Furuyama Takehito, Dalal Yamini, Henikoff Steven
Fred Hutchinson Cancer Research Center and Howard Hughes Medical Institute, 1100 Fairview Avenue North, Seattle, WA 98109, USA.
Proc Natl Acad Sci U S A. 2006 Apr 18;103(16):6172-7. doi: 10.1073/pnas.0601686103. Epub 2006 Apr 6.
Every eukaryotic chromosome requires a centromere for attachment to spindle microtubules for chromosome segregation. Although centromeric DNA sequences vary greatly among species, centromeres are universally marked by the presence of a centromeric histone variant, centromeric histone 3 (CenH3), which replaces canonical histone H3 in centromeric nucleosomes. Conventional chromatin is maintained in part by histone chaperone complexes, which deposit the S phase-limited (H3) and constitutive (H3.3) forms of histone 3. However, the mechanism that deposits CenH3 specifically at centromeres and faithfully maintains its chromosome location through mitosis and meiosis is unknown. To address this problem, we have biochemically purified a soluble assembly complex that targets tagged CenH3 to centromeres in Drosophila cells. Two different affinity procedures led to purification of the same complex, which consists of CenH3, histone H4, and a single protein chaperone, RbAp48, a highly abundant component of various chromatin assembly, remodeling, and modification complexes. The corresponding CenH3 assembly complex reconstituted in vitro is sufficient for chromatin assembly activity, without requiring additional components. The simple CenH3 assembly complex is in contrast to the multisubunit complexes previously described for H3 and H3.3, suggesting that centromeres are maintained by a passive mechanism that involves exclusion of the complexes that deposit canonical H3s during replication and transcription.
每个真核染色体都需要一个着丝粒，以便附着于纺锤体微管上进行染色体分离。尽管着丝粒DNA序列在不同物种间差异很大，但着丝粒普遍由一种着丝粒组蛋白变体——着丝粒组蛋白3（CenH3）标记，它在着丝粒核小体中取代了经典组蛋白H3。传统染色质部分由组蛋白伴侣复合物维持，这些复合物沉积S期受限的（H3）和组成型的（H3.3）组蛋白3形式。然而，将CenH3特异性沉积在着丝粒并在有丝分裂和减数分裂过程中忠实地维持其染色体位置的机制尚不清楚。为了解决这个问题，我们通过生化方法纯化了一种可溶性组装复合物，该复合物可将标记的CenH3靶向果蝇细胞中的着丝粒。两种不同的亲和方法导致纯化出相同的复合物，它由CenH3、组蛋白H4和一种单一的蛋白伴侣RbAp48组成，RbAp48是各种染色质组装、重塑和修饰复合物中高度丰富的成分。在体外重构的相应CenH3组装复合物足以进行染色质组装活动，无需其他成分。简单的CenH3组装复合物与先前描述的H3和H3.3的多亚基复合物形成对比，这表明着丝粒由一种被动机制维持，该机制涉及在复制和转录过程中排除沉积经典H3的复合物。
