Mapping of lysine methylation and acetylation in core histones of Neurospora crassa.

The eukaryotic nucleosome, the fundamental unit of chromatin, plays an important role in packaging and organizing the genetic material (1). Each nucleosome consists of 146 bp of DNA wrapped around an octameric core histone complex consisting of two H2A-H2B dimers flanking a (H3-H4)2 tetramer (1, 2). All core histones have a basic N-terminal domain, a globular domain organized by the histone fold and a C-terminal tail (1, 2). The histone N-terminal tails, which extend out from the core particle, are involved in the establishment of chromatin structural states, whereas their histone fold domains mediate histone-histone and histone-DNA interactions (2). Core histones are susceptible to a variety of post-translational modifications (PTMs), which include methylation, acetylation, phosphorylation, ubiquitination, SUMOylation and ADP-ribosylation. These modifications, which occur mainly on the N-terminal tails (3, 4), can affect interactions of nucleosomes with trans-acting factors, and are thought to play a role in the assembly and disassembly of chromatin states, and can control the accessibility of DNA for important cellular processes including transcription, replication, gene silencing and DNA repair (5-9). Distinct modifications of the histone tails can recruit specific chromatin-binding proteins, and modifications on the same or different histone tails may be interdependent and generatevarious combinations on any individual nucleosome (10-12). Mass spectrometry has been widely used for assessing histone PTMs. It provides direct information about the sites and types of modifications, differentiates isobaric modifications (e.g., acetylation vs. tri-methylation) (13), and allows for quantitative analysis (14). PTM information obtained by mass spectrometric analysis facilitates genome-wide functional studies and provides the foundation for studies of modifications in specific regions of genomes using chromatin immunoprecipitation (ChIP) with antibodies recognizing specifically modified histones (15-17). Neurospora crassa is a convenient model eukaryote, showing genomic features lacking in some other simple eukaryotes (e.g. yeast), including DNA methylation and certain histone modifications (e.g. H3K27 methylation). Studies using this organism have contributed significantly to the fundamental understanding of circadian rhythms, DNA methylation, genome defense systems, mitochondrial protein import, post-transcriptional gene silencing, DNA repair and other processes (18). Being a multicellular filamentous fungus, Neurospora also provides a system to study cellular differentiation and development (19). However, no comprehensive investigation of the PTMs of Neurospora core histones has been reported. In the present study, we extracted core histones from N. crassa and systematically mapped histone methylation and acetylation, using a combination of digestion with various proteases and analyses with multiple types of mass spectrometers. Our results allowed for the identification of acetylation and methylation sites of lysine residues that were found previously in Arabidopsis thaliana, Saccharomyces cerevisiae and humans. More importantly, we identified several unique acetylation and methylation sites in core histones of Neurospora. Our analysis on core histone PTMs provides a foundation for examining the regulation of histone modifications and for genome-wide functional studies in this model organism.