Epigenetics and gene-editing
Lecture 1 15.03.2021
Organization DNA
- Coding DNA for proteins
o 21 000 genes
- Noncoding DNA
o Introns
o Promotors
o Regulate gene expression
- Chromosomes
o Compact during mitosis (2 μm)
o Aligned during interphase (30 μm)
o Parts of chromosome
Telomeres (2 per chromosome)
Replication origins
Centromere – keeping two chromatids together
- Genome size is not equal to protein coding number
Histones
- Highly conserved among species
o Pea vs dog only 2 amino acids different in histone 4
- Post-translational modifications
- H2A, H2B, H3, H4
- Nucleosome: octamer of histones
o 2x H3, 2x H4, 2xH2A and 2x H2B (two times dimer)
H3-H4
H2A – H2B
o 147 bp DNA
o 1x linker DNA (80 bp)
Determines “nucleosome-outgoing” DNA
1 linker histone H1 present per nucleosome core
o > 142 hydrogen bonds per nucleosome indicates DNA interaction with the
nucleosomes
o 1/5 of the nucleosomes are positively/neutral charged indicates interaction with
negatively charged DNA
- Chromatin = DNA + histones + non-histone proteins 1:1:1
o Heterochromatin: closed
o Euchromatin: open
o Accessibility is independent of ATP – “breathing”
Occurs 4 times per second
- Chromatin remodeling = ATP dependent
o Nucleosome sliding (pulling nucleosome core along the DNA double helix), which is
chaperone-independent
o Using chaperons
Histone exchange
Nucleosome core removal
Nucleosome core exchange
- Chromatin structure is opened by
o Breathing (spontaneous) expose part of DNA, allowing proteins to bind
o Nucleosome binding (regulated)
, o Nucleosome core removal/replacement
o Histone replacement
o Histone tail alterations
- Different proteins are needed for chromatin opening
o ATP dependent chromatin remodelers
o Histone chaperones
o Histone modifiers (writers, erasers)
- Zigzag structure
o Compacted DNA
o 30 nm
- Beads on a string
o Open DNA
- Histone tail
o They assist in compaction
o Interaction through tails (particular histone 4)
o Linker histone H1
1 molecule present per nucleosome core (out of the 8 histones, one is H1)
Binds DNA and protein
Larger than other core histone
Less conserved
Determines the direction of nucleosome outgoing DNA
Chromatin is dynamically regulated
- It can switch off genes (silencing)
- Euchromatin contains a barrier that prevents the heterochromatin
spreading event
- Provides the cell with a cellular memory (open/closed structure is
passed on to the next generation)
o Cellular memory epigenetic inheritance
- Spreading effect
o Active gene is translocated to a heterochromatin area inactive (silencing) =
position effect
o Position effect variegation: different appearances in one tissue – like in the eye of the
Drosophila fly. A part of the heterochromatin DNA is put into the euchromatin part
and is thus expressed
o Another important example is X-inactivation
Most common histone modifications – histone tail posttranslational modifications
Deacetylation and methylation repress transcription
H3 is mostly post-translational modified
- Acetylation: removing positive charge results in a more open structure of the chromatin
, o Always acetylation of the lysine – the lysine loses its positive charge since it gets an
acetyl group
o Open chromatin structure thus more DNA transcription
o HDAC inhibitors can be used to re-express tumour suppressor genes in cancer
- Methylation
o Also methylation of the lysine
o Mono, di or tri-methylated
o Can then not be acetylated
- The processes are mutually exclusive: the same lysine cannot be both methylated and
acetylated
- Phosphorylation
o Of the serine
o Introduces negative charges (PO4 is negatively charged)
o DNA is more open since the DNA is also negatively charged and thus repelled
o Transcription factors can thus bind more easily
Other histone variants
- Chaperons can exchange/remove part of the nucleosome core
- There are no variants for histone 4
- They are expressed in a lesser amount than the normal histones in a nucleosome
- The variants act to loosening the chromatin structure
- Histone 3 variants
o H3.3 MyoD promotor differentiates normal cells into muscle cells
How can the cell read the histone modifications?
- The histone-tail modifications are position-dependent (it is dependent on which amino acid
they act whether they repress or activate DNA transcription)
o In histone tail 3
o Trimethylation on lysine 9 silencing by formation of heterochromatin (=H3K9Me3)
o Trimethylation on lysine 4 gene expression
o Trimethylation on lysine 27 silencing by recruiting the Polycomb complex
- Depends on mono vs di vs tri-methylation
o That determines what type of complex will recognize the methylation
o Trimethylation is recognized by the ING-PHD domain
- Depends on other modifications
o A reader protein can only bind to specific modification
o Code-reader complex = multiple reader proteins only a specific combination of
modifications leads to strong binding of this complex
, o Example: the NurF (nucleosome remodeling factor) can only bind if there are also
two other type of modifications present on the tail
- There are also erasers that remove the modification
o HDAC inhibitors remove the acetylation
Chromatin spreading marks
- Initiated by a regulatory protein that binds to an area and recruits an histone-modifying
enzyme (=writer protein)
o The writer makes a mark lysine 9 methylation by histone methyltransferase
- This recruits a reader protein
- This recruits a writer protein
o Writes the k9 methylation on the neighbouring nucleosome
- This recruits again a reader protein
- This initiates ATP-dependent chromatin modelling on a larger area that makes the accessible
DNA into highly condensed DNA
Stop of spreading by
1. Binding of chromatin to core structures
2. Binding to positive chromatin
3. Recruit activating histone writers
Guest lecture Bruggeman
Histone variants and cancer
Types of histones
- Canonical histones
o Most common
o DNA replication coupled
o Do not contain introns
o No poly-A signal
o Genes in clusters on chromosomes
o Function in basic organisation of the chromatin
o Function is to speed up transcription
- Variant histones
o Replication-independent
o Exist for H2A, H2B, H1 and H3
o Contain introns and poly-A signal
o Several functions (transcription, enhancer activation, DNA damage repair,
heterochromatinization, centromeres, telomeres)
o Only in histone 4 there are no variants
Histone chaperones
- Help the correct folding of histone proteins and prevents unspecific binding to negatively
charged proteins
- Bind histones in the cytoplasm
- Facilitate dimer formation
- Upon entry in the nucleus, chaperones bind the dimers and facilitate deposition into nuclear
bodies or chromatin
Nucleosome dynamics
- Active during DNA replication and transcription
- Temporal unwinding of the DNA helix
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