McArthur M, Thomas J O
Cambridge Center for Molecular Recognition, University of Cambridge, UK.
EMBO J. 1996 Apr 1;15(7):1705-14.
We have identified a clear preference of histone H1 for CpG-methylated DNA, irrespective of DNA sequence. The conditions under which this preference is observed allowed cooperative binding of H1; the H1-DNA complexes formed were shown earlier to be 'tramlines' of two DNA duplexes bridged by an array of H1 molecules, and multiples of these. The preference for methylated DNA is clear in sedimentation assays, which also show that the preference is greater with increased methylation level, and in gel retardation assays with an oligonucleotide containing a single methyl-CpG pair; it is shared by the globular domain which also binds cooperatively to DNA. A small intrinsic preference of H1 for methylated DNA is also apparent in Southwestern assays where the immobilized H1 presumably cannot bind cooperatively. Methylated DNA in H1-DNA complexes was partially protected (relative to unmethylated DNA) against digestion by MspI but not by enzymes whose cutting sites were not methylated, consistent with a direct interaction of H1 with methylated nucleotides; this was also true of GH1-DNA complexes. H1 variants (spH1 and H5) from transcriptionally repressed nuclei have a stronger preference than H1 for methylated DNA, suggesting that this may be relevant to the stabilization of chromatin higher order structure and transcriptional repression.
我们已经确定，无论DNA序列如何，组蛋白H1对CpG甲基化的DNA都有明显的偏好。观察到这种偏好的条件允许H1进行协同结合；先前显示形成的H1-DNA复合物是由一系列H1分子桥接的两条DNA双链体的“轨道”，以及这些轨道的倍数。在沉降分析中，对甲基化DNA的偏好很明显，这也表明随着甲基化水平的增加，偏好性更强，并且在含有单个甲基-CpG对的寡核苷酸的凝胶阻滞分析中也是如此；球状结构域也与DNA协同结合，也表现出这种偏好。在免疫印迹分析中，固定化的H1可能无法协同结合，H1对甲基化DNA的微小内在偏好也很明显。H1-DNA复合物中的甲基化DNA相对于未甲基化DNA对MspI消化有部分保护作用，但对切割位点未甲基化的酶没有保护作用，这与H1与甲基化核苷酸的直接相互作用一致；GH1-DNA复合物也是如此。来自转录抑制细胞核的H1变体（spH1和H5）对甲基化DNA的偏好比H1更强，这表明这可能与染色质高级结构的稳定和转录抑制有关。
