Download Genes can be switched on and off by the protein CTCF

Genes can be switched on and off by the protein CTCF
Kristina Jonas
The complex development of mammals requires that the majority of all genes can be switched off at
any given time. Cell and tissue-specific inactivation is a characteristic feature of many developmentally
regulated genes. Inactivation and reactivation of genes is often associated with chemical modifications
of the DNA, or with factors that can bind to the DNA. The protein CTCF is one of these factors; it can
bind to DNA and controls the expression of certain genes. In cells where CTCF is mutated and
therefore can't fulfil its function, genes are expressed at the wrong time. That can lead to cancer.
I examined the regulator functions of CTCF. How strongly CTCF can bind to DNA depends on
how the DNA is modified. Therefore it is important to investigate the conditions under which CTCF can
function properly.
I injected DNA into mouse embryos, using a special reporting system to see if CTCF was active
or not. Dependent upon whether the DNA contained a binding site for CTCF so that CTCF could bind
and function, or contained no such binding site, different signals were expected. However, I could not
observe any signals. Therefor the set-up of this experiment has to be improved.
I also wanted to find out something about the properties of the DNA where CTCF could bind,
and about at what stages of development CTCF bound to these target sites. Beside CTCF another
protein with similar functions was examined, called BORIS. The fragments of DNA where CTCF was
bound were identified using a method called PCR (polymerase chain reaction). The results showed
that CTCF and BORIS could bind to the same target, and suggested they did so at different stages of
development. This might indicate that CTCF and BORIS are involved in the organisation of the change
of chemical modifications during development.
Another interesting property of CTCF is the way its gene is inherited. After many repetitions of
cross experiments, in which two mice heterozygous for the mutated CTCF (i.e. carrying one normal
and one mutated CTCF gene) were mated, an individual that failed to express CTCF was never
observed. These results were unexpected since according to Mendel’s law 25% of the descendants
should lack functional CTCF. This special pattern of inheritance is another sign of the important
functions of CTCF, making sure that all descendants can express the CTCF protein and no individuals
lack this crucial protein. I assume that when the chromosomes are segregated after fertilization
unusual events take place. I wanted to confirm earlier results and find an explanation by visualising the
organisation of chromosomes and the events in meiosis, using a method that made it possible to label
certain parts of the chromosome, for example the CTCF gene, with a fluorescent dye, so that the gene
could be seen on the chromosome in a microscope. It is important to know where the gene is located
to be able to interpret the previous cross experiments properly. My first results were not reliable since
unspecific binding led to a lot of background fluorescence. The conditions for the experiment have to
be optimized in the future.
Degree project in Biology, 10 p, spring 2002
Department of Developmental Biology, Uppsala University
Supervisor: Joanne Whitehead and Rolf Ohlsson