Determining the Molecular Architecture of Centromeric Chromatin

Cells are constantly dividing in our organism. From a single cell, formed upon fusion of an egg and a sperm, we develop into a complex organism through numerous cell divisions. Once organism is fully developed, its cells continue dividing to enable growth and to regenerate tissues. It is estimated that nearly two trillion cells divide in our organism every day. Thus, it is extremely important for an organism to keep cell divisions accurate ensuring that the genetic material, that defines those very cells, does not get lost in the passage from mother to daughter cells. Chromosomes, the carriers of genetic information, once duplicated in the mother cell, must be equally distributed to daughter cells. A specialized region on the chromosome, called centromere, plays a crucial role in this process. The absence of a functional centromere results in cells with an abnormal chromosome number. This is either detrimental to the cell faith, or is associated with cancer and congenital diseases, such as Downs syndrome. Although the protein and DNA composition of the centromere is known, the molecular organisation within this important chromosome locus is still ambiguous. We are employing state-of-art biochemical and biophysical approaches in combination with cell biology, to obtain atomic resolution view of a functional centromere. The knowledge from our basic research will help in engineering centromeres for gene therapies and it will contribute to a better understanding of diseases connected to alteration in chromosome numbers.

The equal partitioning of chromosomes during cell division depends on a region of the chromosome called the centromere. The centromere serves as the foundation for a large mitotic protein complex, the kinetochore. Through the kinetochore, chromosomes attach to microtubules that then pull them into new daughter cells during cell division. The absence of a functional centromere results in cells with an abnormal chromosome number (aneuploidy) that is associated with cancer and congenital diseases, such as Downs syndrome. Our research is aimed at answering how the centromeric chromatin, which is a constitutive part of the chromosome, differs from the rest of the chromatin. In higher eukaryotes the centromere is defined epigenetically by the presence of histone H3 variant, CENP-A. The CENP-A containing nucleosome(s) are able to recruit the Constitutive Centromere Associated Network (CCAN), a complex consisting of 16 different proteins required to obtain a functional centromere. The main aim of the present proposal is to elucidate the biochemical and structural architecture of the complete CENP-A-CCAN complex, including all CCAN components and underlining DNA sequences. We will assemble complex with in vitro purified protein components and human centromeric DNA. We will use biophysical (cryoEM, mass spectrometry), cell-based (cell imaging and yeast genetics) and computational (molecular dynamics) approach to deconvolve architecture of centromeric chromatin. Our preliminary data revealed an exciting new discovery of a specialised chromatin structure directed by centromere-specific proteins. Together, our work will provide a high-resolution structure of the centromeric chromatin, a megadalton protein-DNA complex that is at the heart of the propagation of life. We expect the knowledge from our basic research to help in engineering centromeres for gene therapies and to contribute to a better understanding of diseases connected to cell aneuploidy.