I am working on a research proposal for the study of telomere length
regulation. Since telomerase activity is constant in germ cells, stem
cells, and in a perhaps abnormal way in certain cancer cells, the
question arises as to what prevents telomeres from extending indefinitely.
Telomerase activity in the above mentioned cell types appears to be
independent of cell cycle position, with telomerase activity throughout S
and M phase. Therefore, cell cycle regulatory proteins do not appear to
affect telomerase transcription levels...so how are telomeres maintained
at a rather stable length? (All within a heterogenous but rather narrow
range of several kb)
My thoughts:
1) The level of telomerase transcription is under two layers of control:
self regulated by feedback loop and developmentally regulated - for
permanent silencing upon differentiation of a stem cell into a somatic
tissue cell. If telomerase levels are low in general in, say, stem
cells, then kinetically the enzyme may be constantly operating in a
balance with cell division so that shortening due to division and normal
degredation is balanced by extension. Thus telomerase is limiting.
If this is the case, then the process is saturable so that if a lot of
telomeric seed sequence is added to such a cell, then all telomeres
should shorten and ultimately stabilize at a shorter length.
2) Telomere binding factors (proteins and ribonucleoproteins) act to
regulate telomere length. I see that this could take place in two
ways: Telomerase is a multisubunit enzyme. If any of its subunits are
limiting, then the overall activity of the enzyme is limited by subunit
levels as in 1 above. Alternatively, if such telomere binding species
act to protect telomeres from degradation, and they are produced at a
constant level so that an equilibrium is attained, then extension of
telomere beyond a point at which there is enough binding factors to bind
and protect the sequence would be automatically degraded back to
protection levels. This would be a dynamic process in which telomerase
constantly adds sequence but it get degraded back to set point. This is
closely related to 1 above.
3) All telomeric sequence repeats from all species examined thus far
appear capable of forming very stable G-quartets. Numerous studies with
different sequences and various SHORT lengths (containing 1 to 4 tandem
repeats - vs thousands of tandem repeats in actual telomeres) confirm
that under physiological conditions, telomeric repeats can form stable
G-quartet structures in vitro. In 1991, Alan M. Zahler, James R.
Williamson, Thomas Cech, and David Prescott found that G-quartet DNA
structures can inhibit telomerase activity - that is, telomerase cannot
use such quartets as primer for extension.
If telomeric sequences in vivo do form G-quartets, then it must be
regulated somehow or as soon as 4 tandem repeats were produced, further
extension would be inhibited since a G-quartet could form. Obviously,
such early inhibition doesn't occur.
Thus comes my research proposal. I seek to confirm that telomeres, in
vivo, do in fact contain G-quartets (the regulation of their formation is
not the subject of _this_ particular proposal). To confirm this, I intend
to remove and seperate telomeric DNA from chromosomes in Oxytricha (since
they contain many thousands of telomeres), treat with Proteinase K and
RNAse, and check the CD on the DNA to check for G-quartet content.
Another part of the study would be in S. cerevisiae in which I wish to
knockout the TLC1 gene, which codes for the yeast ribonucleioprotein
telomerase template component of the holoenzyme, with a version
containing alterations to the template such that G-quartets could not
form. If G-quartets are important for telomere length regulation,
then the inability to produce G-quartets should lead to increased length
of telomeres in so treated yeast.
Alternatively, I could use a MoMLV vector to introduce a similarly
altered telomerase template gene into a mouse cell line, overexpress it
with a CMV promoter to favor mutant holoenzyme formation, and check for
telomere length alterations vs G-quartet content (alternatively, I could
go for a knockout). This latter (mouse) is more complicated but has the
advantage to being more applicable to mammalian telomere systems.
Any comments or suggestions? Have I missed something obvious that might
go easier or quicker?
Patrick
