Abstract
The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and several recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read out, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing the effects of asymmetric histone tail point mutants with those of symmetric double mutants revealed that a single methylated H3K36 per nucleosome was sufficient to silence cryptic transcription in vivo. We also demonstrate the utility of this system for analysis of histone modification crosstalk, using mass spectrometry to separately identify modifications on each H3 molecule within asymmetric nucleosomes. The ability to generate asymmetric nucleosomes in vivo and in vitro provides a powerful and generalizable tool to probe the mechanisms by which H3 tails are read out by effector proteins in the cell.
Highlights
Packaging of eukaryotic genomes into chromatin has widespread consequences for genomic function, as nucleosomes generally impede the access of proteins to the underlying DNA (1)
The histone proteins are subject to a huge variety of covalent modifications, which affect genomic function in ways ranging from direct alterations in the biophysical nature of the chromatin fiber to recruitment or activation of various regulatory proteins
Immunopurification and mass spectrometry of nucleosomes marked with H3K27me3 revealed a mixture of symmetrically-modified nucleosomes as well as asymmetric nucleosomes modified on only one H3 tail (3)
Summary
Packaging of eukaryotic genomes into chromatin has widespread consequences for genomic function, as nucleosomes generally impede the access of proteins to the underlying DNA (1). The symmetry inherent to the histone octamer has long been appreciated, and it has been suggested that chromatin function may be regulated at the level of histone modification symmetry – that K4me3/K4me0 and. K4me3/K4me nucleosomes might serve distinct functions in vivo, for example. Increasing evidence supports the existence of asymmetrically-modified nucleosomes in vivo. Immunopurification and mass spectrometry of nucleosomes marked with H3K27me revealed a mixture of symmetrically-modified nucleosomes as well as asymmetric nucleosomes modified on only one H3 tail (3). More recent single molecule studies of H3K4me3/K27me3-marked “bivalent” nucleosomes reveal that the vast majority of these nucleosomes are asymmetrically modified, with each H3 tail bearing one of the two methylation marks, rather than both marks co-occurring on the same H3 molecule (4). ChIP-Exo and chemical mapping studies identified a subset of nucleosomes that exhibit asymmetric contacts with the underlying genomic
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