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.

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.[Keywords: RNA interference; H3K27 methylation; Polycomb group proteins; heterochromatin; DNA elimination] Supplemental material is available at http://www.genesdev.org.

We describe the design and performance of a prototype high performance hybrid mass spectrometer. This instrument consists of a linear quadrupole ion trap (QLT) coupled to a Fourier transform ion cyclotron resonance mass analyzer (FTMS). This configuration provides rapid and automated MS and MS/MS analyses, similar to the "data dependent scanning" found on standard 3-D Paul traps, but with substantially improved internal scan dynamic range, mass measurement accuracy, mass resolution, and detection limits. Sequence analysis of peptides at the zeptomole level is described. The recently released, commercial version of this instrument operates in the LC/MS mode (1 s/scan) with a mass resolution of 100 000 and is equipped with automatic gain control to provide mass measurement accuracy of 1-2 ppm without internal standard. Methodology is described that uses this instrument to compare the post-translational modifications present on histone H3 isolated from asynchronously growing cells and cells arrested in mitosis.

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 1 . RCC1 associates dynamically with chromatin through binding to histones H2A and/or H2B in a Ran-regulated manner 2,3 . Here, we report that, unexpectedly, the amino-terminal serine or proline residue of RCC1 is uniquely methylated on its α-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 α-N-methylation-dependent manner. These data provide the first known function for N-terminal protein methylation.RCC1 has a propeller-like structure, one face of which binds to Ran 4 , whereas the other face binds chromatin. A flexible N-terminal tail contains a nuclear localization signal (NLS) 5,6 . Although the propeller seems rigid, Ran allosterically modulates RCC1 association with chromatin 3 , and the RCC1 D182A mutant, which does not detectably bind Ran, shows reduced chromatin association 7,8 . Removal of the N-terminal tail also leads to exclusion of RCC1 from chromosomes 7 , whereas phosphorylation on Ser 2 and Ser 11 in human RCC1 reportedly permits stable association with mitotic chromosomes 8,9 . These phosphorylations have been proposed to reduce the affinity of the NLS for its nuclear transport receptor, Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ 4 Correspondence should be addressed to I.G.M. (igm9c@virginia.edu).Note: Supplementary Information is available on the Nature Cell Biology website. AUTHOR CONTRIBUTIONST.C. made the RCC1 mutant proteins, identified the methyltransferase activity and its recognition motif, and performed the assays to identify its function. T.L.M. and J.S. performed the mass spectrometry. C.E.S.-T. made the antibodies against methylated RCC1 and performed the experiments with them. D.F.H. directed the mass spectrometry. I.G.M. directed the biochemical and cell biological studies. T.C. and I.G.M. wrote the paper. All authors discussed results and contributed to the manuscript. COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript importin-α3, which, when bound to RCC1, interferes with chromatin attachment 9 . However, Ser 2 is found only in simians and is a proline residue in other mammalian species.The N-terminus of RCC1 resembles histone tails, which also contain NLSs, and which are phosphorylated, acetylated and methylated in complex patterns that comprise a histone code 10,11 . We asked whether ...

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.