Methylace (a methylome)
dědičnost - obratlovci - CpG (C phosphate G) páry - přenos na komplementární GC enzymem "maintenance methylatransferase" (tvorba 3-methylcytosinu)
- 75% CpG methylováno, nemethylované CpG islands - 5' regulační konce mnoha genů (při jejich patologické hypermethylaci pak hrozí gene silencing... např. v onkogenezi)
- mechanismus funkce methylace -
1) vazba methyl-CpG-binding domain proteins (MBDs) - ten pak váže další proteiny - jako histone deacetylasy, histon remodelační proteiny - modifikace histonů - vznik heterochromatinu
2) znemožnění přístupu enzymů k DNA
Methylace a vývoj
- po fertilizaci rozsáhlá demethylace - nebo spíš hydroxylace methylových skupin (pokles aktivity MM nebo specifická demethyláza, nicméně následně opět methylace de novo methylatransferázami
- 75% CpG methylováno, nemethylované CpG islands - 5' regulační konce mnoha genů (při jejich patologické hypermethylaci pak hrozí gene silencing... např. v onkogenezi)
- mechanismus funkce methylace -
1) vazba methyl-CpG-binding domain proteins (MBDs) - ten pak váže další proteiny - jako histone deacetylasy, histon remodelační proteiny - modifikace histonů - vznik heterochromatinu
2) znemožnění přístupu enzymů k DNA
- co to dělá?
1) regulace exprese - vypínání nepotřebných genů pro danou buněčnou linii - funkční ale i informační - identita buněčného typu (jsem neuron..)
2) inaktivace X
3) genomický imprinting
4) vypíná inkorporované virové geny
5) dlouhodobá paměť - neurony
Baktérie
- methylace i adenosinu (N6-methyladenosin)
Detekce methylace DNA
- methylation specific PCR (MSP)
sodium bisulfite (1:1 směs hydrogensiřičitanu sodného NaHSO3 a disiřičitanu sodného Na2S2O5 (HS2O5 =HSO3 -+SO2))
- nemethylované se mění na uracil,
- whole genom bisulfite sequencing (BS-seq)
- HELP Assay
is an abbreviation for HpaII tiny fragment Enrichment by Ligation-mediated PCR.
HpaII and by MspI digestion of the genome followed by ligation-mediated PCR. HpaII only digests 5'-CCGG-3' sites when the cytosine in the central CG dinucleotide is unmethylated, the HpaII representation is enriched for the hypomethylated fraction of the genome. The MspI representation is a control for copy number changes and PCR amplification difficulties.
is an abbreviation for HpaII tiny fragment Enrichment by Ligation-mediated PCR.
HpaII and by MspI digestion of the genome followed by ligation-mediated PCR. HpaII only digests 5'-CCGG-3' sites when the cytosine in the central CG dinucleotide is unmethylated, the HpaII representation is enriched for the hypomethylated fraction of the genome. The MspI representation is a control for copy number changes and PCR amplification difficulties.
HUMAN EPIGENETICS
Showing your age
Hannum et al. carried out microarray-based genome-wide DNA methylation analysis of the blood of 482 individuals ranging from ages 19 to 101. They found 71 sites for which the methylation status was highly correlated with age, allowing age prediction on the basis of this methylation signature. Prediction was accurate to within ~5 years, both in this cohort and in an independent cohort of 174 individuals. Furthermore, most of the markers were located near to genes linked to ageing-related processes and diseases.
These blood-derived signatures were also applicable to various other normal tissues, implying that molecular ageing at the level of DNA methylation is similar across tissue types.
The authors calculated apparent methylomic ageing rates (AMARs), which is the ratio of the methylation-derived molecular age to the actual chronological age, and looked for determinants of this ageing rate. As expected, males tended to age faster than females. Also, exome sequencing of a subset of the cohort identified three genetic variants that influence the ageing rate, seemingly by altering the methylation state of nearby age-dependent methylation sites.
ORIGINAL RESEARCH PAPER Hannum, G. et al. Genome-wide methylation profiles reveal quantitative views of human aging rates. Mol. Cell 21 Nov 2012 (doi:10.1016/j.molcel.2012.10.016)
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