Global chromatin interactions in post-mitotic neurons.

Cell division poses one of the central questions of biology, namely, how does a single cell manage to replicate and separate its entire genome ensuring that both new cells receive an exact copy of each chromosome. To do this, a specialized group of proteins, known as the cohesins, play a central role ensuring that newly replicated DNA molecules do not prematurely separate, resulting in abnormal chromosome distribution and ultimately cell death. However, recent studies have shown that cohesin proteins do more then simply hold DNA strands together. Evidence from human diseases and animal models have suggested cohesins also control gene expression in the cell. My research will focus on how cohesin proteins, with their unique ability to hold DNA molecules together, influence gene expression during cellular development and if this can be explained by their role in establishing the correct 3-dimensional organization of genes within the cell nucleus.

The evolutionarily conserved cohesin complex has fundamental roles in chromosome biology. These include sister chromatid cohesion, post-replicative DNA repair and the control of transcription. We propose to address fundamental questions concerning the underlying mechanisms used by cohesin proteins to mediate their effects on gene expression.

This project will focus on understanding the general principles governing cohesin-based chromatin organization during interphase. To do this, we will use Chromatin Interaction Analysis using Paired-End Tag sequencing. ChIA-PET is a novel technique which combines 3C and next-generation sequencing technologies to discover all long-range chromatin interactions in the nucleus. It is currently the most effective and powerful method for measuring the interaction of functional elements in a genome-wide and un-biased approach.

We will use protocols for the differentiation of mouse ES cells into self-renewing but lineage restricted neural stem (NS) cells. These cells can be further differentiated into an unlimited resource of post-mitotic astrocytes and can be used to investigate the effects of cohesin depletion on chromatin organization under conditions where cohesins are not essential for cell division.

Insight into how cohesin proteins work will lead to a better understanding of many aspects of chromosome biology as well as a deeper knowledge of developmental genetic diseases such as Roberts and Cornelia de Lange syndromes (caused by mutations in cohesin subunits) and cancer, where gene deregulation plays an important role.