Slides + nota’s + remote work
EPIGENETICS
2024-2025
,Table of Contents
Lecture 1: Introduction to Epigenetics (EG)
1. Epigenetics: Scope and Definitions ................................................................................. - 1 -
2. Substrates for Epigenetic Information ............................................................................. - 2 -
2.1. Histone and Nucleosome Structure........................................................................................... - 2 -
2.2. Histone Variants ..................................................................................................................... - 5 -
2.2.1. Histone H3.3 .................................................................................................................................................. - 5 -
2.2.2. Histone CENP-A ............................................................................................................................................ - 6 -
2.2.3. Histone H2A.X ............................................................................................................................................... - 6 -
2.2.4. Histone MacroH2A ........................................................................................................................................ - 7 -
2.3. Histone Modifications (= overview, see Lecture 2) ...................................................................... - 7 -
2.4. DNA Modifications ((De)methylation) (= see Lecture 3) .............................................................. - 7 -
2.5. Chromatin Remodeling (=see Lecture 4) .................................................................................... - 7 -
Lecture 2: Histone Modifications and ncRNA
1. Overview ................................................................................................................... - 8 -
2. Histone Modifications ................................................................................................... - 8 -
2.1. A Concept of Writers, Readers and Erasers of Histone Modifications ........................................... - 8 -
2.2. High Resolution Modifications .................................................................................................. - 9 -
3. Histone Acetylation...................................................................................................... - 9 -
3.1. General process ....................................................................................................................... - 9 -
3.2. Mediators (HATs), Readers and Erasers (HDACs)....................................................................... - 10 -
3.2.1. Mediators (HATs)......................................................................................................................................... - 10 -
3.2.2. Readers ....................................................................................................................................................... - 10 -
3.2.3. Erasers (HDACs) .......................................................................................................................................... - 11 -
4. Histone Methylation .................................................................................................... - 11 -
4.1. General Process:..................................................................................................................... - 11 -
4.2. PEV and Su(VAR)s .................................................................................................................. - 11 -
4.3. Extra Paper: Regulation of Chromatin Structure by Site-specific Histone H3 HMTs ...................... - 11 -
4.3.1. Main Points of Paper .................................................................................................................................... - 12 -
4.3.2. Why is this process an exemplar? ................................................................................................................. - 13 -
4.4. Function: a Matter of Context ................................................................................................. - 13 -
4.4.1. Overview ..................................................................................................................................................... - 13 -
4.4.2. Histone Methylation in Gene Activation....................................................................................................... - 13 -
4.4.3. Histone Methylation in Gene Repression ..................................................................................................... - 13 -
4.4.4. Ambivalent Gene Regulation ....................................................................................................................... - 15 -
4.5. Histone Methylation not as Stable as Thought ......................................................................... - 15 -
4.5.1. Iron and Oxygen Dependent KDMs .............................................................................................................. - 15 -
I
,5. Histone Phosphorylation ..............................................................................................- 16 -
5.1. Overview .............................................................................................................................. - 16 -
6. Overview of Modifications to Remember ........................................................................ - 17 -
7. EG Marks and Other Factors ........................................................................................ - 18 -
7.1. Chromatin States .................................................................................................................. - 18 -
7.2. ENCODE ............................................................................................................................... - 18 -
8. Non-coding RNAs ....................................................................................................... - 19 -
8.1. Introduction .......................................................................................................................... - 19 -
8.1.1. How to Define/Identify non-coding RNA? .................................................................................................... - 19 -
8.2. LncRNAs in Action .................................................................................................................. - 20 -
8.2.1. Categories Based on Mechanisms of Action ................................................................................................. - 20 -
8.2.2. Examples ..................................................................................................................................................... - 20 -
8.3. LncRNA Identification ............................................................................................................ - 22 -
8.4. Discovery of RNA-Based Silencing Mechanisms ........................................................................ - 22 -
8.4.1. Components of RNA-Based Silencing Mechanisms ...................................................................................... - 22 -
8.5. Post-Transcriptional Gene Silencing (PTGS) ............................................................................. - 23 -
8.5.1. Biogenesis and Function of microRNAs........................................................................................................ - 23 -
8.6. Transcriptional Gene Silencing (TGS) .......................................................................................- 24 -
8.7. Circular RNAs – New in TGS and PTGS .....................................................................................- 24 -
Lecture 3: DNA Modifications
1. Methodical Analysis of DNA Modifications .................................................................... - 26 -
2. DNA Methylation ....................................................................................................... - 26 -
2.1. Mechanism ............................................................................................................................ - 27 -
2.1.1. CpG Context ................................................................................................................................................ - 27 -
2.1.2. DNA Methyltransferases (DNMTs) ............................................................................................................... - 27 -
2.1.3. Non-CG methylation.................................................................................................................................... - 28 -
2.2. Additional Factors and Impact on Gene Expression .................................................................. - 29 -
3. DNA Demethylation/hydroxymethylation ..................................................................... - 29 -
3.1. Active ................................................................................................................................... - 29 -
3.1.1. In Gene Expression ...................................................................................................................................... - 30 -
3.2. Passive .................................................................................................................................. - 30 -
3.3. Link Between Both ................................................................................................................. - 30 -
3.3.1. 5mC and 5hmC as Biomarkers ..................................................................................................................... - 30 -
3.4. Genetic Determination and Environmental Influence ................................................................ - 31 -
3.4.1. Non-shared Environment ............................................................................................................................ - 31 -
3.4.2. Heritability................................................................................................................................................... - 31 -
3.5. DNA Methylation in a Medical Setting ..................................................................................... - 32 -
II
,Lecture 4: Chromatin Dynamics
1. Chromatin Dynamics ................................................................................................... - 33 -
2. Chromatin Remodeling Complexes ................................................................................ - 33 -
3. Cellular Memory ......................................................................................................... - 34 -
3.1. PcG/TrxG System: Maintaining Cellular Memory .......................................................................- 34 -
3.2. Molecular Mechanisms of PcG/TrxG Proteins ............................................................................- 34 -
4. Dosage Compensation Systems ..................................................................................... - 35 -
4.1. Definition .............................................................................................................................. - 35 -
4.1.1. Consequences of Gene Dosage Differences Arising from Sex Chromosome Erosion .................................... - 35 -
4.2. Drosophila melanogaster (image) ............................................................................................ - 35 -
4.3. Mammals: X Chromosome Inactivation (XCI) ............................................................................ - 35 -
4.3.1. Discovery ..................................................................................................................................................... - 35 -
4.3.2. General Overview ........................................................................................................................................ - 36 -
4.3.3. Consequences.............................................................................................................................................. - 36 -
4.3.4. Which XCI is Discussed: The XCI Cycle ......................................................................................................... - 36 -
4.3.5. Mechanism of XCI ........................................................................................................................................ - 37 -
4.3.6. XCI is not always Random, it can be Skewed ................................................................................................ - 41 -
4.3.7. Escaping from XCI........................................................................................................................................ - 42 -
4.3.8. Mosaic Females ........................................................................................................................................... - 43 -
5. Chromatin Dynamics During Reprogramming to iPSCs ..................................................... - 43 -
6. ATAC-sequencing....................................................................................................... - 44 -
Clinical Applications in Epigenetics
1. Methods In Research ...................................................................................................- 45 -
1.1. Single Cell Analysis .................................................................................................................- 45 -
2. Clinical Application: Epigenetics in cancer ......................................................................- 45 -
2.1. In General: What Constitutes Cancer? ......................................................................................- 45 -
2.2. Histone Modifications in Cancers ............................................................................................ - 46 -
2.3. Histone (De)acetylation in Cancers ......................................................................................... - 46 -
2.3.1. Overview of Mechanism and Components ................................................................................................... - 46 -
2.3.2. Alterations of HATs in Cancer ...................................................................................................................... - 46 -
2.3.3. Acetyl-Lysine Recognition Proteins and Cancer ........................................................................................... - 47 -
2.3.4. Alterations of HDAC in Cancer ..................................................................................................................... - 47 -
2.3.5. HAT and HDAC Inhibitors in Cancer Therapy ............................................................................................... - 48 -
2.4. DNA Methylation in Cancer .................................................................................................... - 48 -
2.4.1. DNA Hypermethylation in Cancer ................................................................................................................ - 49 -
2.4.2. DNA Hypomethylation in Cancer ................................................................................................................. - 49 -
2.4.3. Loss of Imprinting Through Alterations of DNA Methylation ....................................................................... - 50 -
2.4.4. Mutations in the DNA Methylation Modifiers ............................................................................................... - 50 -
2.4.5. Towards DNA Methylation Markers in Clinics .............................................................................................. - 51 -
2.5. Dysregulation of PcG/TrxG Components Lead to Cancers .......................................................... - 52 -
III
,3. Clinical Application: Cardiovascular Epigenetics
♥ .......................................................... - 53 -
3.1. Introduction ........................................................................................................................... - 53 -
3.2. Congenital Heart Disorders (CHDs) .......................................................................................... - 53 -
3.3. Overview of Heart Failure (HF) Mechanisms.............................................................................. - 53 -
3.4. DNA Modifications in the Heart ...............................................................................................- 54 -
3.4.1. DNA Methylation and Hydroxymethylation ................................................................................................. - 54 -
3.4.2. DNA Methylation in Hypertrophy ................................................................................................................ - 54 -
3.4.3. DNA Hydroxymethylation in Hypertrophy ................................................................................................... - 54 -
3.4.4. DNA Modifications in Fibrosis ...................................................................................................................... - 55 -
3.5. Histone Modifications in the Heart........................................................................................... - 55 -
3.5.1. Overview ..................................................................................................................................................... - 55 -
3.5.2. Histone Acetylation in Cardiac Hypertrophy ................................................................................................ - 55 -
3.5.3. Histone Methylation in Hypertrophy ............................................................................................................ - 56 -
3.5.4. Histone Modifications in Fibrosis ................................................................................................................. - 57 -
3.6. Chromatin Architecture and lncRNA ........................................................................................ - 57 -
3.6.1. Chromatin Architecture in HF ...................................................................................................................... - 57 -
3.6.2. LncRNA and the Epigenome in Hypertrophy................................................................................................ - 57 -
3.7. Towards an EG-based Therapy for HF ..................................................................................... - 58 -
3.8. Overview .............................................................................................................................. - 58 -
4. Clinical Application: Making Sense of the Ageing Methylome ........................................... - 59 -
4.1. DNA Methylation in Development ...........................................................................................- 59 -
4.2. The Epigenetic Clock ............................................................................................................. - 60 -
5. Clinical Application: X-linked Diseases ...........................................................................- 61 -
5.1. Genetic Overview .................................................................................................................. - 61 -
5.2. RETT Syndrome (RTT) ........................................................................................................... - 61 -
5.2.1. Therapeutic Application for Xi-reactivation.................................................................................................. - 61 -
6. Clinical Applications: Other Diseases .............................................................................- 61 -
6.1. ICF Syndrome ....................................................................................................................... - 61 -
6.2. Prader-Willi Syndrome (PWS)................................................................................................. - 61 -
Appendix: Examen Questions + Answers
IV
,Lecture 1: Introduction to Epigenetics (EG)
1. Epigenetics: Scope and Definitions
• 27 June 2000: The genetic code to life deciphered!
• 15 February 2001: The genetic code to life published!
o We thought we knew everything, but alas knowing how it works not…
o E.g. phenotypic differences possible in monozygotic twins
§ Genomic DNA identical (slight differences possible)
§ Mitochondrial DNA identical
§ Due to environmental differences?
§ Differenct X-inactivation pattern?
o E.g. phenotypic differences in inbred/cloned animals
§ Same genome, many individuals
§ Obese to lean, combination of the same pairs gives the same result in offspring
o E.g. phenotypic differences of a chromosome
We see two identical X chromosomes inside a
female cell. The highlighted chromosome is coated
by XIST (X inactive specific transcript) RNA (=
lncRNA) which silences the gene expression. Also
called X chromosome inactivation (XCI).
o E.g. phenotypic differences of cell types (hundreds of cell types, one genome)
Multiple epigenomes define which functions will be used. The
epigenome refers to the collection of chemical modifications on
DNA and histones that regulate gene expression. While every cell
inherits the same genetic blueprint, its epigenetic modifications
dictate which genes are turned on or off—effectively shaping its
identity and function. It’s remembered during cell division but not
necessarily fixed.
• A modern analogy: Waddington’s EG landscape
o Old version: deterministic train tracks
§ Problem: no switching of tracks (= we can push cells in a certain
direction without reverting them back to stem cells)
§ Problem: we can’t go backwards (= we can dedifferentiate cells)
o New version: sideway and backward pushes are possible
• A genome alone cannot create an organism; it requires a reader, such as a
cell or fertilized egg from the same species, to activate molecular
processes in the nucleus and around the DNA.
• Three big domains: developmental, stochastic or age-dependent and exogenous or environmental epigenetics
-1-
,(Debated) definitions of epigenetics:
• Holliday: Mechanisms of temporal and spatial control of gene activity during the
development of complex organisms
§ Pos: aspect of time and space
§ Neg: doesn’t mention EG beyond development, heredity or chromatin modifications
• Riggs: Mitotically/meiotically heritable changes in gene function that cannot be
explained by changes in DNA sequence
§ Pos: covers somatic and germline distinction
§ Neg: ++ heritable changes, no transient epigenetic modifications, would imply that cells that
do not divide would not have an epigenome but e.g. neurons have also an epigenome
• Bird: Structural adaptation of chromosomal regions so as to register, signal or
perpetuate altered activity states
§ Pos: acknowledges signal and a memory system
§ Neg: too vague
• Roadmap: Heritable changes in gene activity and expression and also stable, long- term
alterations in the transcriptional potential of cells that aren’t necessarily heritable
§ Pos: acknowledges heritable and non-heritable aspects + transcriptional potential
§ Neg: heritable vs. not necessarily heritable
• GRC1: Stably heritable phenotype resulting from changes in a chromosome without
alterations in the DNA sequence
§ Pos: captures key principle (no change to DNA sequence)
§ Neg: too restrictive, focus on phenotypes
2. Substrates for Epigenetic Information
2.1. Histone and Nucleosome Structure
• For an overview of the structure of DNA (macro molecules, bindings, helix…) see ‘Molecular Biology’ (Ba1)
• DNA code exists out of 3 billion (=3 miljard) bp’s ≈ 2 meters
o Problem: needs to be squished into a nucleus of 10"m
o Analogue: thread to the moon and back inside a football
• Discovery of nucleosomal structures (Olin & Olin)
o Electron microscopy observations (1974):
§ Linear arrays of spherical particles, suggesting a repeating structure
o Nuclease Digestion Experiments:
§ Chromatin partially digested with nucleases => DNA fragments at regular intervals of ~150 bp’s
§ DNA wrapped around protected structures, with nuclease-sensitive regions in between
1
Genome Reference Consortium is responsible for maintaining and improving the human reference genome, as well as reference
genomes for other species.
-2-
, • Nucleosome = single repeat unit
o = ESI2 shows equal weights of protein and DNA
o = 8 histone proteins = octamer complex
§ 2x H3-H4 dimer as core à forms a tertramer
§ 2x H2A-H2B dimer more loosely associated
§ +charged => basic AA side chains
o = 146 core bp + 50 linker bp of DNA
§ -charged => phosphate groups of DNA backbone
§ wrapped around octamer in two left-handed turns (see figure3)
§ DNA dyad = locus of single strand part
• 5 types of histones:
o Overview:
Name Length (AA) Genes (N) N --> C structure
H1 ≈215 (1) 10 (5) (3) N-tail – Winged helix – (1) C-tail
H2A ≈130 (3) 22 (1) (1) N-tail – HLHLH – (2) C-tail
H2B ≈125 (4) 19 (2) (4) N-tail – HLHLH – (3) C-tail
H3 ≈135 (2) 11 (4) (1) N-tail – HLHLH – (5) C-tail
H4 ≈100 (5) 13 (3) (2) N-tail – HLHLH – (4) C-tail
o HLHLH (helix-loop-helix-loop-helix) = Histone fold domain
o flexible N-terminal tails are accessible for PTM
o Histone H1:
§ Not always present
§ Structurally dissimilar
§ Max 1 histone H1 per nucleosome
§ Located outside the core nucleosome particle
§ Drives higher-order chromatin compaction => 30nm fiber
§ Stabilizes linker DNA + locks both ends in close vicinity
• Nucleosome positions (see also L4, Error! Reference source not found.):
o DNA sequence-independent positioning o DNA sequence-guided (FALSE)
§ Implies flexible nucleosome positions § Different histones for different DNA motifs
§ Nucleosomes positions are sequence-independent § Like TF
because histones interact with the phosphate § ++ #histone variants
backbone of the DNA and not with the actual bases § Implies too stable/rigid and predetermined
≠ from TF (transcription factors) chromatin + fixed position
§ Requires 1 set of histones
2
(ESI = electron scanning imaging) is a technique used in mass spectrometry to study the structure and composition of
biomolecules. It is widely used in proteomics, epigenetics, and metabolomics to analyze molecular modifications, including post-
translational modifications and epigenetic marks.
3
At a distance of approximately 70 bp, the two DNA loops face the same surface of the nucleosome. Two hypothetical factor
binding- sites are indicated by the boxed “A”. As one turn of the DNA is completed every 10 bp, both “A” boxes can be facing the
surface through the major grove, depending on the position of the nucleosome. Nucleosome positioning along the DNA thereby
allows for changes in binding-site geometry that can have a regulatory function
-3-
, o DNA: one full turn every 10 bp => same side outwards every 10 bp
o At the dyad: strong bonds needed => G/C-rich (3 H-bonds)
o Distant from dyad: WW periodicity (Weak-Weak = A/T) every 10 bp
§ Facilitates bending DNA around nucleosome
§ Associate with core histones
o Nucleosome position = flexible ≠ completely random, predictable
o Nucleosome positions affect mutational periodicity
§ Lower mutation frequency in linker DNA
– More access to repair mechanisms (NER, BER4…)
– Higher repair efficiency
– No shielding from damage recognition by nucleosome core
§ Higher mutation frequency at minor groove facing histones (every 10bp)
– Reduced DNA repair access (= tightly bound to histone)
– Bending of DNA is structural deformation => +likelihood mutations
– Replication and transcription hindrance (minor groove-facing = +stable histone bound)
• RC vs. RI nucleosome assembly:
o Replication-Coupled o Replication-Independent
§ During S phase => rapid stability § Throughout cell cycle
§ Canonical histones § Histone variants
– H1, H2A, H2B, H3(.1, .2), H4 – H2A.Z, H3.3, CENP-A, macroH2A…
– Intronless – Polyadenylated, spliced
– mRNAs: not poly-adenylated, 26b stem – Not encoded in gene clusters
loop instead at 3’ UTR => + efficient § Histone replacement, chromatin remodeling,
– Encoded by 2 gene clusters (e.g. on chr. 6) transcription regulation => dynamic
nucleosome
o Process of RC assembly (see also Error! Reference source not found.):
4
Nucleotide excision repair and Base excision repair
-4-
, o Nucleosome (dis)assembly dynamics during S phase (±8 hours => time-efficiency!) (see L4, Error!
Reference source not found.)
§ Dynamic and reversible process
§ ~30,000,000 histone octamers need to be removed
§ ~60,000,000 histone octamers need to be assembled
§ Parental nucleosomes are either recycled (= most common) or degraded
§ New histones are generated and integrated
§ Recycling = essential to copy epigenetic information to daughter cells
– CAF1 is involved in assembling new histones. – FACT complex helps in disassembling and reassembling nucleosomes.
– ASF1 assists by delivering histones. – MCM2 can bind evicted H3-H4 tetramers and, together with ASF1, help
recycle histone dimers.
§ Inheritance needed to maintain epigenetic memory
§ Incorporation of RC histones to maintain DNA chromatinized
§ Mostly random distribution of paternal histones (= from paternal DNA strand)
– Some notable exceptions, e.g. asymmetrical distribution in germline stem cells of Drosophila
§ Mechanistic details remain incompletely understood.
2.2. Histone Variants
• Evolved from archaeal histone (tetramers, ~tetrasome, • Each have distinct function
single wrap) • Some stabilize or destabilize nucleosomes
• More variants of H3 and H2A, fewer of H4 or H2B • AA changes => different PTMs
• Some differ in only a few AA from core histones • Found in specialized chromatin domains
• Deposited in a RI manner (e.g. centromere or telomere)
2.2.1. Histone H3.3
• RI production and deposition
• 4 AA difference from H3 (3 at α2 for UBN1 recognition, 1 at the tail)
In transcriptionally active chromatin In repressed chromatin
Enhancers, promotors, gene bodies Telomeres, pericentric regions, short tandem
repeats, retrotransposons
High dynamic turn-over Low dynamic turn-over
Deposition: Deposition by ATRX-DAXX
• New H3.3 bound and recycled by ASF1-HIRA complex To resolve RI H3.1 loss due to transcription, DNA
• Old H3.3 bound and recycled by UBN1-HIRA complex damage, spontaneous unwrapping, nucleosome-
≠ CAF1 as with H3 inhibitory sequences (telomeric repeats), aging,...
-5-
EPIGENETICS
2024-2025
,Table of Contents
Lecture 1: Introduction to Epigenetics (EG)
1. Epigenetics: Scope and Definitions ................................................................................. - 1 -
2. Substrates for Epigenetic Information ............................................................................. - 2 -
2.1. Histone and Nucleosome Structure........................................................................................... - 2 -
2.2. Histone Variants ..................................................................................................................... - 5 -
2.2.1. Histone H3.3 .................................................................................................................................................. - 5 -
2.2.2. Histone CENP-A ............................................................................................................................................ - 6 -
2.2.3. Histone H2A.X ............................................................................................................................................... - 6 -
2.2.4. Histone MacroH2A ........................................................................................................................................ - 7 -
2.3. Histone Modifications (= overview, see Lecture 2) ...................................................................... - 7 -
2.4. DNA Modifications ((De)methylation) (= see Lecture 3) .............................................................. - 7 -
2.5. Chromatin Remodeling (=see Lecture 4) .................................................................................... - 7 -
Lecture 2: Histone Modifications and ncRNA
1. Overview ................................................................................................................... - 8 -
2. Histone Modifications ................................................................................................... - 8 -
2.1. A Concept of Writers, Readers and Erasers of Histone Modifications ........................................... - 8 -
2.2. High Resolution Modifications .................................................................................................. - 9 -
3. Histone Acetylation...................................................................................................... - 9 -
3.1. General process ....................................................................................................................... - 9 -
3.2. Mediators (HATs), Readers and Erasers (HDACs)....................................................................... - 10 -
3.2.1. Mediators (HATs)......................................................................................................................................... - 10 -
3.2.2. Readers ....................................................................................................................................................... - 10 -
3.2.3. Erasers (HDACs) .......................................................................................................................................... - 11 -
4. Histone Methylation .................................................................................................... - 11 -
4.1. General Process:..................................................................................................................... - 11 -
4.2. PEV and Su(VAR)s .................................................................................................................. - 11 -
4.3. Extra Paper: Regulation of Chromatin Structure by Site-specific Histone H3 HMTs ...................... - 11 -
4.3.1. Main Points of Paper .................................................................................................................................... - 12 -
4.3.2. Why is this process an exemplar? ................................................................................................................. - 13 -
4.4. Function: a Matter of Context ................................................................................................. - 13 -
4.4.1. Overview ..................................................................................................................................................... - 13 -
4.4.2. Histone Methylation in Gene Activation....................................................................................................... - 13 -
4.4.3. Histone Methylation in Gene Repression ..................................................................................................... - 13 -
4.4.4. Ambivalent Gene Regulation ....................................................................................................................... - 15 -
4.5. Histone Methylation not as Stable as Thought ......................................................................... - 15 -
4.5.1. Iron and Oxygen Dependent KDMs .............................................................................................................. - 15 -
I
,5. Histone Phosphorylation ..............................................................................................- 16 -
5.1. Overview .............................................................................................................................. - 16 -
6. Overview of Modifications to Remember ........................................................................ - 17 -
7. EG Marks and Other Factors ........................................................................................ - 18 -
7.1. Chromatin States .................................................................................................................. - 18 -
7.2. ENCODE ............................................................................................................................... - 18 -
8. Non-coding RNAs ....................................................................................................... - 19 -
8.1. Introduction .......................................................................................................................... - 19 -
8.1.1. How to Define/Identify non-coding RNA? .................................................................................................... - 19 -
8.2. LncRNAs in Action .................................................................................................................. - 20 -
8.2.1. Categories Based on Mechanisms of Action ................................................................................................. - 20 -
8.2.2. Examples ..................................................................................................................................................... - 20 -
8.3. LncRNA Identification ............................................................................................................ - 22 -
8.4. Discovery of RNA-Based Silencing Mechanisms ........................................................................ - 22 -
8.4.1. Components of RNA-Based Silencing Mechanisms ...................................................................................... - 22 -
8.5. Post-Transcriptional Gene Silencing (PTGS) ............................................................................. - 23 -
8.5.1. Biogenesis and Function of microRNAs........................................................................................................ - 23 -
8.6. Transcriptional Gene Silencing (TGS) .......................................................................................- 24 -
8.7. Circular RNAs – New in TGS and PTGS .....................................................................................- 24 -
Lecture 3: DNA Modifications
1. Methodical Analysis of DNA Modifications .................................................................... - 26 -
2. DNA Methylation ....................................................................................................... - 26 -
2.1. Mechanism ............................................................................................................................ - 27 -
2.1.1. CpG Context ................................................................................................................................................ - 27 -
2.1.2. DNA Methyltransferases (DNMTs) ............................................................................................................... - 27 -
2.1.3. Non-CG methylation.................................................................................................................................... - 28 -
2.2. Additional Factors and Impact on Gene Expression .................................................................. - 29 -
3. DNA Demethylation/hydroxymethylation ..................................................................... - 29 -
3.1. Active ................................................................................................................................... - 29 -
3.1.1. In Gene Expression ...................................................................................................................................... - 30 -
3.2. Passive .................................................................................................................................. - 30 -
3.3. Link Between Both ................................................................................................................. - 30 -
3.3.1. 5mC and 5hmC as Biomarkers ..................................................................................................................... - 30 -
3.4. Genetic Determination and Environmental Influence ................................................................ - 31 -
3.4.1. Non-shared Environment ............................................................................................................................ - 31 -
3.4.2. Heritability................................................................................................................................................... - 31 -
3.5. DNA Methylation in a Medical Setting ..................................................................................... - 32 -
II
,Lecture 4: Chromatin Dynamics
1. Chromatin Dynamics ................................................................................................... - 33 -
2. Chromatin Remodeling Complexes ................................................................................ - 33 -
3. Cellular Memory ......................................................................................................... - 34 -
3.1. PcG/TrxG System: Maintaining Cellular Memory .......................................................................- 34 -
3.2. Molecular Mechanisms of PcG/TrxG Proteins ............................................................................- 34 -
4. Dosage Compensation Systems ..................................................................................... - 35 -
4.1. Definition .............................................................................................................................. - 35 -
4.1.1. Consequences of Gene Dosage Differences Arising from Sex Chromosome Erosion .................................... - 35 -
4.2. Drosophila melanogaster (image) ............................................................................................ - 35 -
4.3. Mammals: X Chromosome Inactivation (XCI) ............................................................................ - 35 -
4.3.1. Discovery ..................................................................................................................................................... - 35 -
4.3.2. General Overview ........................................................................................................................................ - 36 -
4.3.3. Consequences.............................................................................................................................................. - 36 -
4.3.4. Which XCI is Discussed: The XCI Cycle ......................................................................................................... - 36 -
4.3.5. Mechanism of XCI ........................................................................................................................................ - 37 -
4.3.6. XCI is not always Random, it can be Skewed ................................................................................................ - 41 -
4.3.7. Escaping from XCI........................................................................................................................................ - 42 -
4.3.8. Mosaic Females ........................................................................................................................................... - 43 -
5. Chromatin Dynamics During Reprogramming to iPSCs ..................................................... - 43 -
6. ATAC-sequencing....................................................................................................... - 44 -
Clinical Applications in Epigenetics
1. Methods In Research ...................................................................................................- 45 -
1.1. Single Cell Analysis .................................................................................................................- 45 -
2. Clinical Application: Epigenetics in cancer ......................................................................- 45 -
2.1. In General: What Constitutes Cancer? ......................................................................................- 45 -
2.2. Histone Modifications in Cancers ............................................................................................ - 46 -
2.3. Histone (De)acetylation in Cancers ......................................................................................... - 46 -
2.3.1. Overview of Mechanism and Components ................................................................................................... - 46 -
2.3.2. Alterations of HATs in Cancer ...................................................................................................................... - 46 -
2.3.3. Acetyl-Lysine Recognition Proteins and Cancer ........................................................................................... - 47 -
2.3.4. Alterations of HDAC in Cancer ..................................................................................................................... - 47 -
2.3.5. HAT and HDAC Inhibitors in Cancer Therapy ............................................................................................... - 48 -
2.4. DNA Methylation in Cancer .................................................................................................... - 48 -
2.4.1. DNA Hypermethylation in Cancer ................................................................................................................ - 49 -
2.4.2. DNA Hypomethylation in Cancer ................................................................................................................. - 49 -
2.4.3. Loss of Imprinting Through Alterations of DNA Methylation ....................................................................... - 50 -
2.4.4. Mutations in the DNA Methylation Modifiers ............................................................................................... - 50 -
2.4.5. Towards DNA Methylation Markers in Clinics .............................................................................................. - 51 -
2.5. Dysregulation of PcG/TrxG Components Lead to Cancers .......................................................... - 52 -
III
,3. Clinical Application: Cardiovascular Epigenetics
♥ .......................................................... - 53 -
3.1. Introduction ........................................................................................................................... - 53 -
3.2. Congenital Heart Disorders (CHDs) .......................................................................................... - 53 -
3.3. Overview of Heart Failure (HF) Mechanisms.............................................................................. - 53 -
3.4. DNA Modifications in the Heart ...............................................................................................- 54 -
3.4.1. DNA Methylation and Hydroxymethylation ................................................................................................. - 54 -
3.4.2. DNA Methylation in Hypertrophy ................................................................................................................ - 54 -
3.4.3. DNA Hydroxymethylation in Hypertrophy ................................................................................................... - 54 -
3.4.4. DNA Modifications in Fibrosis ...................................................................................................................... - 55 -
3.5. Histone Modifications in the Heart........................................................................................... - 55 -
3.5.1. Overview ..................................................................................................................................................... - 55 -
3.5.2. Histone Acetylation in Cardiac Hypertrophy ................................................................................................ - 55 -
3.5.3. Histone Methylation in Hypertrophy ............................................................................................................ - 56 -
3.5.4. Histone Modifications in Fibrosis ................................................................................................................. - 57 -
3.6. Chromatin Architecture and lncRNA ........................................................................................ - 57 -
3.6.1. Chromatin Architecture in HF ...................................................................................................................... - 57 -
3.6.2. LncRNA and the Epigenome in Hypertrophy................................................................................................ - 57 -
3.7. Towards an EG-based Therapy for HF ..................................................................................... - 58 -
3.8. Overview .............................................................................................................................. - 58 -
4. Clinical Application: Making Sense of the Ageing Methylome ........................................... - 59 -
4.1. DNA Methylation in Development ...........................................................................................- 59 -
4.2. The Epigenetic Clock ............................................................................................................. - 60 -
5. Clinical Application: X-linked Diseases ...........................................................................- 61 -
5.1. Genetic Overview .................................................................................................................. - 61 -
5.2. RETT Syndrome (RTT) ........................................................................................................... - 61 -
5.2.1. Therapeutic Application for Xi-reactivation.................................................................................................. - 61 -
6. Clinical Applications: Other Diseases .............................................................................- 61 -
6.1. ICF Syndrome ....................................................................................................................... - 61 -
6.2. Prader-Willi Syndrome (PWS)................................................................................................. - 61 -
Appendix: Examen Questions + Answers
IV
,Lecture 1: Introduction to Epigenetics (EG)
1. Epigenetics: Scope and Definitions
• 27 June 2000: The genetic code to life deciphered!
• 15 February 2001: The genetic code to life published!
o We thought we knew everything, but alas knowing how it works not…
o E.g. phenotypic differences possible in monozygotic twins
§ Genomic DNA identical (slight differences possible)
§ Mitochondrial DNA identical
§ Due to environmental differences?
§ Differenct X-inactivation pattern?
o E.g. phenotypic differences in inbred/cloned animals
§ Same genome, many individuals
§ Obese to lean, combination of the same pairs gives the same result in offspring
o E.g. phenotypic differences of a chromosome
We see two identical X chromosomes inside a
female cell. The highlighted chromosome is coated
by XIST (X inactive specific transcript) RNA (=
lncRNA) which silences the gene expression. Also
called X chromosome inactivation (XCI).
o E.g. phenotypic differences of cell types (hundreds of cell types, one genome)
Multiple epigenomes define which functions will be used. The
epigenome refers to the collection of chemical modifications on
DNA and histones that regulate gene expression. While every cell
inherits the same genetic blueprint, its epigenetic modifications
dictate which genes are turned on or off—effectively shaping its
identity and function. It’s remembered during cell division but not
necessarily fixed.
• A modern analogy: Waddington’s EG landscape
o Old version: deterministic train tracks
§ Problem: no switching of tracks (= we can push cells in a certain
direction without reverting them back to stem cells)
§ Problem: we can’t go backwards (= we can dedifferentiate cells)
o New version: sideway and backward pushes are possible
• A genome alone cannot create an organism; it requires a reader, such as a
cell or fertilized egg from the same species, to activate molecular
processes in the nucleus and around the DNA.
• Three big domains: developmental, stochastic or age-dependent and exogenous or environmental epigenetics
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,(Debated) definitions of epigenetics:
• Holliday: Mechanisms of temporal and spatial control of gene activity during the
development of complex organisms
§ Pos: aspect of time and space
§ Neg: doesn’t mention EG beyond development, heredity or chromatin modifications
• Riggs: Mitotically/meiotically heritable changes in gene function that cannot be
explained by changes in DNA sequence
§ Pos: covers somatic and germline distinction
§ Neg: ++ heritable changes, no transient epigenetic modifications, would imply that cells that
do not divide would not have an epigenome but e.g. neurons have also an epigenome
• Bird: Structural adaptation of chromosomal regions so as to register, signal or
perpetuate altered activity states
§ Pos: acknowledges signal and a memory system
§ Neg: too vague
• Roadmap: Heritable changes in gene activity and expression and also stable, long- term
alterations in the transcriptional potential of cells that aren’t necessarily heritable
§ Pos: acknowledges heritable and non-heritable aspects + transcriptional potential
§ Neg: heritable vs. not necessarily heritable
• GRC1: Stably heritable phenotype resulting from changes in a chromosome without
alterations in the DNA sequence
§ Pos: captures key principle (no change to DNA sequence)
§ Neg: too restrictive, focus on phenotypes
2. Substrates for Epigenetic Information
2.1. Histone and Nucleosome Structure
• For an overview of the structure of DNA (macro molecules, bindings, helix…) see ‘Molecular Biology’ (Ba1)
• DNA code exists out of 3 billion (=3 miljard) bp’s ≈ 2 meters
o Problem: needs to be squished into a nucleus of 10"m
o Analogue: thread to the moon and back inside a football
• Discovery of nucleosomal structures (Olin & Olin)
o Electron microscopy observations (1974):
§ Linear arrays of spherical particles, suggesting a repeating structure
o Nuclease Digestion Experiments:
§ Chromatin partially digested with nucleases => DNA fragments at regular intervals of ~150 bp’s
§ DNA wrapped around protected structures, with nuclease-sensitive regions in between
1
Genome Reference Consortium is responsible for maintaining and improving the human reference genome, as well as reference
genomes for other species.
-2-
, • Nucleosome = single repeat unit
o = ESI2 shows equal weights of protein and DNA
o = 8 histone proteins = octamer complex
§ 2x H3-H4 dimer as core à forms a tertramer
§ 2x H2A-H2B dimer more loosely associated
§ +charged => basic AA side chains
o = 146 core bp + 50 linker bp of DNA
§ -charged => phosphate groups of DNA backbone
§ wrapped around octamer in two left-handed turns (see figure3)
§ DNA dyad = locus of single strand part
• 5 types of histones:
o Overview:
Name Length (AA) Genes (N) N --> C structure
H1 ≈215 (1) 10 (5) (3) N-tail – Winged helix – (1) C-tail
H2A ≈130 (3) 22 (1) (1) N-tail – HLHLH – (2) C-tail
H2B ≈125 (4) 19 (2) (4) N-tail – HLHLH – (3) C-tail
H3 ≈135 (2) 11 (4) (1) N-tail – HLHLH – (5) C-tail
H4 ≈100 (5) 13 (3) (2) N-tail – HLHLH – (4) C-tail
o HLHLH (helix-loop-helix-loop-helix) = Histone fold domain
o flexible N-terminal tails are accessible for PTM
o Histone H1:
§ Not always present
§ Structurally dissimilar
§ Max 1 histone H1 per nucleosome
§ Located outside the core nucleosome particle
§ Drives higher-order chromatin compaction => 30nm fiber
§ Stabilizes linker DNA + locks both ends in close vicinity
• Nucleosome positions (see also L4, Error! Reference source not found.):
o DNA sequence-independent positioning o DNA sequence-guided (FALSE)
§ Implies flexible nucleosome positions § Different histones for different DNA motifs
§ Nucleosomes positions are sequence-independent § Like TF
because histones interact with the phosphate § ++ #histone variants
backbone of the DNA and not with the actual bases § Implies too stable/rigid and predetermined
≠ from TF (transcription factors) chromatin + fixed position
§ Requires 1 set of histones
2
(ESI = electron scanning imaging) is a technique used in mass spectrometry to study the structure and composition of
biomolecules. It is widely used in proteomics, epigenetics, and metabolomics to analyze molecular modifications, including post-
translational modifications and epigenetic marks.
3
At a distance of approximately 70 bp, the two DNA loops face the same surface of the nucleosome. Two hypothetical factor
binding- sites are indicated by the boxed “A”. As one turn of the DNA is completed every 10 bp, both “A” boxes can be facing the
surface through the major grove, depending on the position of the nucleosome. Nucleosome positioning along the DNA thereby
allows for changes in binding-site geometry that can have a regulatory function
-3-
, o DNA: one full turn every 10 bp => same side outwards every 10 bp
o At the dyad: strong bonds needed => G/C-rich (3 H-bonds)
o Distant from dyad: WW periodicity (Weak-Weak = A/T) every 10 bp
§ Facilitates bending DNA around nucleosome
§ Associate with core histones
o Nucleosome position = flexible ≠ completely random, predictable
o Nucleosome positions affect mutational periodicity
§ Lower mutation frequency in linker DNA
– More access to repair mechanisms (NER, BER4…)
– Higher repair efficiency
– No shielding from damage recognition by nucleosome core
§ Higher mutation frequency at minor groove facing histones (every 10bp)
– Reduced DNA repair access (= tightly bound to histone)
– Bending of DNA is structural deformation => +likelihood mutations
– Replication and transcription hindrance (minor groove-facing = +stable histone bound)
• RC vs. RI nucleosome assembly:
o Replication-Coupled o Replication-Independent
§ During S phase => rapid stability § Throughout cell cycle
§ Canonical histones § Histone variants
– H1, H2A, H2B, H3(.1, .2), H4 – H2A.Z, H3.3, CENP-A, macroH2A…
– Intronless – Polyadenylated, spliced
– mRNAs: not poly-adenylated, 26b stem – Not encoded in gene clusters
loop instead at 3’ UTR => + efficient § Histone replacement, chromatin remodeling,
– Encoded by 2 gene clusters (e.g. on chr. 6) transcription regulation => dynamic
nucleosome
o Process of RC assembly (see also Error! Reference source not found.):
4
Nucleotide excision repair and Base excision repair
-4-
, o Nucleosome (dis)assembly dynamics during S phase (±8 hours => time-efficiency!) (see L4, Error!
Reference source not found.)
§ Dynamic and reversible process
§ ~30,000,000 histone octamers need to be removed
§ ~60,000,000 histone octamers need to be assembled
§ Parental nucleosomes are either recycled (= most common) or degraded
§ New histones are generated and integrated
§ Recycling = essential to copy epigenetic information to daughter cells
– CAF1 is involved in assembling new histones. – FACT complex helps in disassembling and reassembling nucleosomes.
– ASF1 assists by delivering histones. – MCM2 can bind evicted H3-H4 tetramers and, together with ASF1, help
recycle histone dimers.
§ Inheritance needed to maintain epigenetic memory
§ Incorporation of RC histones to maintain DNA chromatinized
§ Mostly random distribution of paternal histones (= from paternal DNA strand)
– Some notable exceptions, e.g. asymmetrical distribution in germline stem cells of Drosophila
§ Mechanistic details remain incompletely understood.
2.2. Histone Variants
• Evolved from archaeal histone (tetramers, ~tetrasome, • Each have distinct function
single wrap) • Some stabilize or destabilize nucleosomes
• More variants of H3 and H2A, fewer of H4 or H2B • AA changes => different PTMs
• Some differ in only a few AA from core histones • Found in specialized chromatin domains
• Deposited in a RI manner (e.g. centromere or telomere)
2.2.1. Histone H3.3
• RI production and deposition
• 4 AA difference from H3 (3 at α2 for UBN1 recognition, 1 at the tail)
In transcriptionally active chromatin In repressed chromatin
Enhancers, promotors, gene bodies Telomeres, pericentric regions, short tandem
repeats, retrotransposons
High dynamic turn-over Low dynamic turn-over
Deposition: Deposition by ATRX-DAXX
• New H3.3 bound and recycled by ASF1-HIRA complex To resolve RI H3.1 loss due to transcription, DNA
• Old H3.3 bound and recycled by UBN1-HIRA complex damage, spontaneous unwrapping, nucleosome-
≠ CAF1 as with H3 inhibitory sequences (telomeric repeats), aging,...
-5-
