Abstract
In higher eukaryotic cells, DNA molecules are present as chromatin fibers, complexes of DNA with various types of proteins; chromatin fibers are highly condensed in metaphase chromosomes during mitosis. Although the formation of the metaphase chromosome structure is essential for the equal segregation of replicated chromosomal DNA into the daughter cells, the mechanism involved in the organization of metaphase chromosomes is poorly understood. To identify proteins involved in the formation and/or maintenance of metaphase chromosomes, we examined proteins that dissociated from isolated human metaphase chromosomes by 0.4 m NaCl treatment; this treatment led to significant chromosome decondensation, but the structure retained the core histones. One of the proteins identified, HP1-BP74 (heterochromatin protein 1-binding protein 74), composed of 553 amino acid residues, was further characterized. HP1-BP74 middle region (BP74Md), composed of 178 amino acid residues (Lys(97)-Lys(274)), formed a chromatosome-like structure with reconstituted mononucleosomes and protected the linker DNA from micrococcal nuclease digestion by approximately 25 bp. The solution structure determined by NMR revealed that the globular domain (Met(153)-Thr(237)) located within BP74Md possesses a structure similar to that of the globular domain of linker histones, which underlies its nucleosome binding properties. Moreover, we confirmed that BP74Md and full-length HP1-BP74 directly binds to HP1 (heterochromatin protein 1) and identified the exact sites responsible for this interaction. Thus, we discovered that HP1-BP74 directly binds to HP1, and its middle region associates with linker DNA at the entry/exit site of nucleosomal DNA in vitro.
Highlights
The proteins involved in chromosome organization can be further divided into two different groups, according to how they contribute to the formation and/or maintenance of chromosome structure, namely proteins responsible for organizing chromosome structure and proteins that play roles in the regulation of chromatin structure
We demonstrate that a globular domain (BP74GD) located in the middle region of HP1-BP74 (BP74Md) has a linker histone-like structure and that BP74 middle region (BP74Md) binds to nucleosomes and protects extra DNA from micrococcal nuclease (MNase) digestion; BP74Md interacts with HP1 in vitro through a newly identified binding site located at its unstructured region
GFP-fused HP1-BP74 Is Co-localized with Chromatin throughout the Cell Cycle—As described above, we demonstrated that BP74Md possesses structural and some functional similarities with the GD of linker histones
Summary
The proteins involved in chromosome organization can be further divided into two different groups, according to how they contribute to the formation and/or maintenance of chromosome structure, namely proteins responsible for organizing chromosome structure and proteins that play roles in the regulation of chromatin structure. HP1-BP74 Middle Region Binds to Nucleosome and HP1 linker histones, were reported to be involved in the regulation of chromatin structure [15].
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