The role of non-histone proteins in chromosome structure and function during mitosis

I propose to study how a set of key proteins (condensins I/II, cohesin, Kif4A, topo IIα) mold the chromatin fibre into its characteristic rod-shaped helix of loops in mitosis.

Using a transformative system allowing us to obtain cultures entering mitosis with near-perfect synchrony we will adopt an interdisciplinary approach involving genomics (Hi-C, Capture-C, cut&run), proteomics, crosslinking, microscopy and molecular modelling to resolve changes in chromatin fibre and chromosomal protein organisation on a minute-by-minute basis during mitotic entry.

Using non-toxic synchrony methods plus acute (and reversible) protein depletion with auxin degrons, we will determine when, where, how and with whom these proteins act to shape mitotic chromosomes.

Access to the single copy chicken Z chromosome with its unique-sequence centromere allows us unprecedented mapping of the chromatin folding and protein distribution at a vertebrate centromere during this process.

Following our discovery that RNAs mediate assembly of the mitotic chromosome periphery compartment (MCPC) downstream of Ki-67, we will initiate a new research direction identifying the proteins and RNAs involved, and determining their functional significance for chromosome segregation.

Lastly, we will optimise methods for isolating human artificial chromosomes (HACs), avoiding unwanted DNA rearrangements that endanger future synthetic genome efforts.