ABSTRACT
Methylated H3K27 is an important mark for Polycomb group (PcG) protein-mediated transcriptional gene silencing (TGS) in multicellular eukaryotes. Here a Drosophila E(z) homolog, EZL1, is characterized in the ciliated protozoan Tetrahymena thermophila and is shown to be responsible for H3K27 methylation associated with developmentally regulated heterochromatin formation and DNA elimination. Importantly, Ezl1p-catalyzed H3K27 methylation occurs in an RNA interference (RNAi)-dependent manner. H3K27 methylation also regulates H3K9 methylation in these processes. Furthermore, an "effector" of programmed DNA elimination, the chromodomain protein Pdd1p, is shown to bind both K27- and K9-methylated H3. These studies provide a framework for an RNAi-dependent, Polycomb group protein-mediated heterochromatin formation pathway in Tetrahymena and underscore the connection between the two highly conserved machineries in eukaryotes.
Subject(s)
Histones/metabolism , Tetrahymena thermophila/genetics , Tetrahymena thermophila/metabolism , Animals , Chromosome Breakage , DNA, Protozoan/genetics , DNA, Protozoan/metabolism , Heterochromatin/genetics , Heterochromatin/metabolism , Histones/chemistry , Histones/genetics , Methylation , Methyltransferases/genetics , Methyltransferases/metabolism , Models, Biological , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Polycomb-Group Proteins , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , RNA Interference , Repressor Proteins/genetics , Repressor Proteins/metabolismABSTRACT
Regulator of chromatin condensation 1 (RCC1) is the only known guanine nucleotide-exchange factor for the Ran GTPase and has pivotal roles in nucleo-cytoplasmic transport, mitosis, and nuclear-envelope assembly. RCC1 associates dynamically with chromatin through binding to histones H2A and/or H2B in a Ran-regulated manner. Here, we report that, unexpectedly, the amino-terminal serine or proline residue of RCC1 is uniquely methylated on its alpha-amino group. Methylation requires removal of the initiating methionine, and the presence of proline and lysine at positions 3 and 4, respectively. Methylation-defective mutants of RCC1 bind less effectively than wild-type protein to chromatin during mitosis, which causes spindle-pole defects. We propose a bimodal attachment mechanism for RCC1 in which the tail promotes stable RCC1 association with chromatin through DNA binding in an alpha-N-methylation-dependent manner. These data provide the first known function for N-terminal protein methylation.
Subject(s)
Cell Cycle Proteins/metabolism , Chromatin Assembly and Disassembly , Chromatin/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Mitosis/physiology , Nuclear Proteins/metabolism , Protein Methyltransferases/metabolism , Protein Processing, Post-Translational , Animals , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cloning, Molecular , DNA/metabolism , Dogs , Guanine Nucleotide Exchange Factors/chemistry , Guanine Nucleotide Exchange Factors/genetics , HeLa Cells , Histones/metabolism , Humans , Kinetics , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Methionine/chemistry , Methylation , Mutation , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Proline/metabolism , Protein Binding , Protein Conformation , Recombinant Fusion Proteins/metabolism , Serine/metabolism , ran GTP-Binding Protein/metabolismABSTRACT
Histone post-translational modifications have been recently intensely studied owing to their role in regulating gene expression. Here, we describe protocols for the characterization of histone modifications in both qualitative and semiquantitative manners using chemical derivatization and tandem mass spectrometry. In these procedures, extracted histones are first derivatized using propionic anhydride to neutralize charge and block lysine residues, and are subsequently digested using trypsin, which, under these conditions, cleaves only the arginine residues. The generated peptides can be easily analyzed using online LC-electrospray ionization-tandem mass spectrometry to identify the modification site. In addition, a stable isotope-labeling step can be included to modify carboxylic acid groups allowing for relative quantification of histone modifications. This methodology has the advantage of producing a small number of predicted peptides from highly modified proteins. The protocol should take approximately 15-19 h to complete, including all chemical reactions, enzymatic digestion and mass spectrometry experiments.
Subject(s)
Histones/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Tandem Mass Spectrometry/methods , Amino Acid Sequence , Histones/isolation & purification , Histones/metabolism , Protein Processing, Post-TranslationalABSTRACT
Chromatin is regulated at many different levels, from higher-order packing to individual nucleosome placement. Recent studies have shown that individual histone modifications, and combinations thereof, play a key role in modulating chromatin structure and gene activity. Reported here is an analysis of Arabidopsis histone H3 modifications by nanoflow-HPLC coupled to electrospray ionization on a hybrid linear ion trap-Fourier transform mass spectrometer (LTQ/FTMS). We find that the sites of acetylation and methylation, in general, correlate well with other plants and animals. Two well-studied modifications, dimethylation of Lys-9 (correlated with silencing) and acetylation of Lys-14 (correlated with active chromatin) while abundant by themselves were rarely found on the same histone H3 tail. In contrast, dimethylation at Lys-27 and monomethylation at Lys-36 were commonly found together. Interestingly, acetylation at Lys-9 was found only in a low percentage of histones while acetylation of Lys-14 was very abundant. The two histone H3 variants, H3.1 and H3.2, also differ in the abundance of silencing and activating marks confirming other studies showing that the replication-independent histone H3 is enriched in active chromatin.
