1. Telomeres and epigenetics

2. The structure of mammalian telomeres
Telomeres contain a double-stranded DNA region of TTAGGG repeats (green arrows)
Telomeres are characterized by a 150–200-nt long single-stranded overhang of the G-rich strand (G-strand overhang; blue arrows)
Telomerase recognizes the 3' OH at the end of the G-strand overhang, leading to telomere elongation.
Telomere structure and telomerase activity.   A | The structure of mammalian telomeres. Telomeres contain a double-stranded DNA region of TTAGGG repeats (green arrows) which is typically 10–15-kb long in humans and 25–40-kb long in mice. Telomeres are characterized by a 150–200-nt long single-stranded overhang of the G-rich strand (G-strand overhang; blue arrows). Note that the length of telomere repeats is not drawn to scale. Telomerase recognizes the 3' OH at the end of the G-strand overhang, leading to telomere elongation. Two main protein complexes are bound to telomeres, the telomere repeat binding factor 1 and 2 complexes, TRF1 and TRF2. B | A telomere in a T-loop conformation. Strand invasion of the G-strand overhang is highlighted in red. This conformation might prevent the access of telomerase to the 3' OH at the chromosome end. The components of the telomere repeat binding factor 1 (TRF1) (Ca) and 2 (TRF2) (Cb) complexes and the telomerase enzyme (Cc) are shown. The human diseases in which expression of these components has been shown to be altered are indicated. Note that the way that the complexes are shown is not necessarily an exact structural representation. ATM, ataxia telangiectasia mutated; BLM, Bloom syndrome; DKC1, dyskeratosis congenita 1, dyskerin; ERCC1, excision repair cross-complementing 1; KU86, Ku86 autoantigen related protein 1 (also known as XRCC5); MRE11, meiotic recombination 11; NBS1, Nijmegen breakage syndrome 1; POT1, protection of telomeres 1; PTOP, POT1 and TIN2 organizing protein; RAD50, a DNA repair protein; RAP1, repressor/activator protein 1; TANK, tankyrases; TERC, telomerase RNA component; TERT, telomerase reverse transcriptase; TIN2, TRF1-interacting nuclear factor 2.

3. Telomerase enzyme (Cc)
Ribonuleoprotein
TERT, telomerase reverse transcriptase
TERC, telomerase RNA component (single RNA molecule provides an AAUCCC (in mammals) template to guide the insertion of TTAGGG
DKC1, dyskeratosis congenita 1
Telomere structure and telomerase activity.   A | The structure of mammalian telomeres. Telomeres contain a double-stranded DNA region of TTAGGG repeats (green arrows) which is typically 10–15-kb long in humans and 25–40-kb long in mice. Telomeres are characterized by a 150–200-nt long single-stranded overhang of the G-rich strand (G-strand overhang; blue arrows). Note that the length of telomere repeats is not drawn to scale. Telomerase recognizes the 3' OH at the end of the G-strand overhang, leading to telomere elongation. Two main protein complexes are bound to telomeres, the telomere repeat binding factor 1 and 2 complexes, TRF1 and TRF2. B | A telomere in a T-loop conformation. Strand invasion of the G-strand overhang is highlighted in red. This conformation might prevent the access of telomerase to the 3' OH at the chromosome end. The components of the telomere repeat binding factor 1 (TRF1) (Ca) and 2 (TRF2) (Cb) complexes and the telomerase enzyme (Cc) are shown. The human diseases in which expression of these components has been shown to be altered are indicated. Note that the way that the complexes are shown is not necessarily an exact structural representation. ATM, ataxia telangiectasia mutated; BLM, Bloom syndrome; DKC1, dyskeratosis congenita 1, dyskerin; ERCC1, excision repair cross-complementing 1; KU86, Ku86 autoantigen related protein 1 (also known as XRCC5); MRE11, meiotic recombination 11; NBS1, Nijmegen breakage syndrome 1; POT1, protection of telomeres 1; PTOP, POT1 and TIN2 organizing protein; RAD50, a DNA repair protein; RAP1, repressor/activator protein 1; TANK, tankyrases; TERC, telomerase RNA component; TERT, telomerase reverse transcriptase; TIN2, TRF1-interacting nuclear factor 2.

4. Components of telomeric proteins
Two main protein complexes are bound to telomeres, the telomere repeat binding factor 1 and 2 complexes, TRF1 and TRF2
The components of the telomere repeat binding factor 1 (TRF1) (Ca) and 2 (TRF2) (Cb) complexes and are shown.
human diseases in which expression of these components has been shown to be altered are indicated
Telomere structure and telomerase activity.   A | The structure of mammalian telomeres. Telomeres contain a double-stranded DNA region of TTAGGG repeats (green arrows) which is typically 10–15-kb long in humans and 25–40-kb long in mice. Telomeres are characterized by a 150–200-nt long single-stranded overhang of the G-rich strand (G-strand overhang; blue arrows). Note that the length of telomere repeats is not drawn to scale. Telomerase recognizes the 3' OH at the end of the G-strand overhang, leading to telomere elongation. Two main protein complexes are bound to telomeres, the telomere repeat binding factor 1 and 2 complexes, TRF1 and TRF2. B | A telomere in a T-loop conformation. Strand invasion of the G-strand overhang is highlighted in red. This conformation might prevent the access of telomerase to the 3' OH at the chromosome end. The components of the telomere repeat binding factor 1 (TRF1) (Ca) and 2 (TRF2) (Cb) complexes and the telomerase enzyme (Cc) are shown. The human diseases in which expression of these components has been shown to be altered are indicated. Note that the way that the complexes are shown is not necessarily an exact structural representation. ATM, ataxia telangiectasia mutated; BLM, Bloom syndrome; DKC1, dyskeratosis congenita 1, dyskerin; ERCC1, excision repair cross-complementing 1; KU86, Ku86 autoantigen related protein 1 (also known as XRCC5); MRE11, meiotic recombination 11; NBS1, Nijmegen breakage syndrome 1; POT1, protection of telomeres 1; PTOP, POT1 and TIN2 organizing protein; RAD50, a DNA repair protein; RAP1, repressor/activator protein 1; TANK, tankyrases; TERC, telomerase RNA component; TERT, telomerase reverse transcriptase; TIN2, TRF1-interacting nuclear factor 2.

5. A telomere in a T-loop conformation
Strand invasion of the G-strand overhang is highlighted in red
This conformation might prevent the access of telomerase to the 3' OH at the chromosome end.
Telomere structure and telomerase activity.   A | The structure of mammalian telomeres. Telomeres contain a double-stranded DNA region of TTAGGG repeats (green arrows) which is typically 10–15-kb long in humans and 25–40-kb long in mice. Telomeres are characterized by a 150–200-nt long single-stranded overhang of the G-rich strand (G-strand overhang; blue arrows). Note that the length of telomere repeats is not drawn to scale. Telomerase recognizes the 3' OH at the end of the G-strand overhang, leading to telomere elongation. Two main protein complexes are bound to telomeres, the telomere repeat binding factor 1 and 2 complexes, TRF1 and TRF2. B | A telomere in a T-loop conformation. Strand invasion of the G-strand overhang is highlighted in red. This conformation might prevent the access of telomerase to the 3' OH at the chromosome end. The components of the telomere repeat binding factor 1 (TRF1) (Ca) and 2 (TRF2) (Cb) complexes and the telomerase enzyme (Cc) are shown. The human diseases in which expression of these components has been shown to be altered are indicated. Note that the way that the complexes are shown is not necessarily an exact structural representation. ATM, ataxia telangiectasia mutated; BLM, Bloom syndrome; DKC1, dyskeratosis congenita 1, dyskerin; ERCC1, excision repair cross-complementing 1; KU86, Ku86 autoantigen related protein 1 (also known as XRCC5); MRE11, meiotic recombination 11; NBS1, Nijmegen breakage syndrome 1; POT1, protection of telomeres 1; PTOP, POT1 and TIN2 organizing protein; RAD50, a DNA repair protein; RAP1, repressor/activator protein 1; TANK, tankyrases; TERC, telomerase RNA component; TERT, telomerase reverse transcriptase; TIN2, TRF1-interacting nuclear factor 2.

6. Telomerase and telomere length in ageing
Most somatic cells show progressive telomere shortening owing to low or absent telomerase activity
leads to critically short telomeres, which triggers a DNA damage response that results in chromosomal end-to-end fusions or cell arrest and apoptosis.
thought to contribute to the onset of degenerative diseases including human premature ageing syndromes
Changes in telomere length over time during tumour progression, compared with changes in normal tissue. Tumours generally have shorter telomeres than the surrounding normal tissue, owing to the fact that they have had a longer proliferative history in the absence of telomerase activity. This telomere shortening could eventually lead to increased cell death and loss of cell viability within the tumour. However, telomerase is reactivated in more than 90% of all types of human tumour, thereby rescuing short telomeres and perpetuating cells with short telomeres and high chromosomal instability. Similarly, most metastases also contain telomerase-positive cells, which indicates that telomerase is required to sustain their growth. The fact that cancer cells have shorter telomeres than normal cells, together with the fact that cancer growth seems to depend on telomerase reactivation, indicates that therapeutic strategies that are aimed at inhibiting telomerase will preferentially kill tumour cells and have no toxicity on normal cells. The presence or absence of telomerase activity is indicated by the plus symbol and minus symbol respective

7. Telomerase and telomere length in tumourigenesis
Tumour cells have shorter telomeres than normal cells
However, telomerase is reactivated in more than 90% of all types of human tumours
therapeutic strategies aimed at inhibiting telomerase will preferentially kill tumour cells and have no toxicity on normal cells.
Changes in telomere length over time during tumour progression, compared with changes in normal tissue. Tumours generally have shorter telomeres than the surrounding normal tissue, owing to the fact that they have had a longer proliferative history in the absence of telomerase activity. This telomere shortening could eventually lead to increased cell death and loss of cell viability within the tumour. However, telomerase is reactivated in more than 90% of all types of human tumour, thereby rescuing short telomeres and perpetuating cells with short telomeres and high chromosomal instability. Similarly, most metastases also contain telomerase-positive cells, which indicates that telomerase is required to sustain their growth. The fact that cancer cells have shorter telomeres than normal cells, together with the fact that cancer growth seems to depend on telomerase reactivation, indicates that therapeutic strategies that are aimed at inhibiting telomerase will preferentially kill tumour cells and have no toxicity on normal cells. The presence or absence of telomerase activity is indicated by the plus symbol and minus symbol respective

8. Premature ageing syndromes with short telomeres
Ataxia telangiectasia (ATM)
Werner syndrome (WRN);
Bloom syndrome (BLM);
Dyskeratosis congenita (DKC1, Terc);
aplastic anaemia (Terc, Tert);
Fanconi anaemia (FANC genes);
Nijmegen breakage syndrome (NBS); and
ataxia telangiectasia-like disorder (MRE11).

9. Ataxia Telangiectasia
Staggering gait, muscular unco-ordination
Immunodeficiency
Neurodegeneration
premature aging
Skin sensitivity to ionizing radiation
susceptibility to certain types of cancer (breast cancer)
A-T is fatal in the second or third decade of life

10. Ataxia Telangiectasia
Mutations in ATM gene located on chromosome 11q22-q23
encodes large protein kinase involved in cell cycle checkpoint and genotoxic stress responses
Homozygous ATM mutant alleles rare ~ 1/40,000
heterozygous carriers ~ 1-2%
Carriers exhibit intermediate sensitivity to radiation and predisposition to cancer (implications for radiotherapy)
ATM is a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family. All PIKKs have a highly conserved catalytic domain that bears similarity to the catalytic domain of the lipid kinase phosphatidylinositol 3-kinase (PI 3-K). Despite this similarity, all of the PIKK family members exhibit protein rather than lipid kinase activity. ATM and related protein kinases participate in one of the earliest events that occurs in response to DNA insult by phosphorylating the C-terminal tail of the core histone H2AX protein [γ-H2AX (phosphorylated form); Figure 1].6-8 γ-H2AX marks the site of damage and nucleates the formation of damage response and repair complexes.6,8 BRCA1 C-terminal (BRCT) domain-containing proteins are recruited to γ-H2AX creating a protein scaffold for further assembly of signaling complexes that include molecules such as p53-binding protein 1 (53BP1),9-12 mediator of DNA damage checkpoint protein 1 (MDC1),13-15 and the BRCA1 tumor suppressor protein.9 Additionally, the BCRT domain-containing protein Nbs1, the Rad50 and Rad51 DNA repair factors, and others are recruited to the damage site. For many of these proteins, specific details regarding direct protein-protein interactions and order of complex assembly are only beginning to be elucidated. For example, a recent study suggests that Nbs-1 can directly bind to ?-H2AX.16 Furthermore, many of the these proteins are substrates for ATM and related protein kinases, underscoring the critical role of this protein kinase in genotoxic stress responses.3

11. Werner syndrome (WRN short stature (common from childhood on)
wrinkled skin
baldness,
cataracts,
muscular atrophy
tendency to diabetes mellitus
gene WRN mapped to chromosome 8
predicted helicase belonging to the RecQ family

12. FANCONI/BRCA1/BRCA2/ATM/NBS1 PATHWAY
Fanconi Anemia has 8 identified complementation groups (A, B, C, D1, D2, E, F, and G) and genes for at least 7 of these groups have been identified. Fanconi anemia genes, FancB and FancD1, have been identified as the Early Onset Breast Cancer gene BRCA2. Five of the Fanconi Anemia genes (FancA, FancC, FancE, FancF, and FancG) form a complex which interacts with DNA and leads to the mono-ubiquitination of the FancD2 protein. Through an association with BRCA1 and BRCA2 in nuclear loci (represented by the light blue area in the diagram) this leads to activation of the processes that lead to DNA repair. The ATM kinase can be activated by ionizing radiation, which in turn activates many targets. One of these, the FancD2 protein, is phosphorylated by ATM, which then leads to S phase arrest. An additional link in this pathway includes the phosphorylation by ATM of the protein encoded by the gene of the Nijmegen Breakage Syndrome (NBS1) and BRCA1. The NBS1 protein is part of a complex which, in turn, also leads to phosphorylation of FancD2 by ATM. NBS1 appears to have two independent functions, one in inducing S-phase arrest where FancD2 is not required and the second in interacting with FancD2 in promoting DNA repair (see dashed arrow in diagram). Thus, FancD2 is at the cross roads of two pathways -- one leading to S phase arrest which functions from ATM through NBS 1 and associated proteins and the other in response to DNA damage acting through the Fanconi complex

13. Epigenetics
epigenetic modification of DNA in mammals is methylation of cytosine at position C5 in CpG dinucleotides
Other main group is epigenetic post-translational modification of histones

14. Genomic imprinting
defined as an epigenetic modification of a specific parental chromosome in the gamete or zygote that leads to differential expression of the two alleles of a gene in the somatic cells of the offspring.
Differential expression can occur in all cells, or in specific tissues or developmental stages.
About 80 genes are known to be imprinted
Loss of imprinting (LOI)
disruption of imprinted epigenetic marks through gain or loss of DNA methylation, or simply the loss of normal allele-specific gene expression.

15. Epigenetics – differential imprinting
Prader-Willi syndrome
Angelman syndrome
failure to thrive during infancy, hyperphagia and obesity during early childhood, mental retardation, and behavioural problems
molecular defect involves a paternally imprinted domain at 15q11–q13
abnormal gait
speech impairment,
seizures, mental retardation
inappropriate happy demeanor that includes frequent laughing, smiling, excitability
defect lies within the maternally imprinted domain at 15q11–q13

16. Genetic causes Prader-Willi syndrome Angelman syndrome
70% have a deletion of the PWS/AS region on their maternal chromosome 15
7% have paternal uniparental disomy  for chromosome 15 (the individual inherited both chromosomes from the father, and none from the mother)
3% have an imprinting defect
11% have a mutation in UBE3A
1% have a chromosome rearrangement
11% have a unknown genetic cause
70% have a deletion of the PWS/AS region on their paternal chromosome 15
25% have maternal uniparental disomy for chromosome 15 (the individual inherited both chromosomes from the mother, and none from the father)
5% have an imprinting defect
<1% have a chromosome abnormality including the PWS/AS region

17. DNA methylation and cancer
Changes in methylation are early events in tumorigenesis
In tumour cells, repeat-rich heterochromatin becomes hypermethylated and this contributes to genomic instability, a hallmark of tumour cells, through increased mitotic recombination events.
De novo hypomethylation of CpG islands also occurs in cancer cells, and can result in the transcriptional silencing of growth-regulatory genes.
Region of the genome showing repeat-rich, hypermethylated pericentromeric heterochromatin and tumour suppressor gene (TSG) associated with a hypomethylated CpG island (red).

18. References 1) Telomeres and human disease by M Blasco
Nature Reviews Genetics Aug 2005 vol 6 pp611
2) DNA methylation and human disease by KD Robertson
Nature Reviews Genetics Aug 2005 vol 6 pp 597