The human genome, epigenetics, and chromatin remodeling

Question 1

Composition of the human genome

Answer 1

1. Retrotransposons (~42%), mobile eleme nts (RNA
   intermediate)
   * LTR - long terminal repeats
   * LINEs - long interspersed nuclear elements (99%
   inactive)
   * SINEs - short interspersed nuclear elements (Alu)
2. DNA transposons (bacteria-like, small fraciton)
3. Inverted repeats (2 mill)- hairpin structures,
   regulatory roles.
4. Segmental duplications (~5%), blocks of 1-200
   kb, several regions
5. Highly repetitive DNA, >106 copies per genome,
   SSRs/STRs – simple sequence/short tandem (~3%),
   telomeres and centromeres

Question 2

DNA methylation

Answer 2

Cytosine can be methylated
* Stable modification, best studied epigenetic mark
“p” refers to
phosphate group
linking the two
bases
MOL3100: The human genome, epigenetics and chromatin remodeling
C 5-metC
* DNA methylation occurs at C in a CpG context (C before G in the DNA sequence)
* 70-80% of all CpG sites in the human genome is methylated (CpGs are underrepresented)
* Silencing of genes (repression of CpG-rich promoters and transposable elements)
* In females: Inactive X chromosome is heavily DNA methylated

De novo methylation: DNMT3a and DNMT3b

Maintenance methylation: DNMT1

Question 3

DNA methylation by DNA methyltransferase (DNMT)

Answer 3

Cytosine is converted to 5-methylcytosine by DNA methyltransferases (DNMTs)
MOL3100: The human genome, epigenetics and chromatin remodeling
* Methyl donor: S-Adenosyl methionine (SAM)
* De novo DNMTs: put the initial pattern of methyl groups on DNA CpG sites (development)
* Maintenance DNMTs: copy the methylation pattern from the DNA template to the new
strand after replication (somatic cell division)

Question 4

Maintenance DNA methylation by DNMT1

Answer 4

CpG methylation is inherited from mother cell to daughter cells
* DNMT1 (bound to PCNA) recognizes 5-met-C on the parental strand

Question 5

DNA methylation; regulation of gene expression

Answer 5

CpG islands are CG-rich sequences of ~1kb, located in promoters and 5’ region of genes (60% of
human genes)
* CpG islands are usually non-methylated in germ cells, early embryo and most somatic tissue
* CFP1 (CXXC finger protein 1) bind DNA with high unmethylated CG → mark active genes
* Methylation at CpG islands is associated with transcription repression

What causes the silencing effect of DNA methylation?
Methylation of DNA does not affect the charge
* The methyl group may physically prevent the binding of transcription factors
* Methylated DNA recruit proteins that have a methyl-CpG-binding domain, and these proteins will in
turn recruit other chromatin remodeling factors
Methylated DNA function as a signal for other proteins to locate to the locus and
silence genes and form heterochromatin
 Genomic imprinting (only one allele active)
 X chromosome inactivation

Question 6

DNA methylation status during embryonic development:

Answer 6

Se slide 41

Question 7

DNA demethylation;
active and passive mechanisms

Answer 7

Active demethylation: enzymatic process - TET
mediated oxidation of the methyl-group followed by
base excision repair (BER)

Passive demethylation: lack of maintenance
methylation during replication, no DNMT1 or
inhibition of DNMT1

Question 8

DNA demethylation trough stepwise oxidation by the TET (ten-eleven translocase)
enzymes

Answer 8

5hmC, 5fC, 5caC are not
recognized by DNMT1,
→ no methylation of nascent
strand

TDG (thymine DNA glycosylase) can excise T (T:G), 5-fC (formyl) and 5-caC (carboxyl) from DNA,
→ The resulting Abasic sites are repaired by BER (AP-endonuclease, polymerase, ligase)