Transcriptional regulation of centrosomal genes involved in centrosome duplication

The centrosome is the main microtubule organizing center of the cell. Important for microtubule outgrowth in interphase cells, the centrosome organizes the cellular architecture and positioning of cell organelles. In mitotic cells, the centrosome plays a central role in organizing the mitotic spindle to correctly separate the chromosomes. Aberrant centrosomes are often associated with aneuploidy and tumorigenesis. In order to achieve correct chromosome segregation and cell proliferation, duplication of the centrosome and therefore of all the centrosomal components (known as the centrosome cycle) have to be tightly linked to the cell cycle. However, nothing is known about transcriptional control of intrinsic centrosomal components and whether its disturbance is asscociated with tumorigenesis. The purpose of our suggested research project is to fill in this gap.

Objective of the project is the analysis of the transcriptional regulation of genes encoding crucial and intrinsic components of the mammalian centrosome and their putative disturbance in tumor cells. These results will greatly enhance our understanding of the linkage between cell cycle and centrosome cycle which is disturbed in tumour cells. These results moreover will eventually provide us with the tools necessary for the development of anti cancer drugs and the interruption of cancer progression.

By quantitative real time RT-PCR we will first analyse whether endogenous genes encoding centrosomal proteins show cell cycle dependent transcriptionally regulation. For these experiments synchronized cells or cells arrested at specific phases of the cell cycle will be used. We then will proceed with mapping of the promotor region of Odf2 and of centrin-2.. The promotor region will be ligated to firefly luciferase as reporter and expression of the reporter measured by luminescence. By generating deletion constructs which spans the promotor regions, a fine mapping of putative transcription factor binding sites could be achieved. In silico analyses of putative binding sites will be helpful for the identification of conserved binding sites in the promotor regions of other genes encoding centrosomal proteins. Binding of transcription factors to identified putative binding sites will then be verified by electrophoretic mobility shift analyses and eventually by the isolation of those factors which then can be investigated further. Using tumour cells and tumor tissues we will analyse whether transcriptional regulation of genes encoding centrosomal proteins and the location of their proteins are disturbed.

The centrosome (green) is located close to the nucleus (blue).

Prof. Dr. Sigrid Hoyer-Fender