrin controlling pendrin localization, which is disrupted in some pathogenic forms of pendrin. Moreover, point mutations leading to amino acid substitution in the EPHA2 gene are identified from patients bearing mono-allelic mutation of SLC26A4. Ephrin-B2 binds to EphA2 triggering internalization with pendrin inducing EphA2 autophosphorylation weakly. The identified EphA2 mutants attenuate ephrin-B2- but not ephrin-A1-induced EphA2 internalization with pendrin. Our results uncover an unexpected role of the Eph/ephrin system in epithelial function.

Heterochromatin protein 1γ deficiency decreases histone H3K27 methylation in mouse neurosphere neuronal genes.

Heterochromatin protein (HP) 1γ, a component of heterochromatin in eukaryotes, is involved in H3K9 methylation. Although HP1γ is expressed strongly in neural tissues and neural stem cells, its functions are unclear. To elucidate the roles of HP1γ, we analyzed HP1γ -deficient (HP1γ KO) mouse embryonic neurospheres and determined that HP1γ KO neurospheres tended to differentiate after quaternary culture. Several genes normally expressed in neuronal cells were upregulated in HP1γ KO undifferentiated neurospheres, but not in the wild type (WT). Compared to that in the control neurospheres, the occupancy of H3K27me3 was lower around the transcription start sites (TSSs) of these genes in HP1γ KO neurospheres, while H3K9me2/3, H3K4me3, and H3K27ac amounts remained unchanged. Moreover, amounts of the H3K27me2/3 demethylases, UTX, and JMJD3, were increased around the TSSs of these genes. Treatment with GSK-J4, an inhibitor of H3K27 demethylases, decreased the expression of genes upregulated in HP1γ KO neurospheres, along with an increase of H3K27me3 amounts. Therefore, in murine neurospheres, HP1γ protected the promoter sites of differentiated cell-specific genes against H3K27 demethylases to repress the expression of these genes. A better understanding of central cellular processes such as histone methylation will help elucidate critical events such as cell-specific gene expression, epigenetics, and differentiation.

Galactosyl carbohydrate residues on hematopoietic stem/progenitor cells are essential for homing and engraftment to the bone marrow.

The role of carbohydrate chains in leukocyte migration to inflamed sites during inflammation and trafficking to the lymph nodes under physiological conditions has been extensively characterized. Here, we report that carbohydrate chains also mediate the homing and engraftment of hematopoietic stem/progenitor cells (HSPCs) to the bone marrow (BM). In particular, we found that transplanted BM cells deficient in ß-1,4-galactosyltransferase-1 (ß4GalT-1) could not support survival in mice exposed to a lethal dose of irradiation. BM cells obtained from mice deficient in ß4GalT-1 showed normal colony-forming activity and hematopoietic stem cell numbers. However, colony-forming cells were markedly rare in the BM of recipient mice 24 h after transplantation of ß4GalT-1-deficient BM cells, suggesting that ß4GalT-1 deficiency severely impairs homing. Similarly, BM cells with a point mutation in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene, encoding a key enzyme in sialic acid biosynthesis, showed mildly impaired homing and engraftment abilities. These results imply that the galactosyl, but not sialyl residues in glycoproteins, are essential for the homing and engraftment of HSPCs to the BM. These findings suggest the possibility of modifying carbohydrate structures on the surface of HSPCs to improve their homing and engraftment to the BM in clinical application.

Lactosylceramide synthases encoded by B4galt5 and 6 genes are pivotal for neuronal generation and myelin formation in mice.

It is uncertain which ß4-galactosyltransferase (ß4GalT; gene name, B4galt), ß4GalT-5 and/or ß4GalT-6, is responsible for the production of lactosylceramide (LacCer) synthase, which functions in the initial step of ganglioside biosynthesis. Here, we generated conditional B4galt5 knockout (B4galt5 cKO) mice, using Nestin-Cre mice, and crossed these with B4galt6 KO mice to generate B4galt5 and 6 double KO (DKO) mice in the central nervous system (CNS). LacCer synthase activity and major brain gangliosides were completely absent in brain homogenates from the DKO mice, although LacCer synthase activity was about half its normal level in B4galt5 cKO mice and B4galt6 KO mice. The DKO mice were born normally but they showed growth retardation and motor deficits at 2 weeks and died by 4 weeks of age. Histological analyses showed that myelin-associated proteins were rarely found localized in axons in the cerebral cortex, and axonal and myelin formation were remarkably impaired in the spinal cords of the DKO mice. Neuronal cells, differentiated from neurospheres that were prepared from the DKO mice, showed impairments in neurite outgrowth and branch formation, which can be explained by the fact that neurospheres from DKO mice could weakly interact with laminin due to lack of gangliosides, such as GM1a. Furthermore, the neurons were immature and perineuronal nets (PNNs) were poorly formed in DKO cerebral cortices. Our results indicate that LacCer synthase is encoded by B4galt5 and 6 genes in the CNS, and that gangliosides are indispensable for neuronal maturation, PNN formation, and axonal and myelin formation.

Cbx3/HP1γ deficiency confers enhanced tumor-killing capacity on CD8+ T cells.

Cbx3/HP1γ is a histone reader whose function in the immune system is not completely understood. Here, we demonstrate that in CD8+ T cells, Cbx3/HP1γ insufficiency leads to chromatin remodeling accompanied by enhanced Prf1, Gzmb and Ifng expression. In tumors obtained from Cbx3/HP1γ-insufficient mice or wild type mice treated with Cbx3/HP1γ-insufficient CD8+ T cells, there is an increase of CD8+ effector T cells expressing the stimulatory receptor Klrk1/NKG2D, a decrease in CD4+ CD25+ FOXP3+ regulatory T cells (Treg cells) as well as CD25+ CD4+ T cells expressing the inhibitory receptor CTLA4. Together these changes in the tumor immune environment may have mitigated tumor burden in Cbx3/HP1γ-insufficient mice or wild type mice treated with Cbx3/HP1γ-insufficient CD8+ T cells. These findings suggest that targeting Cbx3/HP1γ can represent a rational therapeutic approach to control growth of solid tumors.

New insights into the role of Jmjd3 and Utx in axial skeletal formation in mice.

Jmjd3 and Utx are demethylases specific for lysine 27 of histone H3. Previous reports indicate that Jmjd3 is essential for differentiation of various cell types, such as macrophages and epidermal cells in mice, whereas Utx is involved in cancer and developmental diseases in humans and mice, as well as Hox regulation in zebrafish and nematodes. Here, we report that Jmjd3, but not Utx, is involved in axial skeletal formation in mice. A Jmjd3 mutant embryo (Jmjd3Δ18/Δ18), but not a catalytically inactive Utx truncation mutant (Utx-/y), showed anterior homeotic transformation. Quantitative RT-PCR and microarray analyses showed reduced Hox expression in both Jmjd3Δ18/Δ18 embryos and tailbuds, whereas levels of Hox activators, such as Wnt signaling factors and retinoic acid synthases, did not decrease, which suggests that Jmjd3 plays a regulatory role in Hox expression during axial patterning. Chromatin immunoprecipitation analyses of embryo tailbud tissue showed trimethylated lysine 27 on histone H3 to be at higher levels at the Hox loci in Jmjd3Δ18/Δ18 mutants compared with wild-type tailbuds. In contrast, trimethylated lysine 4 on histone H3 levels were found to be equivalent in wild-type and Jmjd3Δ18/Δ18 tailbuds. Demethylase-inactive Jmjd3 mutant embryos showed the same phenotype as Jmjd3Δ18/Δ18 mice. These results suggest that the demethylase activity of Jmjd3, but not that of Utx, affects mouse axial patterning in concert with alterations in Hox gene expression.-Naruse, C., Shibata, S., Tamura, M., Kawaguchi, T., Abe, K., Sugihara, K., Kato, T., Nishiuchi, T., Wakana, S., Ikawa, M., Asano, M. New insights into the role of Jmjd3 and Utx in axial skeletal formation in mice.

HP-1γ Controls High-Affinity Antibody Response to T-Dependent Antigens.

In vitro observations suggest a role for the mouse heterochromatin protein 1γ (HP-1γ) in the immune system. However, it has not been shown if and how HP-1γ contributes to immunity in vivo. Here we show that in mice, HP-1γ positively regulates the germinal center reaction and high-affinity antibody response to thymus (T)-dependent antigens by limiting the size of CD8(+) regulatory T-cell (Treg) compartment without affecting progenitor B- or T-cell-development. Moreover, HP-1γ does not control cell proliferation or class switch recombination. Haploinsufficiency of cbx-3 (gene encoding HP-1γ) is sufficient to expand the CD8(+) Treg population and impair the immune response in mice despite the presence of wild-type HP-1α and HP-1ß. This is the first in vivo evidence demonstrating the non-redundant role of HP-1γ in immunity.

A replication-dependent passive mechanism modulates DNA demethylation in mouse primordial germ cells.

Germline cells reprogramme extensive epigenetic modifications to ensure the cellular totipotency of subsequent generations and to prevent the accumulation of epimutations. Notably, primordial germ cells (PGCs) erase genome-wide DNA methylation and H3K9 dimethylation marks in a stepwise manner during migration and gonadal periods. In this study, we profiled DNA and histone methylation on transposable elements during PGC development, and examined the role of DNA replication in DNA demethylation in gonadal PGCs. CpGs in short interspersed nuclear elements (SINEs) B1 and B2 were substantially demethylated in migrating PGCs, whereas CpGs in long interspersed nuclear elements (LINEs), such as LINE-1, were resistant to early demethylation. By contrast, CpGs in both LINE-1 and SINEs were rapidly demethylated in gonadal PGCs. Four major modifiers of DNA and histone methylation, Dnmt3a, Dnmt3b, Glp and Uhrf1, were actively repressed at distinct stages of PGC development. DNMT1 was localised at replication foci in nascent PGCs, whereas the efficiency of recruitment of DNMT1 into replication foci was severely impaired in gonadal PGCs. Hairpin bisulphite sequencing analysis showed that strand-specific hemi-methylated CpGs on LINE-1 were predominant in gonadal PGCs. Furthermore, DNA demethylation in SINEs and LINE-1 was impaired in Cbx3-deficient PGCs, indicating abnormalities in G1 to S phase progression. We propose that PGCs employ active and passive mechanisms for efficient and widespread erasure of genomic DNA methylation.

Evidence that the EphA2 receptor exacerbates ischemic brain injury.

Ephrin (Eph) signaling within the central nervous system is known to modulate axon guidance, synaptic plasticity, and to promote long-term potentiation. We investigated the potential involvement of EphA2 receptors in ischemic stroke-induced brain inflammation in a mouse model of focal stroke. Cerebral ischemia was induced in male C57Bl6/J wild-type (WT) and EphA2-deficient (EphA2(-/-)) mice by middle cerebral artery occlusion (MCAO; 60 min), followed by reperfusion (24 or 72 h). Brain infarction was measured using triphenyltetrazolium chloride staining. Neurological deficit scores and brain infarct volumes were significantly less in EphA2(-/-) mice compared with WT controls. This protection by EphA2 deletion was associated with a comparative decrease in brain edema, blood-brain barrier damage, MMP-9 expression and leukocyte infiltration, and higher expression levels of the tight junction protein, zona occludens-1. Moreover, EphA2(-/-) brains had significantly lower levels of the pro-apoptotic proteins, cleaved caspase-3 and BAX, and higher levels of the anti-apoptotic protein, Bcl-2 as compared to WT group. We confirmed that isolated WT cortical neurons express the EphA2 receptor and its ligands (ephrin-A1-A3). Furthermore, expression of all four proteins was increased in WT primary cortical neurons following 24 h of glucose deprivation, and in the brains of WT mice following stroke. Glucose deprivation induced less cell death in primary neurons from EphA2(-/-) compared with WT mice. In conclusion, our data provide the first evidence that the EphA2 receptor directly contributes to blood-brain barrier damage and neuronal death following ischemic stroke.

HP1γ links histone methylation marks to meiotic synapsis in mice.

During meiosis, specific histone modifications at pericentric heterochromatin (PCH), especially histone H3 tri- and dimethylation at lysine 9 (H3K9me3 and H3K9me2, respectively), are required for proper chromosome interactions. However, the molecular mechanism by which H3K9 methylation mediates the synapsis is not yet understood. We have generated a Cbx3-deficient mouse line and performed comparative analysis on Suv39h1/h2-, G9a- and Cbx3-deficient spermatocytes. This study revealed that H3K9me2 at PCH depended on Suv39h1/h2-mediated H3K9me3 and its recognition by the Cbx3 gene product HP1γ. We further found that centromere clustering and synapsis were commonly affected in G9a- and Cbx3-deficient spermatocytes. These genetic observations suggest that HP1γ/G9a-dependent PCH-mediated centromere clustering is an axis for proper chromosome interactions during meiotic prophase. We propose that the role of the HP1γ/G9a axis is to retain centromeric regions of unpaired homologous chromosomes in close alignment and facilitate progression of their pairing in early meiotic prophase. This study also reveals considerable plasticity in the interplay between different histone modifications and suggests that such stepwise and dynamic epigenetic modifications may play a pivotal role in meiosis.

Loss of heterochromatin protein 1 gamma reduces the number of primordial germ cells via impaired cell cycle progression in mice.

Signals from extraembryonic tissues in mice determine which proximal epiblast cells become primordial germ cells (PGCs). After their specification, approximately 40 PGCs appear at the base of the allantoic bud and migrate to the genital ridges, where they expand to about 25 000 cells by Embryonic Day (E)13.5. The heterochromatin protein 1 (HP1) family members HP1alpha, HP1beta, and HP1gamma (CBX5, CBX1, and CBX3, respectively) are thought to induce heterochromatin structure and to regulate gene expression by binding methylated histone H3 lysine 9. We found a dramatic loss of germ cells before meiosis in HP1gamma mutant (HP1gamma(-/-)) mice that we generated previously. The reduction in PGCs in HP1gamma(-/-) embryos was detectable from the early bud stage (E7.25), and the number of HP1gamma(-/-) PGCs was gradually reduced thereafter. Bromodeoxyuridine incorporation into PGCs was significantly reduced in E7.25 and E12.5 HP1gamma(-/-) embryos. Furthermore, a lower proportion of HP1gamma(-/-) PGCs than wild-type PGCs was in S phase, and a higher proportion, respectively, was in G1 phase at E12.5. Moreover, the proportion of p21 (Cip, official symbol CDKN1A)-positive HP1gamma(-/-) PGCs was increased, suggesting that the G1/S phase transition was inhibited. However, no differences were detected between fate determination, migration, apoptosis, or histone modification of PGCs of control embryos and those of HP1gamma(-/-) embryos. Therefore, the reduction in PGCs in HP1gamma(-/-) embryos could be caused by impaired cell cycle in PGCs. These results suggest that HP1gamma plays an important role in keeping enough germ cells by regulating the PGC cell cycle.

Beta4-galactosyltransferase-5 is a lactosylceramide synthase essential for mouse extra-embryonic development.

Glycosphingolipids (GSLs) are important for various biological functions in the nervous system, the immune system, embryogenesis and in other tissues and processes. Lactosylceramide (LacCer), which is synthesized from glucosylceramide (GlcCer) by LacCer synthase, is a core structure of GSLs, including gangliosides. LacCer synthase was reported to be synthesized by the beta4-galactosyltransferase-6 (beta4GalT-6) gene in the rat brain. However, the existence of another LacCer synthase gene was shown in cultured cells lacking beta4GalT-6. Here, we report that LacCer synthase is mainly synthesized by the beta4GalT-5 gene during early mouse embryogenesis, and its disruption is embryonic lethal. beta4GalT-5-deficient embryos showed developmental retardation from E7.5 and died by E10.5 as reported previously. LacCer synthase activity was significantly reduced in beta4GalT-5-deficient embryos and extra-embryonic endoderm (XEN) cells derived from blastocysts, and it was recovered when beta4GalT-5 cDNA was introduced into beta4GalT-5-deficient XEN cells. The amounts of LacCer and GM3 ganglioside were drastically reduced, while GlcCer accumulated in the beta4GalT-5-deficient XEN cells. Hematoma and ectopically accumulated trophoblast giant cells were observed in the anti-mesometrial pole of the extra-embryonic tissues, although all three embryonic layers formed. beta4GalT-5-deficient embryos developed until E12.5 as chimeras with wild-type tetraploid cells, which formed the extra-embryonic membranes, indicating that extra-embryonic defects caused the early embryonic lethality. Our results suggest that beta4GalT-5 is essential for extra-embryonic development during early mouse embryogenesis.

Bidirectional signaling through ephrinA2-EphA2 enhances osteoclastogenesis and suppresses osteoblastogenesis.

Bone is remodeled constantly throughout life by bone-resorbing osteoclasts and bone-forming osteoblasts. To maintain bone volume and quality, differentiation of osteoclasts and osteoblasts is tightly regulated through communication between and within these two cell lineages. Previously we reported that cell-cell interaction mediated by ephrinB2 ligand on osteoclasts and EphB4 receptor on osteoblasts generates bidirectional anti-osteoclastogenic and pro-osteoblastogenic signals into respective cells and presumably facilitates transition from bone resorption to bone formation. Here we show that bidirectional ephrinA2-EphA2 signaling regulates bone remodeling at the initiation phase. EphrinA2 expression was rapidly induced by receptor activator of NF-kappaB ligand in osteoclast precursors; this was dependent on the transcription factor c-Fos but independent of the c-Fos target gene product NFATc1. Receptor EphA2 was expressed in osteoclast precursors and osteoblasts. Overexpression experiments revealed that both ephrinA2 and EphA2 in osteoclast precursors enhanced differentiation of multinucleated osteoclasts and that phospholipase Cgamma2 may mediate ephrinA2 reverse signaling. Moreover, ephrinA2 on osteoclasts was cleaved by metalloproteinases, and ephrinA2 released in the culture medium enhanced osteoclastogenesis. Interestingly, differentiation of osteoblasts lacking EphA2 was enhanced along with alkaline phosphatase, Runx2, and Osterix expression, indicating that EphA2 on osteoblasts generates anti-osteoblastogenic signals presumably by up-regulating RhoA activity. Therefore, ephrinA2-EphA2 interaction facilitates the initiation phase of bone remodeling by enhancing osteoclast differentiation and suppressing osteoblast differentiation.

EphA2 is an essential mediator of UV radiation-induced apoptosis.

One of the physiologic consequences of excessive UV radiation (UVR) exposure is apoptosis. This critical response serves to eliminate genetically injured cells and arises, in part, from activation of DNA damage and p53 signaling. Other contributory pathways, however, likely exist but have not been fully characterized. In a recent global screen of UVR response genes in melanocytes, we identified the receptor tyrosine kinase EPHA2. Using a combination of genetic and pharmacologic approaches, we set out to investigate the upstream regulation of EphA2 by UVR and the functional consequences of this effect. We found that the UVR-associated increase in EphA2 occurs in melanocytes, keratinocytes, and fibroblasts from both human and murine sources. More specifically, UVR effectively up-regulated EphA2 individually in p53-null, p63-null, and p73-null murine embryonic fibroblasts (MEF), suggesting that the p53 family of transcription factors is not essential for the observed effect. However, inhibition of mitogen-activated protein kinase (MAPK) signaling by U0126 and PD98059 significantly reduced the UVR response whereas overexpression of oncogenic NRAS led to an increase in EphA2. These results confirm that UVR induces EphA2 by a p53-independent, but MAPK-dependent, mechanism. In response to UV irradiation, Epha2(-/-) MEFs were highly resistant to UVR-mediated cytotoxicity and apoptosis whereas introduction of EphA2 into both wild-type and p53-null MEFs led to activation of an apoptotic program that can be blocked by caspase-8 inhibition. These functional findings suggest that EphA2 is in fact an essential p53-independent, caspase-8-dependent proapoptotic factor induced by UVR.

Wtap is required for differentiation of endoderm and mesoderm in the mouse embryo.

Wilms' tumor 1-associating protein (WTAP) was previously identified as a protein associated with Wilms' tumor-1 (WT-1) protein that is essential for the development of the genitourinary system. Although WTAP has been suggested to function in alternative splicing, stabilization of mRNA, and cell growth, its in vivo function is still unclear. We generated Wtap mutant mice using a novel gene-trap approach and showed that Wtap mutant embryos exhibited defective egg-cylinder formation at the gastrulation stage and died by embryonic day 10.5. Although they could form extraembryonic tissues and anterior visceral endoderm, Wtap mutant embryos and embryonic stem cells failed to differentiate into endoderm and mesoderm. The chimera analysis showed that Wtap in extraembryonic tissues was required for the formation of mesoderm and endoderm in embryonic tissues. Taken together, our findings indicate that Wtap is indispensable for differentiation of mesoderm and endoderm in the mouse embryo.

A novel beta-myosin heavy chain gene mutation, p.Met531Arg, identified in isolated left ventricular non-compaction in humans, results in left ventricular hypertrophy that progresses to dilation in a mouse model.

Mutations in the betaMHC (beta-myosin heavy chain), a sarcomeric protein are responsible for hypertrophic and dilated cardiomyopathy. However, the mechanisms whereby distinct mutations in the betaMHC gene cause two kinds of cardiomyopathy are still unclear. In the present study we report a novel betaMHC mutation found in a patient with isolated LVNC [LV (left ventricular) non-compaction] and the phenotype of a mouse mutant model carrying the same mutation. To find the mutation responsible, we searched for genomic mutations in 99 unrelated probands with dilated cardiomyopathy and five probands with isolated LVNC, and identified a p.Met531Arg mutation in betaMHC in a 13-year-old girl with isolated LVNC. Next, we generated six lines of transgenic mice carrying a p.Met532Arg mutant alphaMHC gene, which was identical with the p.Met531Arg mutation in the human betaMHC. Among these, two lines with strong expression of the mutant alphaMHC gene were chosen for further studies. Although they did not exhibit the features characteristic of LVNC, approx. 50% and 70% of transgenic mice in each line displayed LVH (LV hypertrophy) by 2-3 months of age. Furthermore, LVD (LV dilation) developed in approx. 25% of transgenic mice by 18 months of age, demonstrating biphasic changes in LV wall thickness. The present study supports the idea that common mechanisms may be involved in LVH and LVD. The novel mouse model generated can provide important information for the understanding of the pathological processes and aetiology of cardiac dilation in humans.

A novel gene trapping for identifying genes expressed under the control of specific transcription factors.

Gene trapping is a powerful method for identifying novel genes and for analyzing their functions. It is, however, difficult to select trapped genes on the basis of their function. To identify genes regulated by transcription factors that are important in the mesodermal formation, we selected trapped ES clones by infection of adenoviral vectors expressing Pax1, Brachyury, and Foxa2. Among 366 trapped genes, seven seemed to be controlled by these transcription factors in the first screening. The trapped genes were identified by 5' RACE, and a Northern blotting revealed that expressions of three trapped genes were regulated by these transcription factors. Expression patterns of Cx43 and HP1gamma implicated their functional relationships to Foxa2 in the formation of the notochord and the neural tube. Furthermore, Wtap mutant mice derived from the trapped clone showed defects in the mesendoderm formation. Our results indicate that trapped ES clones could be selected effectively using transcription factors.

Development of IgA nephropathy-like disease with high serum IgA levels and increased proportion of polymeric IgA in Beta-1,4-galactosyltransferase-deficient mice.

The glycosylation of glycoproteins is important for their biological activity, conformation and stability. Recent studies indicate that aberrant glycosylation causes various human disorders. Here we report that mice lacking beta-1,4-galactosyltransferase-I (beta4GalT-I), which transfers galactose from UDP-Gal to terminal GlcNAc of N- and O-glycans in a beta-1,4- linkage, developed IgA nephropathy (IgAN)-like disease. Urinary albumin levels were significantly increased in the beta4GalT-I-deficient mice. Hematuria was detected in some of the beta4GalT-I-deficient mice, suggesting impaired renal function. Furthermore, histological and immunohistochemical examination showed expanded mesangial matrix, IgA deposition with mesangial pattern and electron-dense deposits in the paramesangial regions in the beta4GalT-Ideficient mice. These results demonstrate that the beta4GalT-I-deficient mice developed IgANlike disease. Furthermore, high serum IgA levels with increased polymeric forms were detected. In humans, serum IgA derived from patients with IgAN has aberrant beta3-galactosylation and sialylation on its O-linked glycans of the hinge region. Mouse IgA does not have O-glycans of the hinge region and has several N-glycans. As expected, beta4-galactosylation on the N-glycans of the serum IgA of the beta4GalT-I-deficient mice was completely absent. This is the first report demonstrating that genetic remodeling of protein glycosylation causes IgAN. We suggest that aberrant beta4-galactosylation of serum IgA participates in the Nishie/Miyaishi/Azuma/Kameyama/Naruse/Hashimoto/Yokoyama/Narimatsu/Wada/Asano 126 development of IgAN, including deposition of IgA, polymerization of IgA, and glomerular injury after IgA deposition.