Higher order chromatin structure is involved in gene regulation by restricting access to the DNA and interfering with processes requiring mobilization of nucleosomes, such as transcription. Compacted chromatin is highly dynamic on biological timescales and its structure can be modulated by histone post-translational modifications – either by altering its intrinsic properties or by generating anchoring points for further structural proteins 43
. To date, intrinsic structural effects have been demonstrated for acetylation of K16 in the H4 tail, leading to fiber decompaction 11
, and for tri-methylation of K20 in H4 resulting in increased folding 44
. Ubiquitylation of H2B has long been proposed to result in fiber decompaction, due to its size and positioning on the nucleosomal surface 23,24
. However, a detailed study of the different effects of uH2B on chromatin structure, dynamics and function has been difficult, as its availability from natural sources is low. Using a defined model system and chemically modified histones we were able to demonstrate that uH2B inhibits both nucleosome array folding, as well as inter-fiber oligomerization, the latter in a cooperative fashion with H4 acetylation on the same array. Consistent with this, nucleosome arrays containing uH2B possess a more biochemically accessible fiber conformation, as demonstrated by our studies using hDot1L. We note, however, that these experiments do not rule out the possibility that binding of hDot1L to ubiquitylated arrays could have an additional effect on local fiber structure.
Several methods have been applied to investigate chromatin higher-order structure formation, including electron microscopy, sedimentation velocity measurements and, most recently, single molecule force spectroscopy 1,3
. These methods are powerful, but either require sample fixation, large sample amounts or are technically challenging. Herein, we present a complementary method based on homo-RET between nucleosomes, which directly reports on inter-nucleosomal distance changes in equilibrium. Applying this method, we were able to show that divalent cation induced chromatin fiber compaction involves conformationally heterogenous intermediates, which are differentially affected by uH2B and acH4.
Based on our data we propose a model for fiber decompaction by H2B ubiquitylation, distinct from the effect of H4 tail acetylation. acH4 affects compaction throughout the folding transition, presumably by weakening H4 tail binding to the H2A acidic patch 12
. This results in a reduction of closely interacting nucleosomes at a given Mg2+
concentration (), and prevents full fiber folding due to counteracting electrostatic repulsion and thermal fluctuations. In contrast, our measurements show that uH2B only interferes with the later stages of compaction. At low ionic strength, transient interactions between nucleosomes in ubiquitylated arrays are not impaired, as reflected by sedimentation coefficients and SSA values comparable to unmodified fibers. This can be attributed to sufficient conformational freedom in these local contacts to accommodate the ubiquitin moiety. However, upon further compaction regular fiber packing is impaired and defects in nucleosome stacking may lead to fiber instability and local unfolding (). The two different modes of action of uH2B and acH4 are further reflected in the cooperativity of these modifications in preventing inter-strand interactions. Finally, we found that the similar sized protein Hub1 could not substitute for ubiquitin in impairing chromatin folding. We therefore hypothesize that specific interactions between ubiquitin and the nucleosomal surface around its anchoring point may be required to prevent escape of ubiquitin from the interface between nucleosomes during compaction. Consistent with this idea, ubiquitylation of H2A, a modification associated with heterochromatin and situated at the opposite side of the nucleosomal surface, does not appear to hinder fiber compaction 45
. It remains to be seen exactly how ubiquitin, when properly localized on the nucleosome, impedes chromatin fiber compaction. We can envision several models for this effect (see Supplementary Fig. S11
) including ubiquitin imposing steric hindrance. Further experiments are required to discriminate between these possible mechanisms. In summary, our results establish a novel function for uH2B in disrupting local chromatin structure and add to the understanding of how combinations of histone post-translational modifications allow fine-tuning of local chromatin fiber compaction as well as higher order structure. By extension, we propose that the increased local accessibility of ubiquitylated nucleosomes might also facilitate chaperone mediated nucleosome dis- and reassembly during transcription 21,22
, in agreement with in vivo
observations on the function of this modification.