VL 27 (Isabel Bäurle) Flashcards
Definition Epigenetics
- any potentially stable, heritable change in gene expression that occurs without DNA sequence change
- describes phenomena in which genetically identical cells/organisms express their genomes differently, causing phenotypic differences
X chromosome inactivation involves epigenetic silencing:
- Color only in female cats
- X-linked gene for fur color
- Expressed → black
- Not expressed→orange
- Inactive one X during
development by epigenetic silencing
Mechanisms of Epigenetic inheritance: DNA Methylation
- DNA methylation→ inactivate gene
- replication → hemimethylated state
- DNA methylase methylates other Cytosine
Mechanisms of Epigenetic inheritance: Prions
prion = a proteinaceous infectious agent that behaves as an inheritable trait, although it contains no nucleic acid.
Examples are:
– PrPSc, the agent of scrapie in sheep and bovine
spongiform encephalopathy, and
– PSI, which confers an inherited state in yeast
Yeast prions show unusual inheritance:
* Sup35
–> in WT soluble form = translation termination factor
–> also as alternative form of oligomeric aggregates, in which it ́s not active in protein synthesis
* spontaneous transition with low frequency
* presence of oligomeric form causes newly synthesized protein to acquire inactive structure
* [PSI+] state forms amyloid fibres
- Purified protein can convert the [psi–] state
of yeast to [PSI+]
Position-effect variegation (PEV)
- first described in drosophila
- PEV in eye color results when white gene is
integrated near heterochromatin - cells with
- inactive white gene
→white patches o active white gene
‘→red patches
Heterochromatin propagates from a nucleation event
- heterochromatin is nucleated at specific sequence, and inactive structure propagates along chromatin fiber
- heterochromatin nucleation caused by protein-binding to specific sequences
- genes within regions of heterochromatin are inactivated
- length of inactive region varies from cell – cell
→ result: gene inaction in this vicinity
→ PEV
Su(var) and E(var) genes
- mutant screens for PEV-suppressor, enhancer
- Su(var) includes HP1, histone methyltransferases HDAC
- E(var) include SWI/SNF chromatin remodelers
HP1: key protein in forming mammalian heterochromatin
- acts by binding to H3K9me → formation of higher-order chromatin structures
Polycomb and trithorax are antagonistic repressors and activators:
- Polycomb group proteins (Pc-G) perpetuate repression state through cell division
- polycomb response protein (PRE)
–> = DNA sequence required for Pc-G action
–> nucleation center from which Pc-G proteins propagate an inactive structure in order to from an epigenetic memory mediated by PREs - trithorax group proteins (TrxG) antagonize Pc-G actions
- Pc-G, TrxG can bind PRE with opposing effects
Picture
* Enhancer of variegation genes → entogonize formation of heterochromatin
* Suppressor of variegation genes → less → more active white gene → positive regulators of heterochromati
* H3K9me → HP1 recognizes → binds → HP1 self-aggregation
* Polycomb found in drosophila; homeotic genes
* H3C/K trimethylation
* Repressors recruit polycomb proteins → repressors vanish
* Repressor proteins independent from polycomb
* Silcencing not maintained in Pc-G mutants → reactivation
DNA methylation
Function:
* reduces DNA-binding of many proteins
* condenses chromatin structure
* binding site for methyl binding proteins
CpG islands are subject to methylation:
- occur in 5 ́ regions of metazoans, contain clustered CpG dinucleotides
- most methyl DNA groups found on cytosine on both CpG doublet strands
In plants DNA methylation occurs in all sequence contexts (see picture)
* plants without CpG islands
* arabidopsis
* heterochromatin near centromere (many transposons,
repetitive sequences)
* methylation in heterochromatin→methylation types
important for keeping heterochromatin state
* no CHG, CHH methylation
* higher CG methylation
CG methylation can be propagated during DNA replication
Asymmetric methylation sites
require additional information
asymmetric methylation sites maintained (+ initiated) by information on associated histones + RNA-based mechanism + RNA-directed DNA methylation (RdDM) → directs DNA methylases to these sites
Some sites are maintained by small
interfering RNAs (siRNAs)
- siRNAs in contact with complexes which have methylases
Method: methylation-sensitive restriction E & Bisulfite Mutagenesis and Sequencing
- no methylation → digestion
- methylation on inner cytosine → digestion by Msp1, Hpa2 doesn ́t digest
➔ southern blot: cleaved or not?
➔ probing membrane with gene specific sequence, which has to be analysed
➔ vert: methylated sequence - DNA denature → Sodiumbisulfate (unmethylated cytosine → uracil)
- amplify treated DNA with PCR
- introduced mutations according to DNA methylation pattern → DNA
sequencing (Sanger-sequencing or Whole genome sequencing)
X-inactivation involves stabilization of Xist RNA, which coats the inactive chromosome:
X inactivation center (Xic)
= cis-acting region on X chromosome
–> necessary, sufficient to ensure that only one X chromosome remains active o includes Xist gene
▪ codes for ncRNA
▪ found on inactive X chromosomes
▪ expressed from inactive X copy
In the late phase, X inactivation is independent of Xist RNA:
