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.
Bibliographical noteFunding Information:
We thank members of the Rando and Kaufman labs for discussions and critical reading of the manuscript. This work was supported by NIH grant R01GM100164 (OJR and PDK), ERC Grant 340712 (NF), and the ISF I-Core on Chromatin and RNA in Gene Regulation (NF). Proteomics experiments were supported by NIH grants P30CA060553 and P41GM108569. AA is grateful to the Azrieli foundation for the award of an Azrieli Fellowship.
© Ichikawa et al.