Yuki Takada

Dilated Cardiomyopathy Caused by Aberrant Endoplasmic Reticulum Quality Control in Mutant KDEL Receptor Transgenic Mice

ABSTRACT Aberrant protein folding beyond the capacity of endoplasmic reticulum (ER) quality control leads to stress response in the ER. The Lys-Asp-Glu-Leu (KDEL) receptor, a retrieval receptor for ER chaperones in the early secretory pathway, contributes to ER quality control. To elucidate the function of the KDEL receptor in vivo, we established transgenic mice expressing a mutant KDEL receptor. We found that the mutant KDEL receptor sensitized cells to ER stress and that the mutant mice developed dilated cardiomyopathy. Ultrastructural analyses revealed expanded sarcoplasmic reticulums and protein aggregates that obstructed the adjacent transverse tubules of the mutant cardiomyocytes. Cardiomyocytes from the mutant mice were sensitive to ER stress when treated with tunicamycin and showed a functional defect in the L-type Ca2+ current. We observed ubiquitinated protein aggregates, enhanced expression of CHOP (a death-related transcriptional factor expressed upon ER stress), and apoptosis in the mutant hearts. These findings suggest that impairment of the KDEL receptor disturbs ER quality control, resulting in accumulation of misfolded proteins in the ER in an in vivo system, and that the dilated cardiomyopathy found in the mutant KDEL receptor transgenic mice is associated with ER stress.

Mammalian Polyhomeotic Homologues Phc2 and Phc1 Act in Synergy To Mediate Polycomb Repression of Hox Genes.

ABSTRACT The Polycomb group (PcG) gene products form multimeric protein complexes and contribute to anterior-posterior (A-P) specification via the transcriptional regulation of Hox cluster genes. The Drosophila polyhomeotic genes and their mammalian orthologues, Phc1, Phc2, and Phc3, encode nuclear proteins that are constituents of evolutionarily conserved protein complexes designated class II PcG complexes. In this study, we describe the generation and phenotypes of Phc2-deficient mice. We show posterior transformations of the axial skeleton and premature senescence of mouse embryonic fibroblasts associated with derepression of Hox cluster genes and Cdkn2a genes, respectively. Synergistic actions of a Phc2 mutation with Phc1 and Rnf110 mutations during A-P specification, coimmunoprecipitation of their products from embryonic extracts, and chromatin immunoprecipitation by anti-Phc2 monoclonal antibodies suggest that Hox repression by Phc2 is mediated through the class II PcG complexes, probably via direct binding to the Hox locus. The genetic interactions further reveal the functional overlap between Phc2 and Phc1 and a strict dose-dependent requirement during A-P specification and embryonic survival. Functional redundancy between Phc2 and Phc1 leads us to hypothesize that the overall level of polyhomeotic orthologues in nuclei is a parameter that is critical in enabling the class II PcG complexes to exert their molecular functions.

Overlapping Roles for Homeodomain-Interacting Protein Kinases Hipk1 and Hipk2 in the Mediation of Cell Growth in Response to Morphogenetic and Genotoxic Signals

ABSTRACT Homeodomain-interacting protein kinase 1 (Hipk1), 2, and 3 genes encode evolutionarily conserved nuclear serine/threonine kinases, which were originally identified as interacting with homeodomain-containing proteins. Hipks have been repeatedly identified as interactors for a vast range of functional proteins, including not only transcriptional regulators and chromatin modifiers but also cytoplasmic signal transducers, transmembrane proteins, and the E2 component of SUMO ligase. Gain-of-function experiments using cultured cells indicate growth regulatory roles for Hipks on receipt of morphogenetic and genotoxic signals. However, Hipk1 and Hipk2 singly deficient mice were grossly normal, and this is expected to be due to a functional redundancy between Hipk1 and Hipk2. Therefore, we addressed the physiological roles of Hipk family proteins by using Hipk1 Hipk2 double mutants. Hipk1 Hipk2 double homozygotes are progressively lost between 9.5 and 12.5 days postcoitus and frequently fail to close the anterior neuropore and exhibit exencephaly. This is most likely due to defective proliferation in the neural fold and underlying paraxial mesoderm, particularly in the ventral region, which may be attributed to decreased responsiveness to Sonic hedgehog signals. The present study indicated the overlapping roles for Hipk1 and Hipk2 in mediating cell proliferation and apoptosis in response to morphogenetic and genotoxic signals during mouse development.

Mammalian Polycomb Scmh1 mediates exclusion of Polycomb complexes from the XY body in the pachytene spermatocytes

The product of the Scmh1 gene, a mammalian homolog of Drosophila Sex comb on midleg, is a constituent of the mammalian Polycomb repressive complexes 1 (Prc1). We have identified Scmh1 as an indispensable component of the Prc1. During progression through pachytene, Scmh1 was shown to be excluded from the XY body at late pachytene, together with other Prc1 components such as Phc1, Phc2, Rnf110 (Pcgf2), Bmi1 and Cbx2. We have identified the role of Scmh1 in mediating the survival of late pachytene spermatocytes. Apoptotic elimination of Scmh1-/- spermatocytes is accompanied by the preceding failure of several specific chromatin modifications at the XY body, whereas synapsis of homologous autosomes is not affected. It is therefore suggested that Scmh1 is involved in regulating the sequential changes in chromatin modifications at the XY chromatin domain of the pachytene spermatocytes. Restoration of defects in Scmh1-/- spermatocytes by Phc2 mutation indicates that Scmh1 exerts its molecular functions via its interaction with Prc1. Therefore, for the first time, we are able to indicate a functional involvement of Prc1 during the meiotic prophase of male germ cells and a regulatory role of Scmh1 for Prc1, which involves sex chromosomes.

topors, a p53 and topoisomerase I-binding RING finger protein, is a coactivator of p53 in growth suppression induced by DNA damage

The RING family zinc-finger protein topors (topoisomerase I-binding protein) binds not only topoisomerase I, but also p53 and the AAV-2 Rep78/68 proteins. topors maps to human chromosome 9p21, which contains candidate tumor suppressor genes implicated in small cell lung cancers. In this study, we isolated the murine counterpart of topors and investigated its impact on p53 function. The deduced amino-acid sequence of mouse topors exhibits extensive similarity to human topors. Overexpressed myc-tagged topors associates with and stabilizes p53, and enhances the p53-dependent transcriptional activities of p21Waf1, MDM2 and Bax promoters and elevates endogenous p21Waf1 mRNA levels. Overexpression of topors consequently results in the suppression of cell growth by cell cycle arrest and/or by the induction of apoptosis. Taken together, these studies identify topors as a positive regulator of p53. The expression of topors is induced by exposure to the genotoxic reagents cisplatin and camptothecin, a DNA topoisomerase I inhibitor. We therefore postulate that topors mediates p53-dependent cellular responses induced by DNA damage, suggesting its physiological role as a tumor suppressor.

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.

Reproductive Isolation in Hybrid Mice Due to Spermatogenesis Defects at Three Meiotic Stages

Early in the process of speciation, reproductive failures occur in hybrid animals between genetically diverged populations. The sterile hybrid animals are often males in mammals and they exhibit spermatogenic disruptions, resulting in decreased number and/or malformation of mature sperms. Despite the generality of this phenomenon, comparative study of phenotypes in hybrid males from various crosses has not been done, and therefore the comprehensive genetic basis of the disruption is still elusive. In this study, we characterized the spermatogenic phenotype especially during meiosis in four different cases of reproductive isolation: B6-ChrXMSM, PGN-ChrXMSM, (B6 × Mus musculus musculus-NJL/Ms) F1, and (B6 × Mus spretus) F1. The first two are consomic strains, both bearing the X chromosome of M. m. molossinus; in B6-ChrXMSM, the genetic background is the laboratory strain C57BL/6J (predominantly M. m. domesticus), while in PGN-ChrXMSM the background is the PGN2/Ms strain purely derived from wild M. m. domesticus. The last two cases are F1 hybrids between mouse subspecies or species. Each of the hybrid males exhibited cell-cycle arrest and/or apoptosis at either one or two of three distinct meiotic stages: premeiotic stage, zygotene-to-pachytene stage of prophase I, and metaphase I. This study shows that the sterility in hybrid males is caused by spermatogenic disruptions at multiple stages, suggesting that the responsible genes function in different cellular processes. Furthermore, the stages with disruptions are not correlated with the genetic distance between the respective parental strains.

Induction of Wnt5a -Expressing Mesenchymal Cells Adjacent to the Cloacal Plate Is an Essential Process for Its Proximodistal Elongation and Subsequent Anorectal Development

Anorectal malformations encompass a broad spectrum of congenital defects and are related to the development of the genital tubercle, including the cloacal plate and urorectal septum. To explore the cellular and molecular basis of anorectal malformations, we analyzed the pathogenetic process using two mouse models: Danforths short tail (Sd) and all-trans retinoic acid (ATRA)-treated mice. Embryologically, the cloacal plate may be divided into distal and proximal parts, with the distal part subdivided into ventral and dorsal parts. In the two mouse models, anorectal malformations occur due to improper development of the proximal part of the cloacal plate. At 10.5 days postcoitus (dpc), in Sd homozygotes, there was a lack of Shh expression only in the cloacal plate and the endoderm around the cloacal plate. In addition, Wnt5a was not expressed in the mesoderm adjacent to the cloacal plate in the two mouse models, and Axin2, which is regulated by Wnt signaling, was not expressed in the dorsal part of the cloacal plate at 12.5 dpc. Based on these results, we suggest that Wnt5a, which is downstream of Shh signaling, and Axin2 affect the development of the proximal part of the cloacal plate.

The mouse Edr2 (Mph2) gene has two forms of mRNA encoding 90- and 36-kDa polypeptides.

The vertebrate Polycomb Group (PcG) genes encode proteins that form large multimeric and chromatin-associated complexes implicated in the stable repression of developmentally essential genes. Here we have isolated a 2.5-kb cDNA for Edr2, a mouse homolog of the Drosophila PcG gene Ph, although it was originally identified as a 3.8-kb cDNA. However, little is known about molecular basis of the 3.8-kb cDNA. Genomic and RNA analyses have shown that Edr2 locates on Chromosome 4 as a single copy gene and is transcribed into at least two transcript isoforms about 3.0 and 4.4 kb in length, most likely corresponding to the 2.5- and 3.8-kb cDNAs, respectively. The largest open reading frames in the 2.5- and 3.8-kb cDNAs encode 36- and 90-kDa polypeptides, respectively. The 36-kDa protein is a truncated form lacking of the N-terminal region of the 90-kDa protein. Interestingly, it has been demonstrated that the 3.0-kb mRNA accumulates at a much higher level than the 3.8-kb mRNA in mouse embryos and mature tissues. Immunostaining assay of mammalian cells has shown that the 36-kDa form tagged with HA colocalizes with the other PcG protein Mel18 in nuclei, suggesting that the smaller protein is capable of forming maltimeric complex with other PcG proteins. Therefore, the 36-kDa protein might function generally as a PcG protein.

Enantioseparation of N-(1-arylethyl)amides and α-[N-(3,5-dinitrobenzoyl)]amino esters by column chromatography. Chiral recognition using a hydrogen bond acceptor centered in a pseudo-C2 symmetric environment. 2

Abstract Enantioseparation of N -(1-arylethyl)amides and α -[ N -(3,5-dinitrobenzoyl)]amino esters was achieved by column chromatography on silica gel modified with a chiral acylurea which has two ( S )-1-(1-naphthyl)ethyl moieties in a pseudo-C 2 symmetric position around an axis of the urea-carbonyl.