Epigenetics & Gene Editing
Lecture 1 – Histones (DNA structure & organisation) (chapter 4: 193-229)
Epigenetics: reprogramming gene expression
● Heritable yet reversible changes in genome functioning
● Not encoded in the DNA seq (incl. DNA methylation, histone modifications)
● Accessibility of the gene → level of expression
● Determines
○ Differentiation and maintenance of cell types
○ Healthy ageing (e.g., epigenetic clock)
○ Disease diagnostics and therapies
● Induced pluripotent stem cells = example of epigenetic editing
Gene editing: rewriting gene sequence
● Make changes to the DNA sequence in a controlled (gene targeted) manner (includes CRISPR)
● Rewriting gene seq → ethical issues
DNA = 3.1 x109 bp (3 billion bp)
● only 1% of is protein-coding
● 45% is transcribed into RNA
● not all genes are expressed in all cell types
● expression profiles are maintained during cell division
● Regulation of gene expression (Ch4, Ch7)
Diseases:
● Heritable diseases is NOT always due to changes in DNA seq, mendelian diseases is only a
small part of the diseases
● many heritable susceptibility = “it runs in the family” → can be genetics but often
epigenetics
● most affected by the environment = “you are what you eat” → epigenetics
DNA: sequences (genes) versus organisation (open)
● Genetics focuses on the sequence of DNA (which genes exist).
○ Quality: What type of RNA/protein is being produced? (e.g., different isoforms
of a protein due to alternative splicing).
● Epigenetics influences how DNA is organized and regulated (which genes are turned on/off).
○ Quantity: How much of a certain RNA/protein is produced? (e.g., higher expression of an enzyme in one
tissue compared to another)
DNA structure
● 4 building blocks, double helix, karyotype → geneticists
● From double helix to karyotypes → steps, really controlled → histones
Gene organisation
● Brown = known genes
● Red = predicted genes (vast majority are still only predicted => importance
of gene editing studies!!)
● Regulatory DNA sequence = determine not the kind of protein but the
amount →
● In a gene: there are introns (non-coding regions) and small part is exon
(coding region)
● Some seq are not coding proteins but are expressed → not without function
(genes that encode RNA only)
,Human genome =
● Length (2.5m): 3.1 x109 bp (3 billion bp)
● protein coding genes: ca 20.000
● non-coding RNAs (ncRNAs): ca 5000 → regulate gene expression rather than producing protein
● Median gene size: 26000 nts
● Mean exon size: 145 nts
○ Only 1% of genome is exons
○ >45% of genome can be transcribed
○ Ca 50% repetitive sequences → may have regulatory/structural
role
If the human genome is 3bill bp long, and only 1% codes for proteins, is the other 99%
junk? NO!!!
Unique junk sequences:
● 40% of junk is unique
● introns (regulation gene expression)
● regulation gene expression (incl. promoters)
● non-coding RNAs (regulation gene expression)
And this 1.5% of genes is not coded in every cell → epigenetics
● How is it regulated?
● How to interfere?
● DNA is really long in a very small place
Functions of the DNA
1. Gene expression → DNA is used as a template for RNA and
protein synthesis.
2. Replication → DNA copies itself before cell division.
a. Both in interphase: chromosomes are long and thin →
more accessibility
3. Cell division → DNA must be properly distributed to daughter
cells.
a. mitotic chromosomes are compact (compacted form is only in m-phase)
→ tight packaging
Minimum parts of a chromosome:
● Kinetochore: epigenetics determine where it binds
● Telomeres (2 per chromosome): replication of the ends protection against “repair”
– marked by sequences and epigenetics
● Replication origins (multiple): start duplication of DNA
● Centromere (1 per chromosome):
○ keeping 2 daughter chromatids together during mitosis
○ human: approx. 100,000 nts
Keynotes: Chromosomes transition between an open, accessible state (interphase=duplication) and a
highly compacted state (mitosis) to balance gene function with proper inheritance. Often the
chromosomes are in the interphase – chromatin leaking out of lysed during interphase. In M-phase =
chromosomes condense, and the kinetochore forms at the centromere to attach to the mitotic
spindle → accurate chromosome segregation to ensure each daughter cell receives the correct
number of chromosomes
DNA organisation
● DNA is not ‘naked’ double helix
● DNA is made up of nucleotides and is compacted to fit in the nucleus
● Compaction:
, ○ Beads on a string formation → not how DNA likes to be → basic compaction of
DNA (step 1 formation)
○ Chromatin fiber of packed nucleosomes
○ Fiber in loops → entire mitotic chromosome
● Histones H2A, H2B, H3, H4
○ also histone variants (e.g. H3.3) → specialised roles
○ Help package DNA into chromatin
○ Histones undergo post-translational modifications (e.g., acetylation, methylation)
to regulate gene expression
● Nucleosome = octamer of histones
○ Contain (2x(H3+H4) + 2x(H2A+H2B))
○ 147 bp are wrapped around it
○ and 1x linker DNA connects adjacent nucleosomes
● Chromatin = DNA + histones + non-histone proteins
○ 1 : 1 : 1 ratio
○ Chromatin further compacts into higher-order structures (e.g., loops, mitotic chromosomes).
○ Interphase chromatin: fibers, roughly 2 types
■ Heterochromatin = dense, closed → inactive genes (gene silencing)
■ Euchromatin = less dense, open → active in gene expression
○ Chromosome compaction levels (example: chr 22 = 48 x 106 nt pairs)
■ Linear double helix → 1.5 cm if fully extended.
■ Interphase chromatin → Compacted 30 µm (~500x shorter than linear DNA).
■ Mitotic chromosome → Highly condensed 2 µm (~10,000x shorter than linear DNA).
○ Dynamic Chromatin Organization:
■ Chromatin switches between euchromatin & heterochromatin depending on:
● Location (specific genes, nuclear regions).
● Timing (gene activation or silencing based on cellular needs).
○ Visual Representations:
■ "Beads on a string" model (electron microscopy): Shows how nucleosomes
(histone-wrapped DNA) form the basic chromatin structure.
■ Open vs. Closed Chromatin Diagram: Illustrates how chromatin compaction affects gene
transcription—only open chromatin allows transcription.
Packing unit = nucleosome
● It consists of 8 histone proteins forming an octamer.
● 147 base pairs of DNA wraps around the histone core ~1.7 times
○ These 2 form the nucleosome core
● Linker DNA (<80 bps) connects adjacent nucleosomes → highly accessible
Histone Composition:
● The histone octamer consists of: 2x H2A, 2x H2B, 2x H3, 2x H4.
● Histones = well conserved proteins in evolution (~60 million copies per type per cell).
● Histone size: 102-135 amino acids (aa).
Nucleosome assembly (histone folds)
● Each histone has:
● An N-terminal tail (subject to modifications that regulate gene expression).
● A histone fold domain (involved in histone-histone interactions and DNA
binding).
○ H3 + H4 dimerize first
○ H2A + H2B dimerize
○ 2 x (H3-H4) = tetramer + 2 (H2A-H2B) = octamer
Nucleosome Disassembly:
, ● Nuclease = digests linker DNA → releasing the nucleosome core.
● High salt concentration causes the histone octamer to dissociate into individual histones and free DNA.
Histone Tails and Their Function:
● All 8 histones have protruding tails
● These tails undergo modifications (e.g., methylation, acetylation) that regulate chromatin structure and gene
expression.
Nucleosomal DNA
● Less accessible than linker DNA
● DNA has no particular sequence preference for nucleosome binding, but
it can be affected by DNA-binding proteins
● Tight association of DNA-histones → stable nucleosome structure
● Despite the tight packaging, transcription/replication/repair possible =
“breathing”, chromatin remodeling
● Chromatin "Breathing" (Spontaneous DNA Unwrapping)
○ Nucleosomal DNA spontaneously unwraps and rewraps →
temporarily accessible for proteins.
○ The cycle occurs 4 times per second, with DNA exposed for
10-50 milliseconds each time.
○ Stable yet dynamic
○ For a short time, during the unwrapping, regulatory DNA
sequence is exposed, regulatory proteins can bind → cellular
processes. When the protein does not bind the DNA keeps on breathing (unwrapping
and rewrapping)
○ This process is ATP-independent and allows for protein binding to regulatory
sequences.
● Histone-DNA Interaction Stability
○ Nucleosomes form 142 hydrogen bonds with DNA, stabilizing their structure.
○ Histones contain many (+)ly charged aa (K=lysine, R=arginine) → interact with the
(-)ly charged DNA backbone.
● Sequence Preferences in DNA Bending
○ A-T rich regions tend to be positioned in the minor groove inside the nucleosome.
○ G-C rich regions are often in the minor groove outside, contributing to nucleosome
stability
Chromatin remodelling (ATP-dependent, controlled)
(fig 4-26) Example: nucleosome sliding = ATP dependent chromatin remodeling complex can
slide the DNA over the histone octane → other seq is accessible.
● Unlike spontaneous chromatin "breathing", chromatin remodeling is ATP-dependent
and actively controlled.
● It involves chromatin remodeling complexes (large protein machines) that:
○ Bind histones and DNA.
○ Contain ATPase subunits (enzymes that hydrolyze ATP).
○ Read histone modifications.
● Remodeling requires multiple rounds of ATP hydrolysis,
often 1 ATP per base pair moved.
Types of ATP-Dependent Chromatin Remodeling:
1. Nucleosome sliding (Fig 4-26): Histone octamer moves
along the DNA to expose or hide regulatory sequences
2. Histone dimer exchange (A) (Fig 4-27): Histone variants
(e.g., H2A-H2B) are swapped in or out by chaperones,
affecting chromatin properties.
Lecture 1 – Histones (DNA structure & organisation) (chapter 4: 193-229)
Epigenetics: reprogramming gene expression
● Heritable yet reversible changes in genome functioning
● Not encoded in the DNA seq (incl. DNA methylation, histone modifications)
● Accessibility of the gene → level of expression
● Determines
○ Differentiation and maintenance of cell types
○ Healthy ageing (e.g., epigenetic clock)
○ Disease diagnostics and therapies
● Induced pluripotent stem cells = example of epigenetic editing
Gene editing: rewriting gene sequence
● Make changes to the DNA sequence in a controlled (gene targeted) manner (includes CRISPR)
● Rewriting gene seq → ethical issues
DNA = 3.1 x109 bp (3 billion bp)
● only 1% of is protein-coding
● 45% is transcribed into RNA
● not all genes are expressed in all cell types
● expression profiles are maintained during cell division
● Regulation of gene expression (Ch4, Ch7)
Diseases:
● Heritable diseases is NOT always due to changes in DNA seq, mendelian diseases is only a
small part of the diseases
● many heritable susceptibility = “it runs in the family” → can be genetics but often
epigenetics
● most affected by the environment = “you are what you eat” → epigenetics
DNA: sequences (genes) versus organisation (open)
● Genetics focuses on the sequence of DNA (which genes exist).
○ Quality: What type of RNA/protein is being produced? (e.g., different isoforms
of a protein due to alternative splicing).
● Epigenetics influences how DNA is organized and regulated (which genes are turned on/off).
○ Quantity: How much of a certain RNA/protein is produced? (e.g., higher expression of an enzyme in one
tissue compared to another)
DNA structure
● 4 building blocks, double helix, karyotype → geneticists
● From double helix to karyotypes → steps, really controlled → histones
Gene organisation
● Brown = known genes
● Red = predicted genes (vast majority are still only predicted => importance
of gene editing studies!!)
● Regulatory DNA sequence = determine not the kind of protein but the
amount →
● In a gene: there are introns (non-coding regions) and small part is exon
(coding region)
● Some seq are not coding proteins but are expressed → not without function
(genes that encode RNA only)
,Human genome =
● Length (2.5m): 3.1 x109 bp (3 billion bp)
● protein coding genes: ca 20.000
● non-coding RNAs (ncRNAs): ca 5000 → regulate gene expression rather than producing protein
● Median gene size: 26000 nts
● Mean exon size: 145 nts
○ Only 1% of genome is exons
○ >45% of genome can be transcribed
○ Ca 50% repetitive sequences → may have regulatory/structural
role
If the human genome is 3bill bp long, and only 1% codes for proteins, is the other 99%
junk? NO!!!
Unique junk sequences:
● 40% of junk is unique
● introns (regulation gene expression)
● regulation gene expression (incl. promoters)
● non-coding RNAs (regulation gene expression)
And this 1.5% of genes is not coded in every cell → epigenetics
● How is it regulated?
● How to interfere?
● DNA is really long in a very small place
Functions of the DNA
1. Gene expression → DNA is used as a template for RNA and
protein synthesis.
2. Replication → DNA copies itself before cell division.
a. Both in interphase: chromosomes are long and thin →
more accessibility
3. Cell division → DNA must be properly distributed to daughter
cells.
a. mitotic chromosomes are compact (compacted form is only in m-phase)
→ tight packaging
Minimum parts of a chromosome:
● Kinetochore: epigenetics determine where it binds
● Telomeres (2 per chromosome): replication of the ends protection against “repair”
– marked by sequences and epigenetics
● Replication origins (multiple): start duplication of DNA
● Centromere (1 per chromosome):
○ keeping 2 daughter chromatids together during mitosis
○ human: approx. 100,000 nts
Keynotes: Chromosomes transition between an open, accessible state (interphase=duplication) and a
highly compacted state (mitosis) to balance gene function with proper inheritance. Often the
chromosomes are in the interphase – chromatin leaking out of lysed during interphase. In M-phase =
chromosomes condense, and the kinetochore forms at the centromere to attach to the mitotic
spindle → accurate chromosome segregation to ensure each daughter cell receives the correct
number of chromosomes
DNA organisation
● DNA is not ‘naked’ double helix
● DNA is made up of nucleotides and is compacted to fit in the nucleus
● Compaction:
, ○ Beads on a string formation → not how DNA likes to be → basic compaction of
DNA (step 1 formation)
○ Chromatin fiber of packed nucleosomes
○ Fiber in loops → entire mitotic chromosome
● Histones H2A, H2B, H3, H4
○ also histone variants (e.g. H3.3) → specialised roles
○ Help package DNA into chromatin
○ Histones undergo post-translational modifications (e.g., acetylation, methylation)
to regulate gene expression
● Nucleosome = octamer of histones
○ Contain (2x(H3+H4) + 2x(H2A+H2B))
○ 147 bp are wrapped around it
○ and 1x linker DNA connects adjacent nucleosomes
● Chromatin = DNA + histones + non-histone proteins
○ 1 : 1 : 1 ratio
○ Chromatin further compacts into higher-order structures (e.g., loops, mitotic chromosomes).
○ Interphase chromatin: fibers, roughly 2 types
■ Heterochromatin = dense, closed → inactive genes (gene silencing)
■ Euchromatin = less dense, open → active in gene expression
○ Chromosome compaction levels (example: chr 22 = 48 x 106 nt pairs)
■ Linear double helix → 1.5 cm if fully extended.
■ Interphase chromatin → Compacted 30 µm (~500x shorter than linear DNA).
■ Mitotic chromosome → Highly condensed 2 µm (~10,000x shorter than linear DNA).
○ Dynamic Chromatin Organization:
■ Chromatin switches between euchromatin & heterochromatin depending on:
● Location (specific genes, nuclear regions).
● Timing (gene activation or silencing based on cellular needs).
○ Visual Representations:
■ "Beads on a string" model (electron microscopy): Shows how nucleosomes
(histone-wrapped DNA) form the basic chromatin structure.
■ Open vs. Closed Chromatin Diagram: Illustrates how chromatin compaction affects gene
transcription—only open chromatin allows transcription.
Packing unit = nucleosome
● It consists of 8 histone proteins forming an octamer.
● 147 base pairs of DNA wraps around the histone core ~1.7 times
○ These 2 form the nucleosome core
● Linker DNA (<80 bps) connects adjacent nucleosomes → highly accessible
Histone Composition:
● The histone octamer consists of: 2x H2A, 2x H2B, 2x H3, 2x H4.
● Histones = well conserved proteins in evolution (~60 million copies per type per cell).
● Histone size: 102-135 amino acids (aa).
Nucleosome assembly (histone folds)
● Each histone has:
● An N-terminal tail (subject to modifications that regulate gene expression).
● A histone fold domain (involved in histone-histone interactions and DNA
binding).
○ H3 + H4 dimerize first
○ H2A + H2B dimerize
○ 2 x (H3-H4) = tetramer + 2 (H2A-H2B) = octamer
Nucleosome Disassembly:
, ● Nuclease = digests linker DNA → releasing the nucleosome core.
● High salt concentration causes the histone octamer to dissociate into individual histones and free DNA.
Histone Tails and Their Function:
● All 8 histones have protruding tails
● These tails undergo modifications (e.g., methylation, acetylation) that regulate chromatin structure and gene
expression.
Nucleosomal DNA
● Less accessible than linker DNA
● DNA has no particular sequence preference for nucleosome binding, but
it can be affected by DNA-binding proteins
● Tight association of DNA-histones → stable nucleosome structure
● Despite the tight packaging, transcription/replication/repair possible =
“breathing”, chromatin remodeling
● Chromatin "Breathing" (Spontaneous DNA Unwrapping)
○ Nucleosomal DNA spontaneously unwraps and rewraps →
temporarily accessible for proteins.
○ The cycle occurs 4 times per second, with DNA exposed for
10-50 milliseconds each time.
○ Stable yet dynamic
○ For a short time, during the unwrapping, regulatory DNA
sequence is exposed, regulatory proteins can bind → cellular
processes. When the protein does not bind the DNA keeps on breathing (unwrapping
and rewrapping)
○ This process is ATP-independent and allows for protein binding to regulatory
sequences.
● Histone-DNA Interaction Stability
○ Nucleosomes form 142 hydrogen bonds with DNA, stabilizing their structure.
○ Histones contain many (+)ly charged aa (K=lysine, R=arginine) → interact with the
(-)ly charged DNA backbone.
● Sequence Preferences in DNA Bending
○ A-T rich regions tend to be positioned in the minor groove inside the nucleosome.
○ G-C rich regions are often in the minor groove outside, contributing to nucleosome
stability
Chromatin remodelling (ATP-dependent, controlled)
(fig 4-26) Example: nucleosome sliding = ATP dependent chromatin remodeling complex can
slide the DNA over the histone octane → other seq is accessible.
● Unlike spontaneous chromatin "breathing", chromatin remodeling is ATP-dependent
and actively controlled.
● It involves chromatin remodeling complexes (large protein machines) that:
○ Bind histones and DNA.
○ Contain ATPase subunits (enzymes that hydrolyze ATP).
○ Read histone modifications.
● Remodeling requires multiple rounds of ATP hydrolysis,
often 1 ATP per base pair moved.
Types of ATP-Dependent Chromatin Remodeling:
1. Nucleosome sliding (Fig 4-26): Histone octamer moves
along the DNA to expose or hide regulatory sequences
2. Histone dimer exchange (A) (Fig 4-27): Histone variants
(e.g., H2A-H2B) are swapped in or out by chaperones,
affecting chromatin properties.
