Abstract
Nucleosome is the basic structural unit of chromatin, and its dynamics plays critical roles in the regulation of genome functions. However, how the nucleosome structure is regulated by histone variants in vivo is still largely uncharacterized. Here, by employing Micrococcal nuclease (MNase) digestion of crosslinked chromatin followed by chromatin immunoprecipitation (ChIP) and paired-end sequencing (MNase-X-ChIP-seq), we mapped unwrapping states of nucleosomes containing histone variant H2A.Z in mouse embryonic stem (ES) cells. We found that H2A.Z nucleosomes are more enriched with unwrapping states compared with canonical nucleosomes. Interestingly, +1 H2A.Z nucleosomes with 30–80 bp DNA is correlated with less active genes compared with +1 H2A.Z nucleosomes with 120–140 bp DNA. We confirmed the unwrapping of H2A.Z nucleosomes under native condition by re-ChIP of H2A.Z and H2A after CTCF CUT&RUN in mouse ES cells. Importantly, we found that depletion of H2A.Z results in decreased unwrapping of H3.3 nucleosomes and increased CTCF binding. Taken together, through MNase-X-ChIP-seq, we showed that histone variant H2A.Z regulates nucleosome unwrapping in vivo and that its function in regulating transcription or CTCF binding is correlated with unwrapping states of H2A.Z nucleosomes.
Highlights
The genome of eukaryotic cells is packaged with histones to form chromatin in the nucleus
Our results showed that H2A.Z is enriched with nucleosome unwrapping compared with canonical nucleosomes, and H2A.Z could function in gene regulation and CTCF binding regulation through modulating the unwrapping states of nucleosomes
Through analysis of fragment length profiles (FLPs) by X-Micrococcal nuclease (MNase)-ChIPseq, we revealed that nucleosomes containing histone variant H2A.Z is more unwrapped than canonical nucleosomes
Summary
The genome of eukaryotic cells is packaged with histones to form chromatin in the nucleus. The crystal structure of the nucleosome core particle showed that the DNA was wrapped on the octamer by about 1.65 superhelix turn in a left-hand manner with periodic interaction with histones [1]. During the nucleosome assembly mediated by salt dialysis in vitro, an (H3H4) tetramer bind DNA first to form a tetrasome wrapping about 80 bp DNA, two H2A–H2B heterodimers were added sequentially to form an intact nucleosome [2]. A dynamic intermediate nucleosome structure called prenucleosome, which consists of a histone octamer wrapped by ∼80 bp of DNA, was reported and it can be converted into intact nucleosomes by histone chaperone [3]. The nucleosomal DNA can unwrap asymmetrically and directionally under tension, which is regulated by the flexibility of DNA sequence and histone chaperone FACT [6,7]. Whereas variations and regulation of nucleosome unwrapping have been demonstrated in vitro, the landscape and regulation of unwrapped nucleosomes in vivo are much less characterized
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