Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
Add more filters










Database
Language
Publication year range
2.
Elife ; 62017 08 01.
Article in English | MEDLINE | ID: mdl-28760201

ABSTRACT

SUV39H is the major histone H3 lysine 9 (H3K9)-specific methyltransferase that targets pericentric regions and is crucial for assembling silent heterochromatin. SUV39H recognizes trimethylated H3K9 (H3K9me3) via its chromodomain (CD), and enriched H3K9me3 allows SUV39H to target specific chromosomal regions. However, the detailed targeting mechanisms, especially for naïve chromatin without preexisting H3K9me3, are poorly understood. Here we show that Suv39h1's CD (Suv39h1-CD) binds nucleic acids, and this binding is important for its function in heterochromatin assembly. Suv39h1-CD had higher binding affinity for RNA than DNA, and its ability to bind nucleic acids was independent of its H3K9me3 recognition. Suv39h1 bound major satellite RNAs in vivo, and knockdown of major satellite RNAs lowered Suv39h1 retention on pericentromere. Suv39h1 mutational studies indicated that both the nucleic acid-binding and H3K9me-binding activities of Suv39h1-CD were crucial for its pericentric heterochromatin assembly. These results suggest that chromatin-bound RNAs contribute to creating SUV39H's target specificity.


Subject(s)
Heterochromatin/metabolism , Histones/metabolism , Methyltransferases/metabolism , Nucleic Acids/metabolism , Repressor Proteins/metabolism , Cell Line , Humans , Protein Binding
3.
Sci Rep ; 6: 24999, 2016 05 16.
Article in English | MEDLINE | ID: mdl-27181506

ABSTRACT

During chromatin-regulated processes, the histone H2A-H2B heterodimer functions dynamically in and out of the nucleosome. Although detailed crystal structures of nucleosomes have been established, that of the isolated full-length H2A-H2B heterodimer has remained elusive. Here, we have determined the solution structure of human H2A-H2B by NMR coupled with CS-Rosetta. H2A and H2B each contain a histone fold, comprising four α-helices and two ß-strands (α1-ß1-α2-ß2-α3-αC), together with the long disordered N- and C-terminal H2A tails and the long N-terminal H2B tail. The N-terminal αN helix, C-terminal ß3 strand, and 310 helix of H2A observed in the H2A-H2B nucleosome structure are disordered in isolated H2A-H2B. In addition, the H2A α1 and H2B αC helices are not well fixed in the heterodimer, and the H2A and H2B tails are not completely random coils. Comparison of hydrogen-deuterium exchange, fast hydrogen exchange, and {(1)H}-(15)N hetero-nuclear NOE data with the CS-Rosetta structure indicates that there is some conformation in the H2A 310 helical and H2B Lys11 regions, while the repression domain of H2B (residues 27-34) exhibits an extended string-like structure. This first structure of the isolated H2A-H2B heterodimer provides insight into its dynamic functions in chromatin.


Subject(s)
Histones/chemistry , Protein Multimerization , Humans , Magnetic Resonance Spectroscopy , Protein Conformation , Protein Folding
4.
Sci Rep ; 6: 22527, 2016 Mar 03.
Article in English | MEDLINE | ID: mdl-26934956

ABSTRACT

The chromodomain of HP1α binds directly to lysine 9-methylated histone H3 (H3K9me). This interaction is enhanced by phosphorylation of serine residues in the N-terminal tail of HP1α by unknown mechanism. Here we show that phosphorylation modulates flexibility of HP1α's N-terminal tail, which strengthens the interaction with H3. NMR analysis of HP1α's chromodomain with N-terminal tail reveals that phosphorylation does not change the overall tertiary structure, but apparently reduces the tail dynamics. Small angle X-ray scattering confirms that phosphorylation contributes to extending HP1α's N-terminal tail. Systematic analysis using deletion mutants and replica exchange molecular dynamics simulations indicate that the phosphorylated serines and following acidic segment behave like an extended string and dynamically bind to H3 basic residues; without phosphorylation, the most N-terminal basic segment of HP1α inhibits interaction of the acidic segment with H3. Thus, the dynamic string-like behavior of HP1α's N-terminal tail underlies the enhancement in H3 binding due to phosphorylation.


Subject(s)
Chromosomal Proteins, Non-Histone/chemistry , Histones/chemistry , Chromobox Protein Homolog 5 , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Histones/genetics , Histones/metabolism , Humans , Lysine , Methylation , Nuclear Magnetic Resonance, Biomolecular , Phosphorylation , Protein Binding , Protein Domains
5.
Mol Cell ; 47(2): 228-41, 2012 Jul 27.
Article in English | MEDLINE | ID: mdl-22727667

ABSTRACT

Centromeric heterochromatin assembly in fission yeast requires the RNAi pathway. Chp1, a chromodomain (CD) protein, forms the Ago1-containing RNA-induced transcriptional silencing (RITS) complex and recruits siRNA-bound RITS to methylated histone H3 lysine 9 (H3K9me) via its CD. Here, we show that the CD of Chp1 (Chp1-CD) possesses unique nucleic acid-binding activities that are essential for heterochromatic gene silencing. Detailed electrophoretic-mobility shift analyses demonstrated that Chp1 binds to RNA via the CD in addition to its central RNA-recognition motif. Interestingly, robust RNA- and DNA-binding activity of Chp1-CD was strongly enhanced when it was bound to H3K9me, which was revealed to involve a positively charged domain within the Chp1-CD by structural analyses. These results demonstrate a role for the CD that provides a link between RNA, DNA, and methylated histone tails to ensure heterochromatic gene silencing.


Subject(s)
Cell Cycle Proteins/genetics , Gene Silencing , Heterochromatin/chemistry , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces/metabolism , Amino Acid Sequence , Argonaute Proteins/metabolism , Chromatin Immunoprecipitation , DNA/chemistry , Dose-Response Relationship, Drug , Eukaryotic Initiation Factors/metabolism , Gene Expression Regulation, Fungal , Kinetics , Methylation , Molecular Sequence Data , Protein Structure, Tertiary , RNA/chemistry , Sequence Homology, Amino Acid
6.
J Mol Biol ; 378(5): 987-1001, 2008 May 16.
Article in English | MEDLINE | ID: mdl-18407291

ABSTRACT

Chromodomains are methylated histone binding modules that have been widely studied. Interestingly, some chromodomains are reported to bind to RNA and/or DNA, although the molecular basis of their RNA/DNA interactions has not been solved. Here we propose a novel binding mode for chromodomain-RNA interactions. Essential Sas-related acetyltransferase 1 (Esa1) contains a presumed chromodomain in addition to a histone acetyltransferase domain. We initially determined the solution structure of the Esa1 presumed chromodomain and showed it to consist of a well-folded structure containing a five-stranded beta-barrel similar to the tudor domain rather than the canonical chromodomain. Furthermore, the domain showed no RNA/DNA binding ability. Because the N-terminus of the protein forms a helical turn, we prepared an N-terminally extended construct, which we surprisingly found to bind to poly(U) and to be critical for in vivo function. This extended protein contains an additional beta-sheet that acts as a knot for the tudor domain and binds to oligo(U) and oligo(C) with greater affinity compared with other oligo-RNAs and DNAs examined thus far. The knot does not cause a global change in the core structure but induces a well-defined loop in the tudor domain itself, which is responsible for RNA binding. We made 47 point mutants in an esa1 mutant gene in yeast in which amino acids of the Esa1 knotted tudor domain were substituted to alanine residues and their functional abilities were examined. Interestingly, the knotted tudor domain mutations that were lethal to the yeast lost poly(U) binding ability. Amino acids that are related to RNA interaction sites, as revealed by both NMR and affinity binding experiments, are found to be important in vivo. These findings are the first demonstration of how the novel structure of the knotted tudor domain impacts on RNA binding and how this influences in vivo function.


Subject(s)
Acetyltransferases/chemistry , Protein Conformation , RNA-Binding Proteins/chemistry , RNA/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Acetyltransferases/genetics , Acetyltransferases/metabolism , Animals , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , RNA/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...