Chromosome

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

• Telomeres and Aging

• Telomeres and Cancer

 Telomeres

• Telomeres are special functional complexes

at the end of eukaryotic chromosomes.

• Telomeres are distinctive structures,

composed of repetitive DNA sequences and
associated proteins, that cap the ends of
linear chromosomes.

• Telomeres are essential for maintaining the

integrity and stability of eukaryotic genomes.
 Telomere Function

• Distinguish intact telomeres from

broken chromosomes

• Facilitate the replication of the very

end of the chromosome

• Transcriptionally repress the genes

near telomeres
 Concept of Telomere

Hermann Muller failed to recover terminally

deleted chromosomes

Muller, H.J. (1938), The remaking of chromosomes. The Collecting

Net 13: 181-195
Barbara McClintock provided
early evidance for the crutial role that
telomeres play in chromosomome
stability
McClintock, B. (1939), The behavior in successive
nuclear divisions of a chromosome broken at meiosis.
PNAS 25: 405-416
McClintock, B. (1941), The stability of broken ends of
chromosomes in Zea mays. Genetics 26: 234-282

"Have been working like hell on an exciting over-all

problem in genetics with wonderful results. It gets
me up early and puts me to bed late!"
Ciliates – the richest source of telomeres

Up to 10 000 000 telomeres

must be generated during
macronuclear development
 Telomere - Simple Repeated Sequence

Blackburn, EH and Gall, JG.

(1978) J Mol Biol 120:33

Tetrahymena - T2G4/C4A2
Oxytricha - T4G4/C4A4

Klobutcher et al, (1981)

PNAS, 78:3015-3019
 Telomere structure

Centromere Subtelomeric region Telomeric repeats

...TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG3’

...AATCCCAATCCCAATCCC5’

Human TTAGGG 2 – 20 kb 150 nt

Plants TTTAGGG 0.5 – 150 kb 30 nt
S. Cerevisiae T(G)1-3 350 bp 16 nt
Oxytricha nova TTTTGGGG 36 nt 16 nt
 S. cerevisiae telomeres
protective
nucleosomes telosome cap

report gene

Rap 1 Tel 1 Cdc 13

Rif 1/2 Ku Stn 1

Sir complex Ten 1

 Telomere structure in mammals

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

Griffith et al., Cell, 1999

 The End Replication Problem
5’ 3’
5’
3’
Leading Lagging
5’ 3’

3’ 5’
Lagging Leading
5’ 3’

Leading Lagging
Lagging Leading
5’
3’
 The End Replication Problem

Olovnikov, AM. (1996) Telomeres, telomerase,

and aging: origin of the theory. Exp Gerontol
31:443

Watson, JD. Origin of concatameric T4 DNA

(1972) Nature New Biol 239:197
 Hypothesis of Telomere Elongation

• 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

Greider and Blackburn (1985), Cell, 43:405-413

 Discovery of Telomerase

• 1985: Telomerase activity of Tetrahymena

• 1989: RNA subunit of Tetrahymena telomerase

• 1994: RNA subunit of S. cerevisiae

• 1995: Telomerase activity of S. cerevisiae

• 1996: Catalytic subunits of Tetrahymena

and S. cerevisiae telomerase

• 1997: Catalytic subunits of human and S. pombe

telomerase, and 1st Telomerase knock mice
 Telomerase

Dyskerin
Anchor site

Catalytic site

--TTAGGGTTAGGGTTAGGGTTAG

hTR

hTERT

hTERT = human telomerase reverse transcriptase

hTR = human telomerase template RNA
 Telomerase in S. cerevisiae

Tlc1 Est3
Est1

Est2 Heloenzyme

Core enzyme

Mutation of EST1, EST2, EST3 or Tlc1 showed

progressive telomere shortening.
 The End Replication Problem
Is Solved by Telomerase
5’ 3’

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

report gene Cdc13

Polα

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

• Telomeres and Aging

• Telomeres and Cancer

 Aging - Senescence
Aging
the progressive loss of function accompanied by
decreasing fertility and increase mortality with
advancing age
Senescence (senex, meaning "old man" or "old age.“)
the state or process of aging.
Replicative Senescence (Cellular senescence)
a state that cells arrest after they undergo a
limited number of cell division
Lifespan
a time period cells (or organism) can live
 Senescent Phenotypes

• Irreversible arrest of cell division (G1 DNA content,

cannot resume proliferation by mitogens).

• Resistant to apoptotic death (for example, human

fibroblasts and T lymphocytes, but not endothelial
cells).

• Selected changes in morphology and metabolism, and

derangements in differentiated functions (cellular
enlargement, increased lysosome biogenesis, and
expression of a β-galactosidase).
Characteristics and Inducers of the Senescent Phenotypes

Telomere shorting
Irreversible
growth
arrest Tumor suppressor
activity
Apoptosis
Others resistance DNA damage

Altered Oncogenic/
differentiated Mitogenic Stimuli
function
Chromatin remodeling

Itahana1K, Dimri G and Campisi J (2001) Regulation of cellular senescence

by p53. Eur. J. Biochem. 268 :2784-2791
 History of cellular aging study

Alexis Carrel: chicken heart fibroblast

cells in culture were established, and grown
for 34 years - vertebrate cells can divide
indefinitely in culture; age is “an attribute of
the multicellular body as a whole”

Witkowski J (1985) Trends Biochem Sci 10:258-260

 Hayflick limit
Hayflick: fibroblast cultures derived from
human skin divide 40 to 50 times, and stop to
undergo “senescence”; cells from older people
undergo fewer division then cells from younger
people.
Carrel’s immortal chicken cell cultures were not
reproducible.

Questions: Does the limited capacity of cells

to divide relate to human aging? What tells cells
to stop dividing?
Hayflick, L. and Morrhead, PS (1961) Exp Cell Res 25:585
What is the connection of telomeres with aging ?

In culture normal human somatic cells exhibit a restricted

lifespan and enter senescence after a set number of
division (~50)

In human somatic cells telomeres shorten by 5-20 repeats

with every cell division

Most somatic cells do not have detectable telomerase

activity

Telomere length shortens with the age of a cell and

eventually cell dies when telomere becomes too short
 Telomeres Shorten With Increased Age
Sperm
Placenta
Fetal brain
Fetal kidney

Colon mucosa (30-65 yrs)

Colon mucosa (65-88 yrs)

Blood (20-39 yrs)

Blood (40-59 yrs)
Blood (60-79 yrs)

0 4 8 12 16 20 (kb)
Tissue Source Telomere Length (kb)

Hastie et al, Nature, 346: 866

Length of telomeres varies among
different cell types

Germline/ES cells (telomeres maintained)

Telomere Length

Pluripotent Stem cells

(intermediate telomere loss)

Normal cells (greatest telomere loss)

Growth
Arrest

Cell Divisions - Replicative Age

 Aging Theory of Telomere Loss

In the absence of telomerase, telomeres shorten

with each cell division. When telomeres reach a
critically short length, normal cells irreversibly
arrest proliferation and acquire a characteristic
enlarged morphology and a variety of altered
functions. This response has been termed
replicative or cellular senescence.
Aging Theory of Telomeres Loss

Keith et al. (2002)

Expert Rev Mol Med

The telomere length is thought to be the “mitotic

clock” that is read to establish the Hayflick limit
when cells cease to divide.

(Mitotic clock: utilize cell divisions as the unit of

time, rather than chronological or metabolic age)
Harley et al. (1990) Nature 345:458
 Short Telomeres are Associated
with pre-mature aging

Image credit: William and Wilkens Publishing Inc.

Hutchinson-Gilford Progeria

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 ?

Cellular senescence causes

telomere shortening ?
 Telomerase activity in stable retinal
pigment epithelial (RPE-hTRT-) clones.

Andrea G. Bodnar et al (1998) Extension of Life-Span by Introduction of Telomerase into

Normal Human Cells. SCIENCE VOL. 279:349
 Telomere length in stable
RPE and BJ (foreskin fibroblast) clones.

Bodnar et al (1998). SCIENCE 279:349

Effect of telomerase expression on cell life-span

Bodnar et al (1998). SCIENCE 279:349

hTERT expression is sufficient to
immortalize normal human cells

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

Sarin et al. (2005) Nature 436:1048

Telomerase Deficient Yeast Senesces

Zhou et al, (2000) Science 289:771

Telomere dysfunction
leads to
defects in plant growth

Riha et al., Science (2001)

G3 Ter knock-out mice are premature ageing

Rudolph et al. (1999) Cell 96:701

 Dyskeratosis Congenita

• Die between the ages of 16 and 50 (premature

greying, early dental loss, bone marrow failure,
liver cirrhosis, pulmonary disease and skin cancer)
• X chromosome: Dyskerin (nucleolus, pseudo-
uridylation of specific residues of RNA.
• Levels of telomerase RNA are low,
• Telomeres are shorter than normal in white blood
cells and fibroblast cells.

Question: what is Dyskerin’s function in

stabilizing telomerase RNA and promoting
telomerase activity?
Tom Vulliamy et al (2001)
The RNA component of
telomerase is mutated in
autosomal dominant
dyskeratosis congenita.
Nature Vol. 413:432
Telomeres are shorter in the cells from DC patient
Summary of telomeres and aging
Telomerase
activity

Telomere
homeostasis

Repress
chromosomal
instability

Extension of life span

• Telomeres and Telomerase

• Telomeres and Aging

• Telomeres and Cancer

 Telomeres and Cancer

• Tumors are caused by mutations, which activate

oncogenes and switch off tumor suppressor genes.

• With few remarkable exceptions, not many human

tumor cells would make it into a full-blown, clinically
relevant tumors without overcoming telomere-
dependent replicative senescence.

• 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)

Normal somatic cells: telomerase negative

Transforming event
M1
5-7 Precrisis cells: no telomerase
M2 Immortal tumor cells
2-4

telomerase activity

M1 M2
Hayflick limit Crisis

Cell Division
Telomerase activity is required for tumorigenic
conversion of human cells

The ectopic expression of the telomerase catalytic

subunit (hTERT) in combination with two
oncogenes (the simian virus 40 large-T oncoprotein
and an oncogenic allele of H-ras) results in direct
tumorigenic conversion of normal human epithelial
and fibroblast cells.
Hahn et al, (1999) Nature
400:464
Alternative Lengthening of Telomeres (ALT)

• Telomerase activity detected 85% of cancer cells.

(Kim et al, 1994 Science)

• Some telomerase-negative tumors (10-15% of cancer)

maintain stable length of their telomeres.

Question
How these cells maintain their telomeres?
No telomerase, no cancer?
Alternative Lengthening of Telomeres (ALT)
est2∆ EST2+

Tlc1

Est2

tlc1 RAD52 tlc1 rad52

Telomerase core enzyme

Teng and Zakian MCB (1999) Lingner et al. Science (1997)

Alternative Lengthening of Telomeres (ALT)

XhoI

X’ Y’ TG1-3

1.3 kb
Tlc1

Est2

Telomerase core enzyme

•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

Resolution of DNA strands

and repair synthesis
Require Rad52
Net telomere elongation
Alternative Lengthening of Telomeres (ALT)

tlc1 RAD52 tlc1 rad52

Teng and Zakian (1999) MCB :8083-93.

 WT (A and B)

NIIDA, et al (2000) Telomere Maintenance in Telomerase-Deficient Mouse Embryonic

Stem Cells: Characterization of an Amplified Telomeric DNA. MCB Vol 20:4115-4127
 DKO741

NIIDA, et al (2000) Telomere Maintenance in Telomerase-Deficient Mouse Embryonic

Stem Cells: Characterization of an Amplified Telomeric DNA. MCB Vol 20:4115-4127
 DKO301

NIIDA, et al (2000) Telomere Maintenance in Telomerase-Deficient Mouse Embryonic

Stem Cells: Characterization of an Amplified Telomeric DNA. MCB Vol 20:4115-4127
Depletion of TRF2 disrupts capping of
chromosome ends

+
-
Telomere
Binding

Cells overexpressing TRF2∆B∆M

Smogorzewska et al., Curr.Biol. (2002)
 Genome instabilities triggered by
chromosome end-to-end fusions

45S rDNA

BAC F11L15

Nondisjunction Chromosome Chromosome Nonreciprocal

loss rupture translocation
Siroky et al., Chromosoma (2003)
 Cellular consequences of telomere dysfunction

Kim et al (2002) Telomeres, aging and cancer: In search of a happy ending.

Oncogene 21, 503 ± 511
Telomerase serves as a target for cancer therapy
T-loop

D-loop
Intact telomere

(TTAGGG)n

Telomerase inhibitor

Stasis (premature senescence)

? 3’
Telomere-based senescence Aging
Apoptosis Death
Increased genomic instability
DNA Damage Signal (engagement of ALT or telomerase revertants)
 Telomerase Inhibitor Approaches

• Catalytic (hTERT) component

– Reverse transcriptase inhibitors
– hTERT promoter/gene therapy
– Dominant-negative or shRNA hTERT gene
therapy
– Small molecule inhibitors
• Template (hTERC) functional RNA component
– Hammerhead ribozymes – hTR template
– Oligonucleotides – telomerase template
antagonist
 Selective killing of telomerase expressing cancer cells
with replication competent viruses

hTERT-promoter Adenovirus

Normal cell

Telomerase - Viral replication

blocked
Viral agent
hTERT adenovirus

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'

hTR/(RNA) • Stable to nucleases

• Forms extremely stable
heteroduplex with RNA (Tm13mer
> 70oC)
• Competitive with telomere
hTERT binding
Catalytic protein
• Binding is base pair-dependent
• When bound, prevents
telomeric substrate binding
 Telomerase Activity and Telomere Length in Cells
Treated with GRN163

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

1 m 3x/wk over 2 months HME50 cells

Summary Telomere
Telomere Shortening
(telomerase inactivation)
Promote genomic
instability
Limits lifespan
Telomere
Stabilization
(telomerase activation)

Immortality
Chromosome
stability

Tumor Tumor
Suppression Promotion

Adapted from Masutomi and Hahn (2003), Cancer Letter 194:163-172

 How do organisms deal with the end
replication problem?
Parvovoruses: Priming via DNA 3’

Adenoviruses: Protein priming Pol pTP

Ser-C-OH
3’ G

Drosophila: Retrotransposition
HeT-A TART

Some plants and insects: Homologous recombination?

Majority of eukaryots: Telomerase

 Telomere evolution
Vertebrates TTAGGG
C.Elegans TTAGGC
Ustilago TTAGGG S. cerevisiae
S.Pombe TTACAGG(G)0-4
Pneumocystis TTAGGG
Neurospora TTAGGG
C.albicans ACGGATGTCTAACTTCTTGGTGT
K.lactis ACGGATTTGATTAGGTATGTGGTGT Human
S.cerevisiae T(G)1-3
Arabidopsis TTTAGGG
Asparagales TTAGGG
Tetrahymena TTGGGG
Slime molds TAGGG T-loops:
Kinetoplastida TTAGGG mammals, protozoa, plants
Giardia TAGGG
Li et al., Cell (2000)
Telomere – telomerase evolution

Nakamura et al., Science (1997

 Humanization of yeast telomeres
Yeast tlc1 strain (T(G)1-3 telomere)

report Cdc13
gene
Sir complex Rap1

Yeast tlc1h strain (TTAGGG telomere)

report Cdc13
gene
Telomere binding factor 1 (TRF1/2 homologue)

Alexander & Zakian, EMBO J. (2003)

 Model for telomere evolution
Circular genome Retrotransponson

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

Telomere capping by T- loops and telomere-specific proteins

de Lange, Nat. Rev. Mol. Cell Biol. (2004)

 Acknowledgements

Ms. Fu Xiao-Hong
Ms. Li Ning

Dr. Wong Jimin

Thank You