Biomedical subjects

Manabu Nakayama

Publications and source records attributed to Manabu Nakayama.

14 recordsLinked to original sources

PubMed2026

Polycomb repressive complex 2 insufficiency underlies myeloid leukemia in Down syndrome.

Children with Down syndrome (DS) have an elevated risk of developing myeloid leukemia (ML; ML in DS [ML-DS]). In addition to mutations in GATA1, which generate the truncated isoform GATA1-short (GATA1s), ML-DS requires additional somatic gene mutations, most frequently in cohesion and Polycomb repressive complex 2 (PRC2) genes. Here, we show that PRC2 insufficiency underlies ML-DS pathogenesis. Transplantation of Gata1s fetal liver cells followed by deletion of the cohesion subunit Stag2 and/or the PRC2 component Ezh2 induced megakaryocyte-biased differentiation and expansion of megakaryocytic progenitors, culminating in lethal myelofibrosis. Mechanistically, loss of Stag2 or Ezh2 reinforced Gata1s-driven reduced chromatin accessibility at erythroid transcription factor target loci in premegakaryocyte/erythroid progenitors (pre-MegEs), thereby promoting megakaryocytic skewing. Ezh2 loss attenuated the Gata1s-mediated global elevation of H3K27 trimethylation in pre-MegEs, resulting in derepression of a broad set of PRC2 target genes and establishing a functionally PRC2-insufficient state. Similarly, Stag2 loss induced a moderate but significant degree of PRC2-insufficient state in Gata1s progenitors. Furthermore, chromosome 21-encoded miR-125b blocked megakaryocytic differentiation of Gata1s progenitors lacking either Stag2 or Ezh2 alone but drove full transformation and expansion of CD150+Sca-1+c-Kit+ leukemic stem cell-like populations only upon concurrent loss of both Stag2 and Ezh2, leading to acute megakaryoblastic leukemia in mice. These findings reveal that cohesin and PRC2 insufficiencies converge on PRC2 dysfunction while exerting distinct epigenetic effects and synergize with trisomy 21 and GATA1s to remodel the epigenetic landscape, driving progression from a preleukemic state to overt leukemia.

Animals↗

PubMed2025

The RORγt ligand-binding domain controls the pathogenicity of IL-17A+ T cells differently in autoimmune diseases of the skin and CNS.

The transcription factor RORγt orchestrates Th17 lineage differentiation, thymic T cell development, and the pathogenesis of several autoimmune disorders. Lipid ligands are required for appropriate regulation of RORγt activity, but it is unclear to what extent lipid recognition controls RORγt function in vivo. Here, we show that the mutation of RORγt alanine-304 in the ligand-binding domain (LBD) to isoleucine (A304I) abrogates lipid-dependent Th17 differentiation and selectively ameliorates γδT17 cell-mediated psoriatic skin inflammation. In contrast, there is no improvement in experimental autoimmune encephalomyelitis in RORγtA304I mice. Consistent with this, the expression of genes characteristic of Th17 cells decreases in RORγtA304I mice, along with a compensatory increase of genes characteristic of Th1-like Th17 cells with pathogenic signatures. Thus, RORγt alanine-304 in the LBD is indispensable for generating γδT17 and conventional Th17 cells and for the suppression of the Th1-like Th17 pathogenic population, which decouples the pathogenicity of skin and CNS autoimmune diseases.

Animals↗

PubMed2006

Absence of DNA polymerase theta results in decreased somatic hypermutation frequency and altered mutation patterns in Ig genes.

Multiple DNA polymerases participate in somatic hypermutation of immunoglobulin (Ig) genes. Mutations at A/T are largely dependent on DNA polymerase eta (POLH) whereas mutations at C/G appear to be generated by several DNA polymerases. We have previously shown that mice expressing a catalytically inactive POLQ (Polq-inactive) have a reduction in C/G mutations. Here we have generated mice that completely lack Polq expression (Polq-null). Polq-null mice have no obvious abnormality in B or T cell differentiation, and their splenic B cells responded normally to various activation signals and underwent normal Ig gene class switching. The mutant mice mounted relatively normal immune responses against a T-dependent antigen although there was a slight decrease in antigen specific antibodies. Polq-null mice exhibited a mild reduction in the overall mutation frequency, however, in contrast to Polq-inactive mice where the reduction mostly affected mutations at C/G, Polq-null mice showed a reduction of both C/G and A/T mutations and there was a significant increase of G to C transversions. These results confirm a role for POLQ in somatic hypermutation and suggest that in the complete absence of POLQ other polymerases may functionally substitute, resulting in a mutation pattern different from that found in Polq-inactive mice.

Animals↗

PubMed2006

A gene-targeting approach for functional characterization of KIAA genes encoding extremely large proteins.

Given that thousands of genes exist in the mammalian genome, criteria are needed to prioritize their functional analysis and to decrease the likelihood of producing gene-targeted mice that lack overt phenotypes. Initial analysis efforts are likely to be fruitful if focused on genes encoding large proteins, since at least some large proteins serve as frameworks for intricate assembly of protein complexes, and their inactivation would render definitive, observable phenotypes. Here, we describe the functional characterization of the murine homologues of five human KIAA genes (KIAA1409, KIAA1440, KIAA1447, KIAA1768, and KIAA1276) that encode large proteins. Gene-targeted mice had phenotypic and developmental defects resulting from the functional deletion of three of these five genes. Mice with targeted disruption of KIAA1409 lacked the ability to drink, and those with targeted disruption of KIAA1447 displayed hind leg motor dysfunction. Disruption of KIAA1440 led to embryonic lethality at the blastocyst stage. The high success rate of our approach demonstrates the rationale for the genome-wide functional examination of large proteins in mice using reverse genetics.

Animals↗

PubMed2005

Preparation of a set of expression-ready clones of mammalian long cDNAs encoding large proteins by the ORF trap cloning method.

Although we have so far identified and sequenced >2000 human long cDNAs, known as KIAA cDNAs, half of them have yet to be functionally annotated. Expression-ready cDNA clones derived from these genes, where the open reading frame (ORF) of the gene of interest is placed under the control of an appropriate promoter, are critical for functional characterization of these gene products. In this study, we attempted to systematically convert original cDNA clones to expression-ready forms for native and fusion proteins. For this purpose, we developed a new method for ORF cloning based on a homologous recombination in Escherichia coli to avoid laborious manipulations and artificial introduction of mutations in ORF. Using 1589 putative full-length ORFs (from 1002 KIAA genes, 119 human known genes and 468 mouse genes) with an average size of 2.8 kb, we successfully prepared expression plasmids for 1463 native proteins and for 1343 fusion proteins by this method. The resultant expression-ready clones were examined using an in vitro transcription/translation system followed by SDS-polyacrylamide gel electrophoresis and by transient expression of GFP-fusion proteins in human embryonic kidney (HEK) 293 cells. This set of expression-ready clones of long cDNAs encoding large proteins would open a new route to experimentally analyze their functions on a proteomic scale, since unavailability of expression-ready clones for mammalian large proteins has been a major obstacle to the functional analysis of these cDNAs.

Animals↗

PubMed2005

Improvement of recombination efficiency by mutation of red proteins.

The Red recombination system of the bacteriophage lambda is a useful tool for engineering bacterial artificial chromosomes (BACs) because desired mutations can be made in any part of the DNA, even in large DNAs, independent of the presence of appropriate restriction enzyme sites. Many have tried changing the inducible promoter or decreasing the copy number of Red genes in Escherichia coli to improve the recombination efficiency. Here we describe a novel method for increasing the recombination efficiency of the Red system by mutating the Red proteins. Mutant Red alpha and Red beta independently increased recombination efficiency. We found that the 18-bp 5' untranslated region (UTR) upstream from the ATG initiation codon of Gam protein is important for recombination efficiency. Although low rates of recombination have been observed in systems using high copy number plasmids, when we performed Red recombination using a high copy number plasmid containing 5'-UTR, mutant Red alpha, and mutant Red beta, recombination increased approximately 145-fold. The novel possibility that mutant Red alpha and Red beta can increase recombination efficiency indicates a new direction for improving recombination efficiency of expression systems that use low or high copy number plasmids or a defective prophage integrated into the E. coli chromosome.

5' Untranslated Regions↗

PubMed2004

Identification of the genes that are expressed in the upper layers of the neocortex.

Laminar specificity is one of the most striking features of neocortical circuitry. To explore the molecular basis of this specificity, particularly in relation to thalamocortical connectivity, we searched for the genes expressed in the upper cortical layers by constructing a subtraction cDNA library that was enriched for genes expressed in layer 4 of perinatal rat somatosensory cortex. Differential screening, sequence analysis and in situ hybridization demonstrated that a new unc5 family member (unc5h4), deltex-like gene, stem cell factor (SCF) and myocyte-specific enhancer factor-2C (MEF-2C) were specifically expressed in layer 4 or layers 2/3-4 at postnatal day 7, by when laminar organization and fundamental cortical circuitries have been established. In terms of regional specificity, unc5h4 and SCF signals were stronger in sensory cortices, whereas MEF-2C and deltex-like gene were expressed rather uniformly in all neocortical regions. Analysis during development demonstrated that expression of these genes was pronounced between late embryonic and early postnatal developmental stages, except for MEF-2C expression, which continued in later stages. These results demonstrate that certain types of molecules including transcription factors, receptor and ligand molecules, are expressed specifically in the upper layers of the developing neocortex, suggesting a role in laminar specification of cortical cells and circuitry.

Amino Acid Sequence↗

PubMed2004

HUGE: a database for human KIAA proteins, a 2004 update integrating HUGEppi and ROUGE.

We have been developing a Human Unidentified Gene-Encoded (HUGE) protein database (http://www.kazusa.or.jp/huge) to summarize results from sequence analysis of human novel large (>4 kb) cDNAs identified in the Kazusa cDNA sequencing project. At present, HUGE contains 2031 cDNA entries (KIAA cDNAs), for each of which a gene/protein characteristic table has been prepared. Since we have been shifting our research attention from the identification and cloning of novel cDNAs to the functional analysis of the proteins encoded by these cDNAs (KIAA proteins), we have not substantially increased the number of cDNA entries in HUGE for some time. Instead, we have manually curated 451 KIAA cDNAs in order to prepare a set of genetic resources to facilitate the functional analysis of KIAA proteins. In addition, we have updated the contents of the corresponding gene/protein characteristic tables in HUGE and have constructed two subsidiary databases, HUGEppi (http://www. kazusa.or.jp/huge/ppi) and ROUGE (http://www. kazusa.or.jp/rouge), to make available the results from our study of KIAA protein function. HUGEppi shows detailed information on protein-protein interactions detected between 84 pairs of KIAA proteins by yeast two-hybrid screening. ROUGE summarizes the results of computer-assisted analyses of approximately 1000 mouse homologues of human large cDNAs that we identified.

Animals↗

PubMed2004

The CAP-Gly domain of CYLD associates with the proline-rich sequence in NEMO/IKKgamma.

CYLD was originally identified as the human familial cylindromatosis tumor suppressor. Recently, it was reported that CYLD directly interacts with NEMO/IKKgamma and TRAF2 in the NF-kappaB signaling pathway. The two proteins bind to a region of CYLD that contains a Cys-box motif and the third cytoskeleton-associated protein-glycine conserved (CAP-Gly) domain. Here we report that the third CAP-Gly domain of CYLD specifically interacts with one of the two proline-rich sequences of NEMO/IKKgamma. The tertiary structure of the CAP-Gly domain shares the five-stranded beta sheet topology with the SH3 domain, which is well known as a proline-rich sequence-recognition domain. However, chemical shift mapping revealed that the peptide binding site of the CAP-Gly domain is formed without the long peptide binding loop characteristic of the SH3 domain. Therefore, CAP-Gly is likely to be a novel proline-rich sequence binding domain with a mechanism different from that of the SH3 domain.

Amino Acid Sequence↗

PubMed2003

Differential profiles of genes expressed in neonatal brain of 129X1/SvJ and C57BL/6J mice: A database to aid in analyzing DNA microarrays using nonisogenic gene-targeted mice.

Strain-specific differences in gene expression have been observed among various inbred mouse strains. Two strains that are commonly used in gene-targeting research today are the 129 substrains, which are used to produce ES cell lines, and C57BL/6J, which is used for the extensive backcrosses required to produce isogenic knockout mice. When F2 nonisogenic littermates are assessed using DNA microarrays, one must determine whether the expression profiles obtained resulted either from specific alteration(s) induced by the targeted gene mutation or from gene expression differences related to the genetic background of the parent mouse strains. In the present study, we report the differential expression profile of genes expressed in neonatal brains and adult spleen and liver of 129X1/SvJ and C57BL/6J strains of mice. These comprehensive profiles were assessed using two types of Agilent Mouse Oligo Microarrays (development and standard) and were compiled into a publicly available database. Researchers can use this database to determine whether their microarray findings represent strain-specific differences in gene expression by comparing their data with those cataloged in our database. This database is useful for effectively analyzing DNA microarray data from nonisogenic littermates, and would help researchers avoid time-consuming backcrosses and confirmatory experiments requiring the use of many mice.

Animals↗

PubMed2003

A system using convertible vectors for screening soluble recombinant proteins produced in Escherichia coli from randomly fragmented cDNAs.

Protein insolubility is a major problem when producing recombinant proteins (e.g., to be used as antigens) from large cDNAs in Escherichia coli. Here, we describe a system using three convertible plasmid vectors to screen for soluble proteins produced in E. coli. This system experimentally identified any random cDNA fragments producing soluble protein domains. Shotgun fragments introduced into any of our three plasmids, which contain Gateway recombination sites, fused in-frame to the ORF of the protein tag. These plasmids produced N-terminal GST- and C-terminal three-frame-adaptive FLAG-tagged proteins, kanamycin-resistant gene-tagged proteins (which were pre-selected for in-frame fused cDNAs), or GFP-tagged fusion proteins. The latter is useful as a fluorescence indicator of protein folding. The Gateway recombination sites promote smooth conversion for enrichment of in-frame clones and facilitate both protein solubility assays and final production of proteins without the C-terminal tag. This high-throughput screening method is particularly useful for procedures that require the handling of many cDNAs in parallel.

Bacterial Proteins↗

PubMed2002

Mammalian fat3: a large protein that contains multiple cadherin and EGF-like motifs.

Using computer-based, motif-trap screening, we have identified a third member of the mammalian fat family, fat3. Human and rat fat3 are also similar to the Drosophila tumor suppressor gene fat. The rat fat3 gene encodes a large protein of 4555 amino acids with 34 cadherin domains, 4 epidermal growth factor (EGF)-like motifs, a laminin A-G motif, and a cytoplasmic domain. Each member of the fat family is differentially expressed in the central nervous system during development. While both fat3 mRNA and fat1 mRNA are abundantly expressed in the fetal brain, the expression of MEGF1/fat2 mRNA is restricted to the postnatal cerebellum. fat3 mRNA and protein expression in the brain peaks at E15 during embryonic development. During this time, robust fat3 immunoreactivity is also observed in the spinal cord. These data suggest that the fat3 protein plays an important role in axon fasciculation and modulation of the extracellular space surrounding axons during embryonic development.

Amino Acid Motifs↗

PubMed2002

MEGF1/fat2 proteins containing extraordinarily large extracellular domains are localized to thin parallel fibers of cerebellar granule cells.

The MEGF1 (protein 1 with multiple EGF-like domains) gene, which was identified using motif-trap screening, encodes an extraordinarily large protein containing two EGF-like and 34 cadherin motifs. In situ hybridization analysis revealed that the MEGF1 gene was specifically expressed in granule cells of the cerebellum. Interestingly, in the developing cerebellum, granule cells in the inner external germinal layer and migrating granule cells expressed MEGF1 mRNA, whereas proliferating cells in the outer external germinal layer did not express MEGF1 mRNA. Expression levels in the internal granule cell layer peaked during the third postnatal week and remained considerably high in the adult cerebellum. MEGF1 protein was detected in only the cerebellum as a single 480-kDa band by immunoblot analyses using polyclonal antibodies against either the N-terminal or the C-terminal region of MEGF1 protein. Using light and electron microscopic immunocytochemistry, specific immunostaining of the MEGF1 protein was observed in the molecular layer of the cerebellum, suggesting that MEGF1 protein was localized in the parallel fibers of cerebellar granule cells. This was corroborated by results from experiments using primary dispersed cultures of cerebellar granule cells and cerebellar microexplant cultures. The homophilic interaction of MEGF1 proteins was confirmed with both a cell aggregation assay and an in vitro copurification assay. Based on these results, a novel function of the enormous protocadherins in cerebellar development, namely, the modulation of the extracellular space surrounding parallel fibers during development, was proposed.

Aging↗

PubMed2002

Protein-protein interactions between large proteins: two-hybrid screening using a functionally classified library composed of long cDNAs.

Large proteins have multiple domains that are potentially capable of binding many kinds of partners. It is conceivable, therefore, that such proteins could function as an intricate framework of assembly protein complexes. To comprehensively study protein-protein interactions between large KIAA proteins, we have constructed a library composed of 1087 KIAA cDNA clones based on prior functional classifications done in silico. We were guided by two principles that raise the success rate for detecting interactions per tested combination: we avoided testing low-probability combinations, and reduced the number of potential false negatives that arise from the fact that large proteins cannot reliably be expressed in yeast. The latter was addressed by constructing a cDNA library comprised of random fragments encoding large proteins. Cytoplasmic domains of KIAA transmembrane proteins (>1000 amino acids) were used as bait for yeast two-hybrid screening. Our analyses reveal that several KIAA proteins bearing a transmembrane region have the capability of binding to other KIAA proteins containing domains (e.g., PDZ, SH3, rhoGEF, and spectrin) known to be localized to highly specialized submembranous sites, indicating that they participate in cellular junction formation, receptor or channel clustering, and intracellular signaling events. Our representative library should be a very useful resource for detecting previously unidentified interactions because it complements conventional expression libraries, which seldom contain large cDNAs.

Brain Chemistry↗