RNAi-dependent H3K27 methylation is required for heterochromatin formation and DNA elimination in Tetrahymena.

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.

Chemical derivatization of histones for facilitated analysis by mass spectrometry.

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.

N-terminal alpha-methylation of RCC1 is necessary for stable chromatin association and normal 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. 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.

Mass spectrometry analysis of Arabidopsis histone H3 reveals distinct combinations of post-translational modifications.

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).