HC7 Epigenetics, chromatin & DNA methylation

Question 1

epigenetic regulation

Answer 1

heritable change in gene expression or genome function, determined by modifications of DNA or chromatin

Question 2

rats lick and groom: effects

Answer 2

DNA methylation will be removed from hippocampal glucocorticoid receptor gene –> increased expression –> low stress levels in pup –> pups will lick and groom their pups.
no licking and grooming: DNA methylation –> high stress levels –> no licking and grooming.

So the behaviour is heritable

Question 3

Nucleosome

Answer 3

DNA wrapped around histones. 147bp. 2x H2A, 2x H2B, 2x H3, 2xH4

Question 4

heterochromatin

Answer 4

condensed DNA: gene expression always off.
H3K9me,
DNA methylation,
transposons, other repetative DNA

Question 5

Euchromatin

Answer 5

gene expression can be active or inactive.

Question 6

inactive euchromatin

Answer 6

H3K27me, polycomb repressive proteins
also called facultative heterochromatin

Question 7

active euchromatin

Answer 7

H3K4me, H3K36me, histone acetylation (H3ac), RNA polymerase II, activator proteins = trithorax proteins.

Question 8

DNA methylation where?

Answer 8

always inactivation of genes.
Mostly at TEs and repetitive elements to prevent insertional mutagenesis.
Also in gene body

Question 9

transposons and DNA stability

Answer 9

they can change position in DNA.
- get excised and dumped elsewhere
- get amplified and amplified regions ends up somewhere else in genome.

It threatens genome function and stability, so they must be silenced.

Question 10

DNA methylation definition,

Answer 10

covalent modification of DNA –> 5 methylcytosine 5mC

Question 11

DNA methylation plants and animals

Answer 11

• animals: mostly 5’-CG-3’ (in ESCs and brain sometimes 5’-CA-3’)
• plants CG, CHG, (symmetric, palindromic) CHH (asymmetric)

Question 12

DNA methylation results in? + how

Answer 12

transcriptional silencing by:
- blocking binding of TFs
recruiting DNA methyl binding proteins (MBPs)–> these recruit repressor proteins (e.g. H3K9 methyltransferases)

DNA methyltransferases bind to histon deacetylases and histone methyltransferases –> transcription inactivation

Question 13

DNA methylation blot analysis: restriction enzymes

Answer 13

methylation sensitive restriction enzymes:
- EcoRI cuts GAATTC no matter if C is methylated or not.
AluI only cuts AGCT is C is unmethylated

Question 14

bisulfite sequencing

Answer 14

dsDNA is denatured –> treat with bisulfite –> unmethylated Cs are changed into Us (deamination).

This is strand specific!

Question 15

MeDIP-chip

Answer 15

immunoprecipitation of 5mC.
Sonicate genomic DNA –> short DNA fragments –> incubate with antibody against 5mC –> precipitate the antibody bound DNA–> attach fluorescent label
Also add random DNA with other label.
You look at average 5mC level over a fragment

Question 16

intragenic DNA methylation (on the gene body)

Answer 16

to prevent cryptic initiation of transcription

when RNA pol II transcribes a gene –> histones get displaced –> SETD2 is associated to RNA pol II–> adds H3K36me to histones behind RNA pol –> DNMT3b binds to H3K36me –> adds DNA methylation to gene body –> prevents cryptic initiation of transcription.
HDAC is also recruited by H3K36me –> acetyl groups behind RNA pol removed –> 5mC added.

5mC prevents RNA pol to randomly bind on a gene and start random transcription