RESUMO
Imprinting, i.e. parent-of-origin expression of alleles, plays an important role in regulating development in mammals and plants. DNA methylation catalyzed by DNA methyltransferases plays a pivotal role in regulating imprinting by silencing parental alleles. DEMETER (DME), a DNA glycosylase functioning in the base-excision DNA repair pathway, can excise 5-methylcytosine from DNA and regulate genomic imprinting in Arabidopsis. DME demethylates the maternal MEDEA (MEA) promoter in endosperm, resulting in expression of the maternal MEA allele. However, it is not known whether DME interacts with other proteins in regulating gene imprinting. Here we report the identification of histone H1.2 as a DME-interacting protein in a yeast two-hybrid screen, and confirmation of their interaction by the in vitro pull-down assay. Genetic analysis of the loss-of-function histone h1 mutant showed that the maternal histone H1 allele is required for DME regulation of MEA, FWA and FIS2 imprinting in Arabidopsis endosperm but the paternal allele is dispensable. Furthermore, we show that mutations in histone H1 result in an increase of DNA methylation in the maternal MEA and FWA promoter in endosperm. Our results suggest that histone H1 is involved in DME-mediated DNA methylation and gene regulation at imprinted loci.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Metilação de DNA , Impressão Genômica , Histonas/metabolismo , N-Glicosil Hidrolases/metabolismo , Transativadores/metabolismo , Arabidopsis/fisiologia , Endosperma/metabolismo , Proteínas de Homeodomínio/metabolismo , Família Multigênica , Mutação , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Early embryogenesis in Arabidopsis (Arabidopsis thaliana) is distinguished by a predictable pattern of cell divisions and is a good system for investigating mechanisms of developmental pattern formation. Here, we identified a gene called LONO1 (LNO1) in Arabidopsis in which mutations can abolish the first asymmetrical cell division of the zygote, alter planes and number of cell divisions in early embryogenesis, and eventually arrest embryo development. LNO1 is highly expressed in anthers of flower buds, stigma papilla of open flowers, and embryo and endosperm during early embryogenesis, which is correlated with its functions in reproductive development. The homozygous lno1-1 seed is not viable. LNO1, a homolog of the nucleoporin NUP214 in human (Homo sapiens) and Nup159 in yeast (Saccharomyces cerevisiae), encodes a nucleoporin protein containing phenylalanine-glycine repeats in Arabidopsis. We demonstrate that LNO1 can functionally complement the defect in the yeast temperature-sensitive nucleoporin mutant nup159. We show that LNO1 specifically interacts with the Arabidopsis DEAD-box helicase/ATPase LOS4 in the yeast two-hybrid assay. Furthermore, mutations in AtGLE1, an Arabidopsis homolog of the yeast Gle1 involved in the same poly(A) mRNA export pathway as Nup159, also result in seed abortion. Our results suggest that LNO1 is a component of the nuclear pore complex required for mature mRNA export from the nucleus to the cytoplasm, which makes LNO1 essential for embryogenesis and seed viability in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Sementes/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Flores/genética , Flores/metabolismo , Teste de Complementação Genética , Glicina/genética , Glicina/metabolismo , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Fenilalanina/genética , Fenilalanina/metabolismo , Mapeamento de Interação de Proteínas , Transporte de RNA , RNA Mensageiro/genética , RNA de Plantas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sementes/genética , Sementes/metabolismo , Especificidade por Substrato , Técnicas do Sistema de Duplo-HíbridoRESUMO
Nucleoporins (Nups) are building blocks of the nuclear pore complex (NPC) that mediate cargo trafficking between the nucleus and the cytoplasm. Although the physical structure of the NPC is well studied in yeast and vertebrates, little is known about the structure of NPCs or the function of most Nups in plants. Recently we demonstrated two Nups in Arabidopsis: LONO1 (LNO1), homolog of human NUP214 and yeast Nup159, and AtGLE1, homolog of yeast Gle1, are required for early embryogenesis and seed development. To identify LNO1 and AtGLE1 homologs in other plant species, we searched the protein databases and identified 30 LNO1-like and 35 AtGLE1-like proteins from lower plant species to higher plants. Furthermore, phylogenetic analyses indicate that the evolutionary trees of these proteins follow expected plant phylogenies. High sequence homology and conserved domain structure of these nucleoporins suggest important functions of these proteins in nucleocytoplasmic transport, growth and development in plants.