A synthetic biology approach to probing nucleosome symmetry.

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.

The architecture and function of the chromatin replication machinery.

Genomic DNA in eukaryotic cells is packaged into nucleosome arrays. During replication, nucleosomes need to be dismantled ahead of the advancing replication fork and reassembled on duplicated DNA. The architecture and function of the core replisome machinery is now beginning to be elucidated, with recent insights shaping our view on DNA replication processes. Simultaneously, breakthroughs in our mechanistic understanding of epigenetic inheritance allow us to build new models of how histone chaperones integrate with the replisome to reshuffle nucleosomes. The emerging picture indicates that the core eukaryotic DNA replication machinery has evolved elements that handle nucleosomes to facilitate chromatin duplication.