No abstract

Regulation of chromatin structure involves histone post-translational modifications which can modulate intrinsic properties of the chromatin fiber to change the chromatin state. We used chemically defined nucleosome arrays to demonstrate that H2B ubiquitylation (uH2B), a modification associated with transcription, interferes with chromatin compaction and leads to an open and biochemically accessible fiber conformation. Importantly, these effects were specific for ubiquitin, as compaction of chromatin modified with a similar ubiquitin-sized protein, Hub1, was only weakly affected. Applying a fluorescence based method we found that uH2B acts through a mechanism distinct from H4 tail acetylation (acH4), a modification known to disrupt chromatin folding. Finally, incorporation of both uH2B and acH4 in nucleosomes resulted in synergistic inhibition of higher order chromatin structure formation, possibly a result of their distinct mode of action.

We have developed a readily accessible disulfide-directed methodology for the site-specific modification of histones by ubiquitin and ubiquitin-like proteins. The disulfide-linked analog of mono-ubiquitylated H2B stimulated the H3K79 methyltransferase activity of hDot1L to a similar extent as the native isopeptide linkage. This permitted structure-activity studies of ubiquitylated mononucleosomes that revealed plasticity in the mechanism of hDot1L stimulation and identified surfaces of ubiquitin important for activation.

The dynamic architecture of chromatin fibers, a key determinant of genome regulation, is poorly understood. Here, we employ multimodal single-molecule Förster resonance energy transfer studies to reveal structural states and their interconversion kinetics in chromatin fibers. We show that nucleosomes engage in short-lived (micro- to milliseconds) stacking interactions with one of their neighbors. This results in discrete tetranucleosome units with distinct interaction registers that interconvert within hundreds of milliseconds. Additionally, we find that dynamic chromatin architecture is modulated by the multivalent architectural protein heterochromatin protein 1α (HP1α), which engages methylated histone tails and thereby transiently stabilizes stacked nucleosomes. This compacted state nevertheless remains dynamic, exhibiting fluctuations on the timescale of HP1α residence times. Overall, this study reveals that exposure of internal DNA sites and nucleosome surfaces in chromatin fibers is governed by an intrinsic dynamic hierarchy from micro- to milliseconds, allowing the gene regulation machinery to access compact chromatin.

Elucidating the molecular details of how chromatin-associated factors deposit, remove and recognize histone posttranslational modification (‘PTM’) signatures remains a daunting task in the epigenetics field. Here, we introduce a versatile platform that greatly accelerates biochemical investigations into chromatin recognition and signaling. This technology is based on the streamlined semi-synthesis of DNA-barcoded nucleosome libraries with distinct combinations of PTMs. Chromatin immunoprecipitation of these libraries treated with purified chromatin effectors or the combined chromatin recognizing and modifying activities of the nuclear proteome is followed by multiplexed DNA-barcode sequencing. This ultrasensitive workflow allowed us to collect thousands of biochemical data points revealing the binding preferences of various nuclear factors for PTM patterns and how pre-existing PTMs, alone or synergistically, affect further PTM deposition via crosstalk mechanisms. We anticipate that the high-throughput and -sensitivity of the technology will help accelerate the decryption of the diverse molecular controls that operate at the level of chromatin.