The H2A.B histone variant is an epigenetic regulator involved in transcriptional upregulation, DNA synthesis, and splicing that functions by replacing the canonical H2A histone in the nucleosome core particle. Introduction of H2A.B results in less compact nucleosome states with increased DNA unwinding and accessibility at the nucleosomal entry and exit sites. Despite being well characterized experimentally, the molecular mechanisms by which H2A.B incorporation alters nucleosome stability and dynamics remain poorly understood. To study the molecular mechanisms of H2A.B, we have performed a series of conventional and enhanced sampling molecular dynamics simulation of H2A.B and canonical H2A containing nucleosomes. Results of conventional simulations show that H2A.B weakens protein/protein and protein/DNA interactions at specific locations throughout the nucleosome. These weakened interactions result in significantly more DNA opening from both the entry and exit sites in enhanced sampling simulations. Furthermore, free energy profiles show that H2A.B containing nucleosomes have significantly broader free wells, and that H2A.B allows for sampling of states with increased DNA breathing, which are shown to be stable on the hundreds of nanoseconds timescale with further conventional simulations. Together, our results show the molecular mechanisms by which H2A.B creates less compacted nucleosome states as a means of increasing genetic accessibility and gene transcription.SIGNIFICANCENature has evolved a plethora of mechanisms for altering the physical and chemical properties of chromatin fibers as a means of controlling gene expression. These epigenetic processes may serve to increase or decrease DNA accessibility, manage the recruitment of remodeling factors, or tune the stability of the nucleosomes that make up chromatin. Here, we have used both conventional and enhanced sampling molecular dynamics simulations to understand how one of these epigenetic mechanisms, the substitution of canonical H2A proteins with the H2A.B variant, exerts its influence on the structures and dynamics of the nucleosome. Results show at the molecular level how this variant alters inter-molecular interactions to increase DNA accessibility as a means of increasing genetic accessibility and gene transcription.
Non-coding RNAs (ncRNAs) are an emerging epigenetic factor and have been recognized as playing a key role in many gene expression pathways. Structurally, binding of ncRNAs to isolated DNA is strongly dependent on sequence complementary, and results in the formation of an RNA.DNA-DNA (RDD) triple helix. However, in vivo DNA is not isolated, but is packed in chromatin fibers, the fundamental unit of which is the nucleosome. Biochemical experiments have shown that ncRNA binding to nucleosomal DNA is elevated at DNA entry and exit sites and is dependent on the presence of the H3 N-terminal tails. However, the structural and dynamical bases for these mechanisms remains unknown. Here, we have examined the mechanisms and effects of RDD formation in the context of the nucleosome using a series of all-atom molecular dynamics simulations. Results highlight the importance of DNA sequence on complex stability, elucidate the effects of the H3 tails on RDD structures, show how RDD formation impacts the structure and dynamics of the H3 tails, and show how RNA alters the local and global DNA double helical structure. Together, our results suggest ncRNAs can modify nucleosome, and potentially higher-order chromatin, structures and dynamics as a means of exerting epigenetic control.SIGNIFICANCENon-coding RNAs (ncRNAs) play an essential role in gene regulation by binding to DNA and forming RNA.DNA-DNA (RDD) triple helices. In the cell, this occurs in the context where DNA is not a free double helix but is instead condensed into chromatin fibers. At the fundamental level, this compaction involves wrapping approximately 147 DNA basepairs around eight histone proteins to form the nucleosome. Here, we have used molecular dynamics simulations to understand the interplay between the structure and dynamics of RDD triple helices with the nucleosome. Results highlight the importance of RNA sequence on RDD stability regardless of its environment and suggest potential mechanisms for cross-talk between epigenetic factors and the effects of ncRNA binding on local and higher-order chromatin structures.
subnucleosomal species lacking the standard number of histones exist in cells, at least as transcriptional and remodeling intermediates. Altering the composition of nucleosomes may serve as a mechanism of chromatin regulation. We recently found that the H3 tails exist in a dynamic ensemble of states within the nucleosome core particle, collapsed onto the core DNA. This results in inhibition of binding by a model histone reader domain in the absence of other nuclear factors. We sought to investigate the effects of removing one or both H2A/H2B dimers on the conformational ensemble of the H3 tails. In this study, we demonstrate that the conformation of the H3 tails is sensitive to the assembly state of the nucleosome core particle (NCP). Solution NMR studies demonstrate that the H3 tails experience distinct environments between octasomes (canonical nucleosomes), hexasomes (lacking one dimer), and tetrasomes (lacking both dimers). These studies additionally demonstrate that the accessibility of the H3 tails is influenced by the assembly state of the NCP. Altogether, these studies suggest that nucleosome assembly state is another mechanism of modulating histone tail conformation and accessibility, with implications in chromatin signaling and remodeling.
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