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The
focus of our research is to understand the molecular basis
of telomere function in human cells. Telomeres, the ends of
chromosomes, consist of long tandem arrays of G-rich repeats
bound to specific proteins.
Telomeres act as protective caps and serve as templates for
replication by the enzyme telomerase. Telomere integrity
is
essential for chromosome stability and maintenance, and telomeres
play a key role in replicative senescence and cancer. To
elucidate
the molecular mechanisms that control telomere length and
chromosome end protection we are investigating the protein
components of the telomeric complex. Telomeres are coated
by sequence specific DNA binding proteins TRF1 and TRF2 and
their associated proteins, TIN2, TPP1, POT1, Tankyrase 1,
and Rap1. We
would like to understand how these proteins regulate access
of telomeres to telomerase and how they protect chromosome
ends from cellular DNA repair systems.
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| Tankyrase
1, Tankyrase 2 and TRF1 |
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Tankyrase 1 is a member of the
poly(ADP-ribose) polymerase (PARP) family of enzymes. Tankyrase1
ADP-ribosylates TRF1 inhibiting its ability to bind to
telomeric DNA.
We have shown that tankyrase 1 is
a positive regulator of telomere length; overexpression
of tankyrase 1 in human tumor cells releases TRF1 from
telomeres allowing access to telomerase and telomere elongation.
We found that after TRF1 is released from telomeric DNA
it becomes accessible to the ubiquitination machinery
and
is degraded by ubiquitin–mediated proteolysis,
thereby preventing its rapid reassociation with telomeres.
Our
findings suggest a novel mechanism of sequential post-translational
modification of TRF1 (ADP-ribosylation and ubiquitination)
for regulating access of telomerase to telomeres.
Tankyrase 2,
a closely related tankyrase 1 human homolog, shares
a number of properties with tankyrase 1, including its
ability to ADP-ribosylate TRF1 and induce telomere elongation.Tankyrase
1 and tankyrase 2 heterodimerize suggesting overlapping
functions.
We have generated tankyrase 2
knockout mice and
will use these mice and tankyrase 2 knockout cell lines
to elucidate the function of tankyrase 2 and to distinguish
the roles of these related proteins at telomeres.
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| Tankyrase
1 and Telomere Cohesion |
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Unexpectedly, tankyrase 1
siRNA knockdown cells arrest in mitosis due to innapropriate
sister telomere associations. The PARP actvity of Tankyrase
1 is required for normal mitotic progression.
Inhibiting tankyrase 1 expression,
by siRNA, in human tumor cells lead to a mitotic arrest.
We found that cells deficient for tankyrase
1 enter mitosis normally; chromosomes align on the metaphase
plate, but
are unable to proceed to anaphase. Fluorescent
in situ hybridization using chromosome specific probes
revealed
that while sister chromatids are separated
at
their centromeres and along their arms, they remain
associated at their telomeres, likely though
proteinacious bridges. Only a catlytically active
tankyrase 1, but not
a PARP dead mutant,
is able to rescue
the
mitotic phenotype.
. We propose that telomeres
require a distinct
tankyrase 1-dependent mechanism for sister chromatid
resolution prior to anaphase. We are currently investigating
if other telomeric proteins are involved in resolution
or establishment of sister telomere associations,
and screening for other interacting proteins which
may also be involved.
We
are also investigating other proteins in the TRF1 complex
that influence
telomere length regulation, including the TRF1/TRF2 interacting
protein TIN2, and the recently identified telomere protein
TPP1.
Recently we have identified a new telomere protein, TPP1
(TINT1, PIP1, PTOP1). We have shown that
TRF1 and TRF2 are linked via TIN2, a previously identified
TRF1-interacting
protein,
and
its
novel
binding
partner TPP1. TPP1 localizes to telomeres via TIN2,
where it functions as a negative regulator of telomerase-mediated
telomere elongation. TIN2 associates with TPP1, and TRF1
or TRF2 throughout the cell cycle, revealing a partially
redundant unit in telomeric chromatin that may provide flexibility
in telomere length control. Our findings suggest a dynamic
cross talk between TRF1 and TRF2 and provide a molecular
mechanism for telomere length
homeostasis by TRF2 in the absence of TRF1. We are further
exploring the function of TPP1 by inhibiting its expression
using siRNA, as well as screening for both TIN2 and TPP1
interacting factors.
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