DeLANGE, Titia TELOMERES Mar 20 2013

introduction by Gilbert Chu:  tells bio of  Cynthia and Alex Tseng:
This is a memorial lecture in their honor;
Alex died in 2006 of GBM =glioblastoma multiforme.
Cynthia also died at age 56 of Lyme disease.

pronounced TICH euh  (not  tishuh);  from Amsterdam...
first woman to win the Heineken prize..

Bev Mitchell introduces Titia:

title:  How Telomeres solve the chromosome end-protection problem...

the chromosome end-protection problem

eukaryotic cells must have a mechanism to distinguish broken DNA ends
 from the natural ends of linear chromosomes...

if  DNA  damage signals pathways were activated,
you would get cell cycle arrest and cell death.

and  DSB (dbl stranded break)  repair pathways...

also the end replication probem:

discovered  by Cal Greider and Liz Blackburn...

end-replication problem: solved by telomerase...

Telomerase is active in the germ line and in cancer...

but in normal cells  get  telomere shortening... DNA damage, p53 activation, senescence/ apoptosis 

telomere shortening limits proliferative capacity...

once Rb/p53  is lost  then get  telomere crisis and  get  weird repairs,
brakage-fusion bridge cycles,
translocations,  get lots of inapprop repair at dysfunc telomeres
telomere damage signaling leading to tetraploidization...

may be  the way that  cancer  is  facilitateed...

Telomeropathies  (lung fibrosis,  dyskeratosis congenita  etc...  idiopathic lung fibrosis
  may be  due to a defic of  telomerase...

 it needs to engage    SHELTERIN  (the object of most of her career!)

the tags are bound by shelterin...

TRF1  and TRF2  are  specific binding proteins...

linked to  TIN2  TPP! and POT1 a/b   discov. in Tom CECH lab...

100 to 1000 copies of shelterin per telomere...

telomere is a highly condensed glob  (not a straight line)

also numerous shelterin assoc proteins...

Mammalian shelterin  is abundant, conserved in mice...

shelterin represses six pathways that threaten chromosome ends...

(v heavily defended  (the ends))

Sfeir and do Lange Science 2012

we use simple  Cre-LoxP  conditional gene deletion in MEFs...

create floxed alleles;  do retroviral Cre

what happens when you  modify or take out  shelterin components  (do expts using retroviral cre)

then u get non-homologous end-joining 

she shows  mouse chromosomes  with  GFP labeled  telomeres...

cells made a terrible mistake...  take DS breaks and ligated them
all together  (tha tis non-homolg  end-joining...huge error!!)

so,,   when we delete  TRF2  from cells in cells that lack the Ku70/8-  heterodimer
then fusion freq is 10 fold lower...

this IS  non-homologous end joining...

TF2  represses  ATM  kinase at telomeres...

MRN finds  DS = double stranded breaks...

ATM  phosphorylates factors near the breaks...

if cells do NOT contain ATM then response does not happen

so  TRF2  represses  classical end-joining  and  ATM  kinase signaling...
NHEJ = non-homolog  end-joining..

TRFs responsible for repression of classical NHEJ and ATM signaling...

but  ATM is not the only kinase that threatens ends...

there also is  ATR kinase... another big threat to our cells...

ATR kinase its own  kinase  Chk1 -P  (check1 phosphorylase)

Removal of POT1a activates ATR kinase signaling at telomeres...
so MUST have POT1a to prevent this...

normally  if you have  POT1a  there is no RPA  but if u get rid off
POT1a then  RPA  accumualtes...

POT1a  competes with RPA  but RPA is v abundant...

but POT1a is  tethered!... so it has a high ON rate cuz its tethered close by (by ss ? dna)

in 2010 we knew  Shelterin represses 4 pathways that threaten chromos ends...

her "first born" was TRF1"  has nothing to do with end protection...
but  TRF1 does protect semi-conserv  replic of  TTAGGG repeat DNA...

if u make a double KO  TRF1 AND TRF2  DKO  double KO mouse  then NO shelterin remains  and u get

telomerases fusing together  upon deletion of TRFs (classical NHEJ)

lengthy expt:

all that remians is DNA with nucleosomes
what remains is telomeric  DNA packaged in nucleosomes...

Sfeir and de Lange Science 2012...

the end-proteciton problem:  now SIX pathways threaten telomeres...

ATM kinase
ATR kinase
classical  NHEJ
HDR
alternativ e  NHEJ
and 5'  end resection...

Next... How does shelterin do its job?

now what have we learned recently abt the funciton of TRF2 ?

TRF2  blocks  ATM kinase signaling and it blocks  Ku70\80 to c-NHEJ.

do how does  simple  DS DNA binding protein  (TRF2) do it?

T-LOOP !!!!  causes folding over...

a model  Jack Griffith did this  1999...

Jack saw lariots... tail and a loop...

it appeared that  15 to 40%  of  telomeres with in t-loop folds... but no data to verify this...

Xiaowei Zhuang   breaks diffraction limit  (great  imaging technique)
 invented STORM imaging...

she compares  conventional  PNA FISH  to  STORM...  incredible resolution!  BUT...
NO LOOP TO BE FOUND!!!!

using STORM...  you see  t-loops and linear strands  etc.

variable insertion points...

she shows  many t-loops detected by STORM...

then grad student...  he looked at fused telomeres and did see major decrease in t-loops...

normally TRF2  deletion leads to telomere fusions....

STORM imaging after TRF2  deletion does lead to disappearance of t-loops...

the key is TRF2  (looks good for this old model (of Griffith)...

without  TRF2  telomeres open up  and are subject to attack... theya re protected when folded in t-loops...
but this is not the only thing that TRF2 does.

she is at Rockefeller...

mouse whisperer..

Ylli Doksani... key  postdoc !!!

Q  why do u need POT1 if u have a t-loop...

telomeres are disorganized in mammalian cells..