Studying and manipulating chromatin motion in mammalian cells

Abstract

Histone modifications such as methylation and acetylation are known to be key determinants in the regulation of gene expression, but little is known about how higher order chromatin structures, and their spatial organisation in the nucleus, can control gene expression. This remains a key question in addressing the role of spatial organisation in genomic function.If changes in nuclear position have a role in gene expression, chromatin within the cell must be able to move distances that would accommodate this. In the first part of my PhD I investigated the range of chromatin motion in living human cells. E.coli Lac operator arrays inserted into the human genome and visualised using Lac repressor protein fused to GFP, are able to move up to 2-3 pm over the period of two hours, distances greater than previously reported and similar to motion observed in yeast. I have also determined whether the position of a locus is conserved from one cell cycle to the next by following cells through mitosis. From this analysis it was concluded that although some aspects of positioning were conserved, loci position was established anew each cell cycle.Although I have shown that chromatin mobility is quite constrained within the nucleus, proteins associated with chromatin have been shown to be highly mobile. I have investigated the effect of different factors that might affect the mobility of linker histones using fluorescence recovery after photobleaching. I have shown that while Su(Var)3-9, responsible for tri-methylation of Lysine 9 on histone 3, and MeCP2, a DNA methylation binding protein, have no effect on linker histone mobility, the methylation of DNA does. In the absence of DNA methylation, linker histones are more tightly bound to the chromatin fibre.In humans it is well established that chromosomes have a gene-density related radial organisation within the cell nucleus. I have mapped the radial position of mouse chromosomes in ES cells to determine if a similar pattern of organisation exists. My results suggest there may be a loose correlation between chromosome size and position within the mouse genome, but not gene density. Furthermore differentiation iii of mouse ES cells, induced changes in the position of some chromosomes, suggesting that gene expression may have a role in chromosome position.Although correlations in nuclear position and expression have been seen in many model organisms, only in budding yeast has there been direct experimental confirmation that position can control gene expression. To determine directly if nuclear position can regulate gene expression in the mouse I aimed to artificially tether a gene to the edge of the mouse nucleus. Arrays of Lac operator sequences were inserted into or near genes. To tether genes to the nuclear periphery, Lac repressor was fused to the integral membrane proteins emerin or LAP2ß. I have shown that these fusion proteins can transiently anchor transfected Lac-operator containing plasmids to the nuclear periphery of mouse cells and that this silences gene expression from these plasmids. Anchoring of endogenous mouse genes was also investigated.