Final Report Summary - CHROMOSOME PERIPHERY (Assembly and function of the chromosome periphery during mitosis)
Executive Summary:
While chromosomes fill the nuclear space as decondensed chromosome territories during interphase, they are dramatically re-structured into compact rod-shaped rigid bodies during mitosis. The aim of my work is to elucidate molecular mechanisms underlying this cell cycle-dependent chromosome re-organization. I focus on the chromosome axis domain and the poorly characterized chromosomal domain defined as “chromosome periphery”. As a first aim I planned to establish a detailed map of the proteins that target to the chromosome periphery and axis in human cells, applying both molecular biology (Chromatin Immunoprecipitation followed by sequencing, ChIP-seq) and imaging approaches.
I performed ChIP-seq experiments of topoisomerase II alpha, a chromosome axis protein, using mitotic and unsynchronized HeLa cells. I identified about 4000 binding sites distributed over all chromosomes. The data will be validated using a tagged version of topo II and by single-cell assays (e. g. Fluorescence In Situ Hybridization, FISH). Furthermore ChIP-seq experiments will be extended to chromosome periphery proteins.
I have imaged eight chromosome periphery proteins in mitotic cells using three-dimensional confocal live-cell microscopy. This yielded a spatio-temporal map of chromosome periphery assembly, which I am planning to extend by further chromosome periphery proteins.
To elucidate the functional relevance of chromosome periphery components in mitotic chromosome morphogenesis, I aim to systematically screen candidate regulators. Solid-state transfection screening plates have been generated in collaboration with Jan Ellenberg (EMBL, Heidelberg) and first pilot experiments indicate feasibility of screening.
Altogether these approaches will yield temporal, spatial and functional data that will give insight into how the chromosome axis and periphery domains contribute to mitotic chromosome formation.