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BACKGROUND:Hydroxymethylation of DNA at the C5 position of cytosine (5hmC) is recognized as an important epigenetic mark. The molecular role of 5hmC in gene regulation, however, is not well understood.METHODS:We studied the effects of 5-hydroxymethylation (5hmC) on nucleosome properties in vitro using a combination of biochemical and fluorescence assays. Competitive reconstitution was used to evaluate the effect of 5hmC on nucleosome formation. The effects of 5hmC on nucleosome compactness and stability were characterized using FRET assays. These findings have also been compared with another important epigenetic mark, the cytosine methylation (5mC) of DNA.RESULTS:We observed that hydroxymethylation increases the binding affinity of DNA for the histone octamer. The formed nucleosome exhibits slightly different conformations based on the sequence and epigenetic context of DNA. DNA hydroxymethylation decreases the stability of formed nucleosomes in salt-induced dissociation processes.CONCLUSION:DNA containing 5hmC is more likely to be incorporated into nucleosomes. Once formed, the 5hmC nucleosomes might be in an open and transcriptionally active state due to the weakened interaction of hydroxymethylated DNA with the H2A-H2B dimers. GENERAL SIGNIFICANCE: Our results reveal the effect of 5hmC on regulating nucleosome compactness and stability in vitro.
Chromatin; DNA hydroxymethylation; FRET; Nucleosome
...DNA fragments containing either methylated or hydroxymethylated cytosines were amplified using a label free DNA template and Taq DNA Polymerase (New England Biolabs). The DNA template was custom synthesized (GenScript)...
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