The aim of this proposal is to characterize the molecular function of the human structural maintenance of chromosome (SMC) protein family in chromosome dynamics. SMC family proteins, which possess predicted myosin-like motor protein structures, are essential in yeast and are found in diverse organisms. Various homologs play critical roles in several fundamental nuclear events that involve structural changes of chromosomal DNA, including chromosome condensation, cohesion, recombination, repair and global repression of transcription. Deregulation of any of these processes could play a role in oncogenesis and developmental abnormalities. Although fundamental to the cell, the molecular mechanisms underlying the structural changes of chromosomes and the machinery that orchestrates them remain elusive. We chose to address these questions in a human system, using the human SMC proteins as molecular tags. We identified and cloned human SMC family proteins and found that they form two distinct heterodimeric complexes in the cell, both of which are involved in mitotic chromosome organization. We obtained evidence supporting the hypotheses that differential interactions with other cellular proteins mediate the functional specificities and cell cycle-dependent effects of the SMC proteins, and that the SMC complexes may have additional roles during interphase. In order to test these hypotheses, we propose to carry out comparative studies of the two SMC complexes using four approaches. First, the precise steps at which the two complexes are required for mitotic chromosome organization will be assessed by blocking the functions of the endogenous proteins using antibody microinjection assays and by the expression of dominant negative mutants in the cell. Second, the interactions of SMC complexes with other cellular factors at different cell cycle stages will be characterized, and the associated proteins will be identified and cloned. The posttranslational modification of the SMC and associated proteins will be compared at different cell cycle stages to address the mechanism of cell cycle regulation of their functions. Third, the domains of these proteins required for specific interactions and cellular localization will be determined in vitro and in vivo using various mutants. Fourth, the specific localization of the SMC complexes in the nucleus will be analyzed by chromatin-crosslinking and immunoprecipitation (CHIP) analysis and by in situ colocalization with the known chromosome-binding factors or genomic sequences. These studies will address the molecular functions of SMC proteins in human cells, and provide further understanding of the mechanism of chromosome structure regulation and human disease associated with chromosome abnormalities.
Showing the most recent 10 out of 13 publications
