Mendonca Agnes, Chang En Hyung, Liu Wenjie, Yuan Chongli
School of Chemical Engineering, Purdue University, West Lafayette, IN 47906, USA.
School of Chemical Engineering, Purdue University, West Lafayette, IN 47906, USA.
Biochim Biophys Acta. 2014 Nov;1839(11):1323-9. doi: 10.1016/j.bbagrm.2014.09.014. Epub 2014 Sep 28.
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.
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.
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.
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.
Our results reveal the effect of 5hmC on regulating nucleosome compactness and stability in vitro.
胞嘧啶C5位的DNA羟甲基化（5hmC）被认为是一种重要的表观遗传标记。然而，5hmC在基因调控中的分子作用尚未得到充分理解。
我们使用生化和荧光检测相结合的方法，在体外研究了5-羟甲基化（5hmC）对核小体特性的影响。采用竞争性重组来评估5hmC对核小体形成的影响。使用荧光共振能量转移（FRET）检测来表征5hmC对核小体紧密性和稳定性的影响。这些发现还与另一种重要的表观遗传标记——DNA的胞嘧啶甲基化（5mC）进行了比较。
我们观察到羟甲基化增加了DNA与组蛋白八聚体的结合亲和力。基于DNA的序列和表观遗传背景，形成的核小体表现出略有不同的构象。DNA羟甲基化在盐诱导的解离过程中降低了形成的核小体的稳定性。
含有5hmC的DNA更有可能被整合到核小体中。一旦形成，由于羟甲基化DNA与H2A-H2B二聚体之间的相互作用减弱，5hmC核小体可能处于开放和转录活跃状态。
我们的结果揭示了5hmC在体外调节核小体紧密性和稳定性的作用。
