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Zhou Chromosome and Chromatin 2
Zhou Chromosome and Chromatin 2
Chromatin
Telomeres
周金秋
jqzhou@sibs.ac.cn
Istitute of Biochemistry and Cell Biology
Shanghai Institutes for Biological Sciences
Chinese Academy of Sciences
Fall, 2005
I. Chromosome and Chromatin
II. Centromere
III. Telomeres
• Telomeres and Telomerase
Tetrahymena - T2G4/C4A2
Oxytricha - T4G4/C4A4
...TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG3’
...AATCCCAATCCCAATCCC5’
report gene
TRF1 TRF2
TTTAGG ? 3’
Pot1
Homodimerization Myb
TRF1
Homodimerization Myb
TRF2
Telomere structure in mammals
Rap 1
TRF 1 Pot 1
? 3’
TTTAGG
TRF 2
T-loop
Ku
D-loop
MRX
Visualization of T-loops in mammals
3’ 5’
Lagging Leading
5’ 3’
Leading Lagging
Lagging Leading
5’
3’
The End Replication Problem
• Homologous recombination?
– De novo telomere addition in the development of
Tetrahymena macronucleus
– yeast telomere sequence (TG1-3) was added in
the YAC with Tetrahymena telomere sequence
(T2G4)
• Enzyme?
Telomerase Activity
Dyskerin
Anchor site
Catalytic site
--TTAGGGTTAGGGTTAGGGTTAG
hTR
hTERT
Tlc1 Est3
Est1
Est2 Heloenzyme
Core enzyme
Leading Lagging
5’ 3’
5’ 3’
3’ Leading Lagging 5’
5’ 3’
3’ 5’
Telomere elongation by Telomerase
Telomerase RNA
TERT
UCCCAAUC
GTTAGGGTTAG
C
Telomere
Telomere elongation by Telomerase
Telomerase RNA
TERT
UCCCAAUC
GTTAGGGTTAGGGTTAG
C AACCC
Telomere
Telomere Replication
Regulation of telomerase at
chromosome termini in yeast
S-phase
telomerease
Est1
Sir2/3/4 complex Ku
Telomere Position Effect
GFP-LacI
Interphase positioning of
telomeres can be achieved
through two partially redundant
mechainnisms. One requires the
heterodimeric yKu complex. The
second requires Silent
information regulators, correlates
with transcriptional repression,
and is specific to S phase.
Hediger et al, (2002)
Current Biology 12:2076
• Telomeres and Telomerase
Telomere shorting
Irreversible
growth
arrest Tumor suppressor
activity
Apoptosis
Others resistance DNA damage
Altered Oncogenic/
differentiated Mitogenic Stimuli
function
Chromatin remodeling
0 4 8 12 16 20 (kb)
Tissue Source Telomere Length (kb)
Growth
Arrest
Telomere length
• Werner’s Syndrome: Wrn, 3’ to 5’
DNA helicase/nuclease deficiency -
telomeres are lost at a greater rate
• Hutchinson-Gilford Progeria: a point
mutation in lamin A, a component of
the inner nuclear membrane. Some
tissues have short telomeres Birth Middle AgeOld Age
Telomere shortening causes
cellular senescence ?
160
140
hTERT +
Population Doublings
120
100
80
60 hTERT -
40
0 50 100 150 200 250 300
Days
Telomerase can promote
proliferation of resting stem cells
Telomere
homeostasis
Repress
chromosomal
instability
• In
humans, telomerase activity detected in many
types of cancer cells, and not detectable in most
somatic cell lineage.
(Kim et al, 1994 Science)
Telomere hypothesis of immortalization
Activation of telomerase activity in cancer cells stabilizes telomere length
Germline cells:
~15 telomerase positive
Stem cells
TRFt length (kb)
telomerase activity
M1 M2
Hayflick limit Crisis
Cell Division
Telomerase activity is required for tumorigenic
conversion of human cells
Question
How these cells maintain their telomeres?
No telomerase, no cancer?
Alternative Lengthening of Telomeres (ALT)
est2∆ EST2+
Tlc1
Est2
XhoI
X’ Y’ TG1-3
1.3 kb
Tlc1
Est2
•Enzyme?
•Homologous recombination? Teng and Zakian (1999) :8083-93.
Alternative Lengthening of Telomeres (ALT)
3’
5’
3’
Homologous recombination: 5’
a DNA strand from one telomere Strand invasion
anneals with the complementary
strand of another telomere,
thereby priming synthesis of new
telomeric DNA using the
Elongation
complementary strand as a copy
template
+
-
Telomere
Binding
45S rDNA
BAC F11L15
D-loop
Intact telomere
(TTAGGG)n
Telomerase inhibitor
hTERT-promoter Adenovirus
Normal cell
Cell destruction
Cancer Viral release
cell Virus spread
Telomerase + Viral
replication
Ly
si
Un s Bu
tr e ff e
ate r
Ve d
cto
DN r con
- hT tr
H1299
ER ol
Un T
tr e
Ve ated
cto
rc
DN on
tro
- hT l
RCC23
Un E RT
tr e
ate
Ve d
cto
r
DN cont
ro
- hT l
DU145
ER
DN-hTERT T
Cre excised
leads to cancer cell death
DN-hTERT
Inhibition of telomerase with DN-hTERT (D869A)
Telomerase RNA (hTR) template as a
target for inhibition
GRN163: 3 ‘NH2-AACAGATTGGGAT-OH 5'
hTER: 5‘…UUGUCUAACCCUAAC…3'
GRN163 GRN163
nM
12 µM
12 nM
1. rol
l
ro
ro
nt
nt
nt
.5
25
5
kb
co
co
co
19
7.7
6.2
• Potential as a universal
4.3 oncology target
3.5 • High tumor specificity
2.7 • Synergy expected with
cytotoxic drugs
1.9 • Systemic delivery possible
1.5
Immortality
Chromosome
stability
Tumor Tumor
Suppression Promotion
Drosophila: Retrotransposition
HeT-A TART
report Cdc13
gene
Sir complex Rap1
report Cdc13
gene
Telomere binding factor 1 (TRF1/2 homologue)
RT
Genomic RNA
Linear genome with terminal repeats AAAAA
T-loop phase
Telomerase
RT
Telomerase phase 5’
Template RNA
3’ 3’
5’
Telomerase-mediated
telomere maintenance
Ms. Fu Xiao-Hong
Ms. Li Ning