Flashcards in Graeme Finlay-2 Deck (21):
What is epigenetic inheritance?
The flow of information between cell generations that is not encoded in the primary sequence of the DNA with epigenetic regulation being mediated in part by changes in histone acetylation, histone methylation and DNA methylation
What is required for DNA to be transcribed?
The appropriate transcription factors are present
Histones are acetylated and appropriately methylated
Cytosines in promoters are unmethylated
What are the marks associated with histone code which allow identification of active and inactive genes?
Active genes include H3 and H4 acetylation and H3K4Me2/3
Inactive genes include H3K9Me2/3 and H3K27Me3
Where does cytosine methylation occur?
Where C is followed by G
A high density of CpG dinucleotides occurs in CpG islands found in gene promoters
Most genes (70%) have a CpG island within -2 to +1 kb of the transcription start site
In normal tissue, most CpGs are methylated except for those in CpG isalnds
How can methylated cytosines be detected?
DNA is reacted with sodium bisulphite, NaHSO3 which converts C to U but leaves 5-MeC unchanged this is followed by
DNA sequencing using PCR primers that avoid CpGs
A methylation specific PCR using PCR primers specific for both C-to-U modifications and the unchanged C sequences
What occurs to the proportion of methylated CpGs in tumour DNA?
There is hypomethylation of CpG islands which leads to the activation of proto-oncogenes (like HRAS, CCND2 encoding cyclin D2) and the multi-drug resistance gene (MDR-1)
What are two of the key sequences in tumour DNA which can be demethylated ?
Pericentromeric satellite sequences which leads to centromeric decondenstaion, mitotic recombination, aneuploidy and chromosomal instability, carcinomas often have chromosomal translocations with pericentromeric breakpoints
Promoters of parasitic elements such as retrotransposons leading to their transposition or to transcription of adjacent genes
What are the mechanisms of hypomethylated that have been suggested?
SW1/SNF complexes use ATP to reposition nucleosomes, SNF activity is required for normal DNA methylation, but components often have deleterious mutations in cancer with SNF5 mutations cause malignant rhabdoid tumours
A splice variant of DNA methyltransferase 3B lacks the methyltransferase catalytic domain and is associated with hypomethylation and liver cancer
Carcinogens may cause hypomethylation, Cadmium inhibits DNA methyltransferase and arsenic leads to hypomethyaltion of proto-oncogenes
Loss of the APC TSG in colon epithelium may deregulate DNA methylase
Diets deficient in methyl donors have been shown to lead to liver cancer in rodents
How may regional hypermethylation fulfil for one of Knudson’s hits?
Regional hypermethylation of CpG islands in the promoters of tumour suppressor genes will induce gene silencing and can therefore fulfil the two hit hypothesis as one allele of the tumour suppressor gene may be mutant, while hypermethylation selectively silences the wild type allele
What can hypermethylation be used for clinically?
For early detection of cancer in pople at risk (cancer families, uranium miners and smokers) and of occult (hidden) lymph node metastases: it occurs in premalignant lesions and it can be detected in serum, saliva, sputum and urine
As a prognostic factor methylation of genes such as CDKN2A is associated with poor outcome
It may be a biomarker of response to chemotherapy as methylation of O6 methylguanine DNA methyltransferase gene predicts response to anticancer drugs that alkylate DNA
It may act as a therapeutic target with decitabine demethylates DNA and reactivavtes TSGs but is non-specific and toxic for normal cells with synergistic effects may be obtained with decitabine and trichostatin A
What are the routes by which hypermethylation may arise?
Mutant ras proteins activate an epigenetic silencing programme as KRAS transformation has been shown to induce Ras epigenetic silencing effectors which silence tumour suppressor genes such as FAS
Chronic inflammation (hepatitis, pancreatitis, H. Pylori-gastritis) which generates DNA damage with neutrophil and eosinophil derived HOCl and HOBr generates 5-Cl or Br- cytosine that directs DNMT1 to methylate the opposite strand at CpGs, repair of the DNA strand breaks recruits methylating enzymes to the repair site
Exposure to carcinogens such as tobacco smoke
What are examples of Ras epigenetic silencing effectors?
Histone methyltransferases such as EZH2
Polycomb group repressor proteins such as Bmi 1
The DNA methylation maintenance enzyme DNMT1
What does knock-down of any one of the Ras-Epigenetic silencing effectors lead to?
Loss of EZH2, DNMT1 and Bmi1 from the FAS promoter
Reduction of FAS gene methylation
De-repression of the FAS gene
Loss of anchorage independence
Suppression of tumorigenicity
What does suppression of gene activity require?
Histone methyltransferases such as EZH2 which establishes the H3K27Me3 mark and recruits polycomb repressor complex-1
DNA methyltransferases such as DNMT3a/b which initiates CpG methylation and DNMT1 recognizes hemimethylated CpG DNA during replications and maintains methylation
Proteins with methyl CpG binding that recruit histone deacetylases which remove acetyl groups from histones
What may cause the altered state of DNA methylation where in normal cells Tumour suppressor genes may be repressed?
Increased expression of EZH2 as a result of genetic deletions affecting microRNA-101
Increased expression of DNMT3a.b as a result of miR-29 silencing
Increased expression of Bmi1 as a result of miR200c silencing
What is genetic imprinting?
This refers to the way in which chromosomes receive a molecu.ar tag when gametes are formed so that maternally and paternally derived alleles are expressed differently
How does the IGF2 gene show genetic imprinting?
In the normal kidney this gene is only expressed from the paternally derived chromosome with the differentially methylated region being methylated and suppressing H19 transcription
A downstream enhancer activates the IGF2 gene, the DMR of the maternally derived allele is demethylated and binds to the CTCF insulator which prevents the enhancer from activating IGF2 so the H19 gene is expressed and generates anti-proliferative miRNAs which helps prevent parthenogenic development in mammals
What occurs in Wilms tumour?
The increased methylation of the maternal DMR silences H19 and activates IGF2, this leads to bi-allelic expression of IGF2 causing a gene dosage effect which suppresses apoptosis
What is the relationship between Beckwith-wiedemann syndrome and wilms tumours?
In individuals with beckwith-weidemann syndrome the relative rsik of wilms tumours is 800 fold with epigenetic abnormalities including loss of the maternal allele and loss of imprinting
What role does loss of imprinting play in sporadic cancers?
About 10% of people show loss of imprinitng at the IGF2 gene un lymphocytes and have a 3-5 fold increase frequency of colon adenomas