ABSTRACT
Interphase chromatin is organized in distinct nuclear sub-compartments, reflecting its degree of compaction and transcriptional status. In Caenorhabditis elegans embryos, H3K9 methylation is necessary to silence and to anchor repeat-rich heterochromatin at the nuclear periphery. In a screen for perinuclear anchors of heterochromatin, we identified a previously uncharacterized C. elegans chromodomain protein, CEC-4. CEC-4 binds preferentially mono-, di-, or tri-methylated H3K9 and localizes at the nuclear envelope independently of H3K9 methylation and nuclear lamin. CEC-4 is necessary for endogenous heterochromatin anchoring, but not for transcriptional repression, in contrast to other known H3K9 methyl-binders in worms, which mediate gene repression but not perinuclear anchoring. When we ectopically induce a muscle differentiation program in embryos, cec-4 mutants fail to commit fully to muscle cell fate. This suggests that perinuclear sequestration of chromatin during development helps restrict cell differentiation programs by stabilizing commitment to a specific cell fate. PAPERCLIP.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/embryology , Caenorhabditis elegans/genetics , Chromosomal Proteins, Non-Histone/metabolism , Embryo, Nonmammalian/cytology , Heterochromatin , Histone Code , Amino Acid Sequence , Animals , Caenorhabditis elegans/cytology , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/genetics , Cell Differentiation , Cell Nucleus/genetics , Cell Nucleus/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , Embryo, Nonmammalian/metabolism , Molecular Sequence Data , Sequence AlignmentABSTRACT
A natural variation is observed at position 4 of the 3'-end of influenza A virus genomes, where U (U4) or C (C4) is present. The replicon activity of C4 was 28% of U4. We compared the transcription and replication activity of U4 [v84(U4)], C4 [v84(C4)] and the complimentary RNA (c84) using the purified influenza virus RNA polymerase in vitro. ApG-primed replication activities of v84(C4) and c84 were 23.8% and 7.8% of v84(U4). Globin mRNA-primed transcription activities of v84(C4) and c84 were 36.9% and 6.81% of v84(U4). De novo replication activities of v84(C4) and c84 were 21.3 and 10.2% of v84(U4). This difference came from their polymerase binding activity. When all the eight genome segments of WSN strain were changed to U4, the virus titer was 760 times higher than the wild type. However, its pathogenicity in mice was lower than the wild type.
Subject(s)
Genome, Viral , Influenza A virus/genetics , Influenza A virus/physiology , Virus Replication/genetics , Virus Replication/physiology , Animals , Base Sequence , Cell Line , DNA-Directed RNA Polymerases/metabolism , Dogs , Female , Gene Expression , Genetic Variation , Humans , Influenza A virus/pathogenicity , Interferon-beta/genetics , Mice , Mice, Inbred BALB C , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Viral/genetics , Replicon , Transcription, Genetic , Viral Nonstructural Proteins/genetics , Virulence/genetics , Virulence/physiologyABSTRACT
The influenza virus RNA polymerase (RdRp) was purified from insect cells (around 0.2mg/l). The RdRp catalyzed all the biochemical reactions of influenza virus transcription and replication in vitro; dinucleotide ApG and globin mRNA-primed transcription, de novo initiation (replication), and polyadenylation. The optimal Mg concentration, pH and temperature were 8mM, 8.0 and 25 degrees C, respectively, which were slightly different from those measured for RdRp of virions. This system is a single-round transcription system. K(m) (microM) were 10.74+/-0.26 (GTP), 33.22+/-3.37 (ATP), 28.93+/-0.48 (CTP) and 22.01+/-1.48 (UTP), and V(max) (fmol nucleotide/pmol RdRp/min) were 2.40+/-0.032 (GTP), 1.95+/-0.17 (ATP), 2.07+/-0.17 (CTP), and 1.52+/-0.38 (UTP), which agreed with high mutation of influenza viruses.
Subject(s)
Influenza A Virus, H1N1 Subtype/enzymology , Influenza A Virus, H5N1 Subtype/enzymology , RNA-Dependent RNA Polymerase/chemistry , Viral Proteins/chemistry , Animals , Cells, Cultured , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/isolation & purification , Influenza A Virus, H5N1 Subtype/genetics , Influenza A Virus, H5N1 Subtype/isolation & purification , Insecta/cytology , Kinetics , RNA-Dependent RNA Polymerase/biosynthesis , RNA-Dependent RNA Polymerase/isolation & purification , Viral Proteins/biosynthesis , Viral Proteins/isolation & purificationABSTRACT
Topological description and matching are committed steps for three-dimensional reconstruction of vessel tree from two angiograms. Binary tree is proposed to describe the two-dimensional vessel tree. "Node Weight" and "Similar Node" are defined in order to get better description. Vessel segments in two images are matched by the preorder traversal of the binary trees, and the method is proved fast and accurate.
Subject(s)
Angiography/methods , Image Processing, Computer-Assisted/methods , Imaging, Three-Dimensional/methods , Models, Biological , Radiographic Image Enhancement/methods , Algorithms , Computer Simulation , Humans , Pattern Recognition, Automated/methodsABSTRACT
RIZ1 (PRDM2) and PRDI-BF1 (PRDM1) are involved in B cell differentiation and the development of B cell lymphomas. These proteins are expressed in two forms that differ by the presence or absence of a PR domain. The protein product that retains the PR domain is anti-tumorigenic while the product that lacks the PR domain is oncogenic and over-expressed in tumor cells. The conserved PR domain is homologous to the SET domain from a family of histone methyltransferases. RIZ1 is also a histone methyltransferase and methylates lysine 9 in histone H3. This activity has been mapped to the PR domain. In the present study, deuterium exchange mass spectrometry was used to define the structural boundaries of the RIZ1 PR domain and to map sites of missense mutations that occur in human cancers and reduce methyltransferase activity. Flexible segments were selectively deleted to produce protein products that crystallize for structural studies. Segments at the carboxyl terminus of the PR domain that are involved in methylation of H3 were shown to be flexible, similar to SET domains, suggesting that the PR and SET methyltransferases may belong to an emerging class of proteins that contain mobile functional regions.
Subject(s)
DNA-Binding Proteins/chemistry , Nuclear Proteins/chemistry , Transcription Factors/chemistry , Amino Acid Sequence , Base Sequence , Crystallization , DNA Primers , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Electrophoresis, Polyacrylamide Gel , Histone-Lysine N-Methyltransferase , Humans , Mass Spectrometry , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Nuclear Proteins/metabolism , Nuclear Proteins/physiology , Protein Conformation , Sequence Homology, Amino Acid , Substrate Specificity , Transcription Factors/metabolism , Transcription Factors/physiologyABSTRACT
Histone methyltransferase (HMT)(1) class enzymes that methylate lysine residues of histones or proteins contain a conserved catalytic core termed the SET domain, which shares sequence homology with an independently described sequence motif, the PR domain. Intact PR or SET sequence is required for tumor suppression functions, but it remains unclear whether it is histone methyltransferase activity that underlies tumor suppression. We now show that tumor suppressor RIZ1 (PRDM2) methylates histone H3 on lysine 9, and this activity is reduced by mutations in the PR domain found in human cancers. Also, S-adenosylhomocysteine or methyl donor deficiency inhibits RIZ1 and other H3 lysine 9 methylation activities. These results support the hypothesis that H3 lysine 9 methylation activities of a PR/SET domain have tumor suppression functions and may underlie carcinogenesis associated with dietary methyl donor deficiency.
