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Roles of clustered genomic organization of Protocadherin-α on individual neuron specific Protocadherin-α choice
Abstracts P1-d01 Involvement of E-protein, HEB, in transcriptional repression of the mouse lysophosphatidic acid receptor-1 gene expression in neocortical neuroblast cells Sung-Oh Huh 1 , N.H. Kim 1 , Y. Sun 1 , S.M. Oh 1 , S.H. Kim 1 , J.S. Hwang 1 , J. Contos 2 , J. Chun 3 1
Dept of Pharm, Col of Med, Hallym University, South Korea; 2 Fred Hutchinson Cancer Research Center, USA; 3 The Scripps Research Institute, USA
LPA1, is one of the five GPCRs for LPA and has been implicated in brain development. Importance of the spatio-temporal regulation of LPA1 has been suggested by a loss-of-function mutation study. However, detailed information on the cis-acting elements involved in the transcription of LPA1 gene has not been elucidated yet. Here, using the 5 RACE analysis, transcription initiation site was identified. Transient transfection of neocortical neuroblast cells, with various 5 deletion constructs of LPA1 gene, revealed that the 5 proximal region harboring SP1 binding sites, serves as the basal promoter for LPA1 transcription. In addition, we have also identified negative regulatory element that control the basal expression level of LPA1. Taken together, we propose, based on our data, that E proteins are required for regulation of LPA1 gene expression during the course of neocortical development. doi:10.1016/j.neures.2009.09.360
Topo II is a member of type II DNA topoisomerases that catalyze the transport of one duplex DNA through another. Previous studies showed that the enzyme plays critical roles in neuronal development. However, the underlying molecular mechanism remains unknown. Here we show a strong correlation between the genomic location of topo II action sites and the genes it regulates. Topo II appears to aid the transcriptional induction of a limited number of genes. These genes (group A1) were functionally biased towards membrane proteins with ion channel, transporter, or receptor activities. Significant proportions of them encode long transcripts and are juxtaposed to a Long AT-rich Intergenic Region (LAIR). We mapped topo II action sites using a functional immunoprecipitation scheme. These sites were concentrated both in A1 gene boundaries and the adjacent LAIR, suggesting a direct link between the action sites and the transcriptional activation. The results also imply a silencing effect imposed by AT-rich intergenic regions, which is abrogated by the enzyme to activate nearby genes. doi:10.1016/j.neures.2009.09.363
P1-d05 A novel splicing variant of STAT5A in the brainstem suppresses STAT5A activity Yoshihisa Watanabe 1 , Masaya Ikegawa 2 , Masaki Tanaka 1 1 2
P1-d02 In silico analysis of NRG1 evolution in vertebrates Chih-Fong Chou, Miwako Ozaki Waseda Research Institute, Japan NRG1 belongs to neuregulin family of EGF-like signaling molecules involved in cellcell communication during development and disease. It plays important roles in developing tissues of nerve, heart, and mammary glands. In neurobiology, NRG1 signaling is associated with synaptic transmission, myelination of Schwann cells and human disease of schizophrenia. NRG1 isoforms are highly sophisticated in biological activities and great diversity of the in vivo functions. The nervous system is a common trait in all bilateria but NRG1 can only be identified in vertebrates. The gene was analyzed for type I to IV coding regions from 10 vertebrate genomes. The loci, gene structures, ClustalW analyses, phylogenetic trees and conserved motifs in ecto- and cytoplasmic domains were compared. Non-mammalian vertebrates mainly carry types I (may have evolved a spacer different from mammalian isoforms), II and III NRG1. Type IV NRG1 can be identified in most mammals however rodents type IV Nrg1 is not fully evolved. Finally, a highly conserved domain (CD59 –CD24 –CD103 ), intracellular phosphorylation sites and bipartite nuclear localization signals postulate physiological significance. doi:10.1016/j.neures.2009.09.361
S89
Dept. Cell Biol., Res. Inst. Geriatr., Kyoto Pref Univ. Med., Kyoto, Japan; Dept. Genomic Med. Sci., Kyoto Pref Univ. Med., Kyoto, Japan
STATs regulate a variety of cellular functions, including differentiation and proliferation. STAT3 and STAT5 are known to play important roles in brain processes, such as energy homeostasis and neuronal development. We isolated a novel splicing variant of STAT5A from a cDNA library of the mouse brainstem. This variant, STAT5A E18, lacked exon 18 and caused a frameshift in the C-terminus, resulting in deletion of a tyrosine phosphorylation site and a transactivation domain. Expression of STAT5A E18 was detected in the mouse brainstem, but not in the mouse cerebrum or cerebellum. STAT5A E18 protein showed dot-like structures in the cytoplasm and could not translocate into the nucleus. STAT5A E18 showed a strong tendency to aggregate, which led to coaggregation with STAT5A full length. This coaggregation inhibited the nuclear transport of STAT5A and suppressed prolactin-induced activation of STAT5A. Collectively, these results suggest that STAT5A E18 may function as a suppressor of STAT5A in restricted tissues. doi:10.1016/j.neures.2009.09.364
P1-d06 Roles of clustered genomic organization of Protocadherin-␣ on individual neuron specific Protocadherin-␣ choice Ryosuke Kaneko 1 , Manabu Abe 2 , Takahiro Hirabayashi 3 , Arikuni Uchimura 3 , Kenji Sakimura 2 , Yuchio Yanagawa 1 , Takeshi Yagi 3 1
P1-d03 Genetic mapping for aggressive behavior using B6-MSM consomic mouse strains Hiroki Sugimoto 1,4 , Aki Takahashi 1 , Toshihiko Shiroishi 2,3 , Tsuyoshi Koide 1,3 1
MGRL, NIG, Japan; 2 MGL, NIG, Japan; Info Sys, Trans Res Integ Cent, Japan
3
SOKENDAI, Japan;
4
Res Org
Aggressive behavior plays an important role in social context. For example, mice show aggressive behavior to compete for mating partner, to protect pups, and to compete for territory and foods. These aggressive behaviors are different among mouse strains due to genetic factors as well as environmental factors. However, genetic mechanisms underlying difference of the aggressive behavior are not fully analyzed. Here, to elucidate the genetic mechanism for aggressive behavior, we performed genetic mapping of aggressive behavior using the consomic mouse strains (an arbitrary chromosome of laboratory mouse C57BL/6J was substituted by a chromosome of wild-derived mouse strain MSM/Ms) and subconsomic mouse strains substituted by a part of chromosome of MSM. The result of mapping by social interaction test and resident–intruder test indicated new aggression increasing genetic locus on chromosome 15. Also, we performed qRT-PCR analysis for candidate genes of aggressive behavior on this locus. We will discuss the probable genetic mechanism for increased aggression. doi:10.1016/j.neures.2009.09.362
P1-d04 DNA topoisomerase II (topo II) activates a subset of neuronal genes Kuniaki Sano 1 , Mary Miyaji-Yamaguchi 1 , Kimiko Tsutsui 1 , Ken Tsutsui 2 1
Neurogenomics, Okayama Univ., Japan;
2
Genome Dynamics, Japan
Gunma University Grad. Sch. Medicine, Gunma, Japan; 2 Niigata University BRI, Niigata, Japan; 3 Osaka University FBS, Osaka, Japan
Diverse protocadherin (Pcdh) isoforms encoded by the Pcdh gene clusters are expressed in nervous systems. For the twelve Pcdh-␣ isoforms (Pcdh-␣1–␣12), a variety of expression patterns have been found in single Purkinje cells, of which the gene regulation is monoallelic and combinatorial. Here we investigated the roles of clustered organization on gene regulation of the Pcdh-␣ isoforms. In combination with targeted integration of loxP sites into Pcdh-␣ locus using ES cells, TT2 and B6-RENKA, and trans-chromosomal Cre-loxP recombination using Sycp1-Cre Tg mouse, the clustered organization of mouse Pcdh-␣ genes was mutated in germ line at frequency of 5.8%. In this mutant mouse, the Pcdh-␣ cluster contained large duplication of the cluster (218 kb). The expression levels of each Pcdh-␣ isoforms were altered in mutant mouse cerebellum. These data indicate that the gene regulation in the Pcdh-␣ cluster is influenced by its clustered genomic organization. doi:10.1016/j.neures.2009.09.365
P1-d07 Influence of lentiviral vector infection on the development of cerebellar Purkinje cells in vivo Yusuke Sawada 1 , Go Kajiwara 1 , Takashi Torashima 1 , Kiyohiko Takayama 1 , Hajime Horiuchi 1,2 , Akira Iizuka 1 , Kazuhiro Mitsumura 1 , Hirokazu Hirai 1,2 1 Department Neurophysiol., Gunma University Grad. Sch. Med., Maebashi, Japan; 2 SORST, JST, Kawaguchi, Japan
Lentiviral vectors are useful tools for transgene delivery to cerebellar Purkinje cells. Here we studied whether infection of high titer lentiviral vectors expressing GFP affected the development of neonatal rat Purkinje cells. Dendritic development of Purkinje cells was examined at postnatal day 21 (P21) by loading a fluorescent dye into infected cells, followed by their morphological analysis. Infected Purkinje cells showed well-differentiated dendrites almost comparably to non-infected Purkinje
Dept of Pharm, Col of Med, Hallym University, South Korea; 2 Fred Hutchinson Cancer Research Center, USA; 3 The Scripps Research Institute, USA
LPA1, is one of the five GPCRs for LPA and has been implicated in brain development. Importance of the spatio-temporal regulation of LPA1 has been suggested by a loss-of-function mutation study. However, detailed information on the cis-acting elements involved in the transcription of LPA1 gene has not been elucidated yet. Here, using the 5 RACE analysis, transcription initiation site was identified. Transient transfection of neocortical neuroblast cells, with various 5 deletion constructs of LPA1 gene, revealed that the 5 proximal region harboring SP1 binding sites, serves as the basal promoter for LPA1 transcription. In addition, we have also identified negative regulatory element that control the basal expression level of LPA1. Taken together, we propose, based on our data, that E proteins are required for regulation of LPA1 gene expression during the course of neocortical development. doi:10.1016/j.neures.2009.09.360
Topo II is a member of type II DNA topoisomerases that catalyze the transport of one duplex DNA through another. Previous studies showed that the enzyme plays critical roles in neuronal development. However, the underlying molecular mechanism remains unknown. Here we show a strong correlation between the genomic location of topo II action sites and the genes it regulates. Topo II appears to aid the transcriptional induction of a limited number of genes. These genes (group A1) were functionally biased towards membrane proteins with ion channel, transporter, or receptor activities. Significant proportions of them encode long transcripts and are juxtaposed to a Long AT-rich Intergenic Region (LAIR). We mapped topo II action sites using a functional immunoprecipitation scheme. These sites were concentrated both in A1 gene boundaries and the adjacent LAIR, suggesting a direct link between the action sites and the transcriptional activation. The results also imply a silencing effect imposed by AT-rich intergenic regions, which is abrogated by the enzyme to activate nearby genes. doi:10.1016/j.neures.2009.09.363
P1-d05 A novel splicing variant of STAT5A in the brainstem suppresses STAT5A activity Yoshihisa Watanabe 1 , Masaya Ikegawa 2 , Masaki Tanaka 1 1 2
P1-d02 In silico analysis of NRG1 evolution in vertebrates Chih-Fong Chou, Miwako Ozaki Waseda Research Institute, Japan NRG1 belongs to neuregulin family of EGF-like signaling molecules involved in cellcell communication during development and disease. It plays important roles in developing tissues of nerve, heart, and mammary glands. In neurobiology, NRG1 signaling is associated with synaptic transmission, myelination of Schwann cells and human disease of schizophrenia. NRG1 isoforms are highly sophisticated in biological activities and great diversity of the in vivo functions. The nervous system is a common trait in all bilateria but NRG1 can only be identified in vertebrates. The gene was analyzed for type I to IV coding regions from 10 vertebrate genomes. The loci, gene structures, ClustalW analyses, phylogenetic trees and conserved motifs in ecto- and cytoplasmic domains were compared. Non-mammalian vertebrates mainly carry types I (may have evolved a spacer different from mammalian isoforms), II and III NRG1. Type IV NRG1 can be identified in most mammals however rodents type IV Nrg1 is not fully evolved. Finally, a highly conserved domain (CD59 –CD24 –CD103 ), intracellular phosphorylation sites and bipartite nuclear localization signals postulate physiological significance. doi:10.1016/j.neures.2009.09.361
S89
Dept. Cell Biol., Res. Inst. Geriatr., Kyoto Pref Univ. Med., Kyoto, Japan; Dept. Genomic Med. Sci., Kyoto Pref Univ. Med., Kyoto, Japan
STATs regulate a variety of cellular functions, including differentiation and proliferation. STAT3 and STAT5 are known to play important roles in brain processes, such as energy homeostasis and neuronal development. We isolated a novel splicing variant of STAT5A from a cDNA library of the mouse brainstem. This variant, STAT5A E18, lacked exon 18 and caused a frameshift in the C-terminus, resulting in deletion of a tyrosine phosphorylation site and a transactivation domain. Expression of STAT5A E18 was detected in the mouse brainstem, but not in the mouse cerebrum or cerebellum. STAT5A E18 protein showed dot-like structures in the cytoplasm and could not translocate into the nucleus. STAT5A E18 showed a strong tendency to aggregate, which led to coaggregation with STAT5A full length. This coaggregation inhibited the nuclear transport of STAT5A and suppressed prolactin-induced activation of STAT5A. Collectively, these results suggest that STAT5A E18 may function as a suppressor of STAT5A in restricted tissues. doi:10.1016/j.neures.2009.09.364
P1-d06 Roles of clustered genomic organization of Protocadherin-␣ on individual neuron specific Protocadherin-␣ choice Ryosuke Kaneko 1 , Manabu Abe 2 , Takahiro Hirabayashi 3 , Arikuni Uchimura 3 , Kenji Sakimura 2 , Yuchio Yanagawa 1 , Takeshi Yagi 3 1
P1-d03 Genetic mapping for aggressive behavior using B6-MSM consomic mouse strains Hiroki Sugimoto 1,4 , Aki Takahashi 1 , Toshihiko Shiroishi 2,3 , Tsuyoshi Koide 1,3 1
MGRL, NIG, Japan; 2 MGL, NIG, Japan; Info Sys, Trans Res Integ Cent, Japan
3
SOKENDAI, Japan;
4
Res Org
Aggressive behavior plays an important role in social context. For example, mice show aggressive behavior to compete for mating partner, to protect pups, and to compete for territory and foods. These aggressive behaviors are different among mouse strains due to genetic factors as well as environmental factors. However, genetic mechanisms underlying difference of the aggressive behavior are not fully analyzed. Here, to elucidate the genetic mechanism for aggressive behavior, we performed genetic mapping of aggressive behavior using the consomic mouse strains (an arbitrary chromosome of laboratory mouse C57BL/6J was substituted by a chromosome of wild-derived mouse strain MSM/Ms) and subconsomic mouse strains substituted by a part of chromosome of MSM. The result of mapping by social interaction test and resident–intruder test indicated new aggression increasing genetic locus on chromosome 15. Also, we performed qRT-PCR analysis for candidate genes of aggressive behavior on this locus. We will discuss the probable genetic mechanism for increased aggression. doi:10.1016/j.neures.2009.09.362
P1-d04 DNA topoisomerase II (topo II) activates a subset of neuronal genes Kuniaki Sano 1 , Mary Miyaji-Yamaguchi 1 , Kimiko Tsutsui 1 , Ken Tsutsui 2 1
Neurogenomics, Okayama Univ., Japan;
2
Genome Dynamics, Japan
Gunma University Grad. Sch. Medicine, Gunma, Japan; 2 Niigata University BRI, Niigata, Japan; 3 Osaka University FBS, Osaka, Japan
Diverse protocadherin (Pcdh) isoforms encoded by the Pcdh gene clusters are expressed in nervous systems. For the twelve Pcdh-␣ isoforms (Pcdh-␣1–␣12), a variety of expression patterns have been found in single Purkinje cells, of which the gene regulation is monoallelic and combinatorial. Here we investigated the roles of clustered organization on gene regulation of the Pcdh-␣ isoforms. In combination with targeted integration of loxP sites into Pcdh-␣ locus using ES cells, TT2 and B6-RENKA, and trans-chromosomal Cre-loxP recombination using Sycp1-Cre Tg mouse, the clustered organization of mouse Pcdh-␣ genes was mutated in germ line at frequency of 5.8%. In this mutant mouse, the Pcdh-␣ cluster contained large duplication of the cluster (218 kb). The expression levels of each Pcdh-␣ isoforms were altered in mutant mouse cerebellum. These data indicate that the gene regulation in the Pcdh-␣ cluster is influenced by its clustered genomic organization. doi:10.1016/j.neures.2009.09.365
P1-d07 Influence of lentiviral vector infection on the development of cerebellar Purkinje cells in vivo Yusuke Sawada 1 , Go Kajiwara 1 , Takashi Torashima 1 , Kiyohiko Takayama 1 , Hajime Horiuchi 1,2 , Akira Iizuka 1 , Kazuhiro Mitsumura 1 , Hirokazu Hirai 1,2 1 Department Neurophysiol., Gunma University Grad. Sch. Med., Maebashi, Japan; 2 SORST, JST, Kawaguchi, Japan
Lentiviral vectors are useful tools for transgene delivery to cerebellar Purkinje cells. Here we studied whether infection of high titer lentiviral vectors expressing GFP affected the development of neonatal rat Purkinje cells. Dendritic development of Purkinje cells was examined at postnatal day 21 (P21) by loading a fluorescent dye into infected cells, followed by their morphological analysis. Infected Purkinje cells showed well-differentiated dendrites almost comparably to non-infected Purkinje