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
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.