Project: PRJNA609170
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 genome-wide unwrapping states of nucleosomes containing histone variant H2A.Z in mouse embryonic stem (ES) cells. We found that H2A.Z is enriched with unwrapped nucleosomes. Interestingly, the function of +1 H2A.Z nucleosome in transcriptional regulation is correlated with the unwrapping states. We further showed that H2A.Z nucleosomes adjacent the CTCF binding sites (CBS) may adopt an open conformation. We confirmed the unwrapping state of H2A.Z nucleosomes under native condition by re-ChIP of H2A.Z after CTCF CUT&RUN in mouse ES cells. Importantly, we found that depletion of H2A.Z results in increased CTCF binding, indicating dynamic competition between the unwrapped H2A.Z nucleosomal intermediates and CTCF at the CBS. Taken together, our results showed that histone variant H2A.Z regulates transcription and CTCF binding through modulating the nucleosome unwrapping. Overall design: Chromatin was first crosslinked with formaldehyde, then digested with MNase and solubilized by minimal sonication. After Chromatin immunoprecipitation (ChIP), the ChIPed DNA was subjected to paired-end sequencing without size selection. The read pairs were mapped to the mouse reference genome (mm9) using Bowtie2, and only read pairs with mapping quality higher than 10 (mapq > 10) were retained for further analyses. We counted the frequency of the length of ChIPed DNA fragments per base pair from 0 to 200 bp to analyze the fragment length profiles of histones.
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