William C. Earnshaw - US grants

The kinetochore is the specialized chromosomal region which forms the attachment point for the microtubules of the mitotic spindle. Little is known about either the composition or mode of action of this structure. This proposal presents a plan for the identification, structural mapping, and functional characterization of kinetochore proteins. These experiments will exploit autoimmune sera from patients with the CREST syndrome of scleroderma. These sera contain antibodies which bind specifically to the centromere region of human chromosomes. The possible significance of these antibodies in the pathogenesis of the CREST syndrome is currently unknown. In immunoblots they react with three minor chromosomal proteins CENP-A (Mr = 17kd), CENP-B (80kd) and CENP-C (140kd), as well as with several other antigens. We have used affinity-purified antibodies to show that the CENP species are found at the centromere. In an effort to obtain biochemical amounts of these extremely minor proteins, we will use the lambda GT11 expression vector system to clone cDNAs encoding the CENP antigens. This will enable us to purify sufficient amounts of CENP: B-galactosidase fusion proteins from bacterial lysogens for injection into rabbits. In this way we will obtain high titer antibodies of known specificity directed against individual CENP species. These antibodies will be used to probe isolated taxol-stabilized mitotic apparatus in experiments designed to show which, if any, of the CENP species interact closely with spindle microtubules. They will also be used in conjunction with an in vitro microtubule assembly assay in order to probe the role of individual CENP species in kinetochore function. The rabbit antibodies will also be used for immunoelectron microscopy to map the distribution of individual CENP proteins in the trilaminar kinetochore structure. In addition, the cDNA clones will be used to determine the pattern of synthesis of the CENP proteins throughout the cell cycle by Northern blotting, the organization of the CENP genes by Southern blotting, and finally, the sequence of the CENP proteins. These data will demonstrate whether the CENP species are the products of a multi-gene family, as suggested by previous immunoblotting experiments. The CENP species are not the only chromosomal proteins recognized by the CREST patient sera. The above experimental design will also be applied to the study of other antigens whose chromosomal location is currently unknown.

The purpose of this project is to understand the role that non-histone proteins play in chromosome structure and condensation. Over the past eight years our efforts have focussed on protein components of the chromosome scaffold fraction. While the precise biological meaning of the scaffold fraction is a subject of ongoing debate, our results suggest that it may contain a number of proteins that have bona fide roles in chromosome structure. In previous experiments funded by this project, we identified the major protein of the scaffold fraction as DNA topoisomerase II. During the upcoming grant period, we will follow two major avenues of research. In the firsT, we will characterize three new scaffold proteins: INCENPs, ScII and ScIII. A new assay will be used to determine if they are tightly associated with chromosomal structures in vivo. Unlike topo II, these are not previously known proteins, and we will thus characterize them in detail at the molecular level by cloning and sequencing their cDNAs. We will also map their distribution in chromosomes by confocal and electron microscopy. Preliminary observations suggest that the binding affinities of the INCENPs change at the metaphase:anaphase transition, and we will therefore study their modification status and stability across the cell cycle. Since the INCENPs, ScII and ScIIl are new proteins, we have no clues as to their function in the chromosome. We will therefore undertake to determine this in vivo using antibody microinjection and transient transfection with partial cDNAs encoding putative isolated protein domains. In addition to these functional studies, we will use our antibody libraries to attempt to identify homologues of these proteins in Drosophila for future collaborative genetic analysis. In vitro studies that complement the in vivo studies are also proposed. We have succeeded in developing a new extract system from somatic cells that causes interphase nuclei to undergo chromosome condensation in vitro. The unique aspect of this system is that unlike frog egg extracts that have similar activity, somatic cell extracts lack large stockpiles of structural proteins. They can thus be used to assay the role of structural proteins in chromosome condensation. Where possible, we will take advantage of our observation that certain of the major chromosomal non-histone proteins such as topoisomerase II are selectively lost during chicken erythropoiesis. We will use late erythroblast nuclei as substrates in mitotic extracts from which specific non-histone proteins have been depleted by immunoprecipitation. This provides a powerful assay for analysis of the role of individual non-histones in chromosome condensation. The molecular and structural characterization of these proteins coupled with our powerful new in vitro assay system should enable us to begin to assign functions to some members of this largely uncharacterized group of chromosomal proteins.

The centromere regulates the movements of the chromosomes in mitosis. Work from this lab, among others, has used antibody probes to show that the centromere is composed of several subdomains in addition to the one prominent subdomain recognized by electron microscopy: the kinetochore. The kinetochore has recently become the focus of wide interest since it may contain the mechanochemical motor responsible for anaphase movement of the sister chromatids to the spindle poles. The ultimate goal of our studies is to construct a structural and functional map of the outer domains of the human centromere. Using human autoantibodies, we identified a family of centromere proteins, CENP-A (17 kDa), CENP-B (80 kDa), and CENP-C (140 kDa) in previous work supported by this grant. We cloned, sequenced, and began an in depth characterization of the molecular structure and biological role of CENP-B. We also obtained preliminary clones of CENP-C. In the experiments proposed for the upcoming grant period we will continue our analysis of CENP-B function, we will carry out a detailed molecular and functional analysis of CENP-C, and we will continue to screen for new components of the centromere and kinetochore. CENP Antigens, General. We will continue to characterize the disruption of chromosome movements in mitosis caused by injection of purified autoantibodies into cultured cells. CENP-B. We will examine the association between CENP-B with alpha-satellite DNA by in vitro binding experiments using cloned proteins and crude chromosomal extracts and by immunocytological investigation of abnormal chromosomes with translocated alpha-satellite domains. We will attempt to determine the role of CENP-B in vivo by creating dominant disruptions of CENP-B function, using transfection and/or injection of cultured cells with plasmid constructs expressing different CENP-B subdomains. Such constructs will also be used to identify those portions of CENP-B required for correct targeting and assembly into the centromere. We will examine the interaction of CENP-B with other chromosomal proteins by chemical crosslinking and by affinity chromatography using various CENP-B subdomains expressed in bacteria. We will characterize the patterns of transcription and posttranslational modification of CENP-B across the cell cycle. CENP-C. We will isolate full-length cDNA clones for CENP-C, obtain the DNA sequence, and use an immunological approach to demonstrate that these clones encode bona fide CENP-C. We will map the distribution of CENP-C in the centromere by immunoelectron microscopy.We will perform analyses similar to those listed above in order to determine the biological role of CENP-C in centromere structure and function. New Centromere Components. We will use a newly designed shotgun cloning method to identify new components of the centromere and kinetochore. Either mitotic chromosome scaffolds or an isolated human minichromosome will be used as antigen. Newly identified antigens will be used to elucidate the network of protein-protein interactions within the centromere. Antigens of suitable interest will be characterized as above for CENPs B and C.