Orthaus S, Klement K, Happel N, Hoischen C, Diekmann S
Leibniz-Institute for Age Research - Fritz Lipmann Institute, Beutenbergstr. 11, D-07745 Jena, Germany.
Nucleic Acids Res. 2009 Jun;37(10):3391-406. doi: 10.1093/nar/gkp199. Epub 2009 Mar 31.
The vertebrate kinetochore complex assembles at the centromere on alpha-satellite DNA. In humans, alpha-satellite DNA has a repeat length of 171 bp slightly longer than the DNA in the chromatosome containing the linker histone H1. The centromere-binding protein CENP-B binds specifically to alpha-satellite DNA with properties of a centromeric-linker histone. Here, we analysed if linker histone H1 is present at or excluded from centromeric chromatin by CENP-B. By immunostaining we detected the presence, but no enrichment or depletion of five different H1 subtypes at centromeric chromatin. The binding dynamics of H1 at centromeric sites were similar to that at other locations in the genome. These dynamics did not change in CENP-B depleted cells, suggesting that CENP-B and H1 co-exist in centromeric chromatin with no or little functional overlap. By bimolecular fluorescence complementation (BiFC) and Förster resonance energy transfer (FRET), we revealed that the linker histone H1 subtypes H1 degrees and H1.2 bind to centromeric chromatin in interphase nuclei in direct neighbourhood to inner kinetochore proteins.
脊椎动物的动粒复合体在α-卫星DNA的着丝粒处组装。在人类中，α-卫星DNA的重复长度为171 bp，略长于含有连接组蛋白H1的核小体中的DNA。着丝粒结合蛋白CENP-B以着丝粒连接组蛋白的特性特异性结合α-卫星DNA。在这里，我们分析了连接组蛋白H1是否通过CENP-B存在于着丝粒染色质中或被排除在外。通过免疫染色，我们在着丝粒染色质中检测到了五种不同H1亚型的存在，但没有富集或缺失。H1在着丝粒位点的结合动力学与基因组中其他位置的相似。这些动力学在CENP-B缺失的细胞中没有改变，表明CENP-B和H1在着丝粒染色质中共存，功能重叠很少或没有。通过双分子荧光互补（BiFC）和荧光共振能量转移（FRET），我们发现连接组蛋白H1亚型H1°和H1.2在间期细胞核中与内动粒蛋白直接相邻的位置结合着丝粒染色质。
