FMR1基因調控:細胞內DNA足印之研究

FMR1 is the gene responsible for the
fragile X syndrome. The cloning of FMR1
make revolutionary changes in the diagnosis
of fragile X syndrome. Both the diagnosis of
patients and carriers, and even prenatal
diagnosis, could be easily and confidently
done by the analysis of CGG repeat and gene
methylation. However, position cloning did
not help the understanding of the function of
FMR1. Till now, the function of FMR1 is
unknown, and there is no effective treatment
for the fragile X syndrome.
We have studied the structure of FMR1
promoter, and proved its methylation
sensitivity (Hwu et al. BBRC 1993; 193:324-
9). We also showed by genetic (study of
mutants) and biochemical (study with
recombinant transcription factor) evidences
that, FMR1 promoter could be activated by
cAMP and CREB (Hwu et al. DNA & Cell
Biology).
In this study, we want to use in vivo
DNA footprinting to study the interactions
between FMR1 promoter (including the
methylation sensitive element, MSE) and
nuclear proteins. These interactions will help
the understanding of the regulation and
function of FMR1 protein. Beside, we will
develop an technique called RNase
protection assay (RPA). RPA is a sensitive,
stable and reliable method to quantitate RNA.
It will be used to detect changes of FMR1
expression in cells under various conditions,
to prove the presence of FMR1 regulation.
2
Unfortunately, we met a great difficulty
in the in vivo DNA footprinting assay. The
FMR1 promoter is extremely GC rich, and it
also contains the CGG repeat. This made
primer extension, the critical step in
footprinting assay, very inefficiency. RPA
assay could not detect any changes in the
cellular FMR1 gene, since the regulation of
FMR1 is very cell specific, and even
subcellular specific. Now we have shifted our
method into animal model study which goes
smoothly recently.