Study On The Roles Of Condensin Complex Involved In Chromosome Condensation In HeLa Cells

Eukaryotic cells transfer their genetic information to the next generation via mitosis. As carrier of gene information, the faithful replication and separation of DNA molecules are vital for the transfer of genetic information. And DNA molecules binds to histones and non-histone proteins to form chromatin to engage in this process. During mitosis, sister chromatids were highly compressed into rod-shaped chromosomes to faciliate separation. Both histones and non-histone proteins play important role during chromatincondenation. Histone octamer composed of two sets of H2A, H2B, H3 and H4 are surrounded by DNA molecule to form nucleosomes. With the help of non-histone proteins, chromatin forms chromsomes during mitosis. As the major non-histone proteins, condensin and topoisomeraseⅡαplay fundamental roles in the cell cycle. Condensin is the main condensining protein on chromosome, a series of series of research on condensin function during chromosome condensation have been done in yeast, fruit fly, xenopus and other species. Howerer, the function of condensin in higher mammalian cells is rarely studied. The effect of overexpression or knockdown of condenin on chromosome separation during mitosis is scarely reported. And topoisomeraseⅡα, participating a series of events duing cell cycle, is also found to engage in chromosome condenation, and prohibiting its function pauses the cell cycle. But the role it plays in chromosome condensation is unclear,and the interaction of topoisomeraseⅡwith other non-histone proteins, such as condensin, is under hot debate.In this paper, a series of experiments has been conducted to unveil the nature of condensin complex in chromosome formation. Overexpression and knockdown of condensin were conducted to check the role of condensin in chromosome condenation. In vitro reconstruction of HeLa nuclei in Xenopus egg extract were done to further investigate the impact of condensin in mammalian chromsome condenation. Co-immunoprecipitation assay were carry out to study the interaction of condensin with topoisomeraseⅡα. Results show that over expression of the C-terminal domain of SMC subunits disturb chromosome condensation, leading to swelled chromosomes, while knockdown of SMCs severely disturb the mitotic chromosome formation, leading to chromatin bridges and multiple nuclei. And in vitro reconstruction of HeLa nuclei show that condensin is vital for chromosome condensation, and rod-shaped chromosomes fail to form when condensin is depleted from the extract and the chromatin remains the same in the extract. Meanwhile, condensin is found to intimately related with topoisomeraseⅡαat certain stages of cell cycle, co-localization and co-immnoprecipitation of the two proteins are found in our study.The results above indicate that disturbance in condensin function or expression affect normal chromosome condensation and influence the mitotic progression in mammalian cells, and condensin is necessary for chromosome condensation. Meanwhile, the interaction of condensin with topoisomeraseⅡαshow that condensin is a active protein, participating in chromosome condensation together with other non-histone proteins.