Histone Modifications and Epigenetics Study Notes

Course Title and Lecturer

Course: Histone Modifications and Epigenetics BS2091
Semester: 2025-26 SEM2
Lecturer: Prof. Thomas Schalch

Intended Learning Outcomes

After studying the material in this lecture, you should be able to:

Define epigenetics and describe its role in biology.
Explain the reader-writer-eraser concept as the foundation of an epigenetic system.
Compare and contrast the chemical nature and biological role of lysine methylation and acetylation.
Associate specific histone marks with genomic features.
Describe the technique of chromatin immunoprecipitation and apply it.
Describe the molecular mechanisms driving constitutive heterochromatin formation.

Definition of Epigenetics

Epigenetic trait: A stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence.
  - Example: One of two X-chromosomes epigenetically silenced.
  - Phenotype visible as bands in diverse blood cells of identical genotype.
  - Example: Metastatic phenotype showing elevated histone H3K18 acetylation.
  - Example of Arabidopsis phenotype rescue by an epigenetic mutant: clk-st and kyp-1, genetically identical but significantly different individuals.
  - Example: Different coat color in genetically identical individuals determined by epigenetic silencing.
References:
- Berger SL et al. (2009). An operational definition of epigenetics. Genes Dev 23, 781-3. DOI.
- C.D. Allis, M.-L. Caparros, T. Jenuwein, D. Reinberg, Epigenetics, (Second Edition), Cold Spring Harbor Press, Cold Spring Harbor (2015).

Chromatin as an Epigenetic System

An epigenetic system should be:
  - Gene-specific: Directly impacts specific genes.
  - Inducible: Can be activated or deactivated.
  - Stable: Provides long-lasting changes.
  - Reversible: Can revert back under certain conditions.
To affect the phenotype, there must be:
- Readout: Downstream pathways must lead to a change in phenotype.
Chromatin is a powerful epigenetic system because it features:
- Histone variants.
- Complex post-translational modifications on histones and DNA.

Histone Variants and Post-Translational Modifications

Histones H3 and H2A:
- Important variant forms exist.
Post-translational modifications (PTMs):
  - Common modifications on nucleosomes include:
    - Methylation (red flags).
    - Acetylation (green lollipops).
    - Phosphorylation (yellow stars).
    - Ubiquitylation (purple balloons).
Histone code hypothesis:
  - Distinct histone modifications act sequentially or in combination to form a 'histone code.'
  - This code is read by other proteins to bring about distinct downstream events, acting as cis-regulatory elements.
  - References:
    - Strahl BD & Allis CD (2000). The language of covalent histone modifications. Nature 403, 41-5. DOI.

Writers, Readers, and Erasers

Roles:
  - Readers: Proteins that recognize and bind to epigenetic marks.
  - Writers: Enzymes that add epigenetic marks.
  - Erasers: Enzymes that remove epigenetic marks.
The histone code hypothesis proposes a system of cellular activities responsible for installation, removal, and reading out of histone modifications.

Mechanisms Driving Epigenetic States

Establishment:
  1. A sequence-specific initiator recruits specific epigenetic writers and erasers.
  2. Writers and erasers establish a specific chromatin modification pattern.
  3. The modification pattern attracts a specific set of readers.
  4. Readers recruit gene silencing or activating machinery.
  5. Result: Change in phenotype.
Maintenance:
  1. Readers drive positive feedback loops to enforce their cognate marks.
  2. The system becomes independent of the initiator.
  3. Progeny inherit the epigenetic state independently of the initiating stimulus.

Chromatin Structure and DNA Folding

Folding of DNA:
  - Organization levels include:
    - 2 nm: DNA double helix.
    - 11 nm: Nucleosome in chromatin fibers.
    - 20-30 nm: Folded chromatin fibers.
    - 700 nm - 1400 nm: Mitotic chromosome structures.
Topological organization:
- Topologically associated domains, compartments, heterochromatin, and euchromatin along with chromosome territories influenced by condensin and cohesin.

Classification of Chromatin

Euchromatin:
  - Region that is decondensed and contains transcriptionally active loci.
  - Early replicating, hyperacetylated histones.
  - Active genes enriched for methylation at Lys4, H3K4, H3K36, and H3K79.
Heterochromatin:
  - Highly compacted chromatin with regions of silenced DNA.
  - Late replicating, hypoacetylated histones, high levels of DNA methylation.
  - Classification:
    - Constitutive heterochromatin: Flanking centromeres.
      - Features include:
        - H3 tri-methyl K9, mono-methyl K27, and H4 tri-methyl K20.
    - Facultative heterochromatin: Can become transcriptionally active (e.g., inactive X chromosome).
      - Marked by H3 tri-methyl K27, di-methyl K9, and H4 mono-methyl K20.

Chemical Nature of Methylation and Acetylation

Lysine methylation:
- Enzymatic action of histone methyltransferases adds methyl groups.
- Creates three states: mono-, di-, and tri-methylation.
Lysine acetylation:
  - Enzymatic action of histone acetyltransferases (HATs) adds acetyl groups, removing positive charge.
  - Associated actions:
    - Histone deacetylases (HDACs): Remove acetyl groups, restoring positive charge.

Chromatin Immunoprecipitation (ChIP)

ChIP technique:
  - A method used to reveal histone modifications and where gene regulatory factors bind to their targets.
  - qPCR is used to measure locus-specific enrichment.
  - ChIP-seq determines genome-wide distribution of a specific factor or post-translational modification (PTM).
  - Steps include cross-linking chromatin and extracting from cells via sonication.

Heterochromatin Variants and RNA Interference

Example of H3K9 methylation:
- Represents a key modification in heterochromatin.
Heterochromatin in S. pombe:
  - Found at centromeres, mating type locus, and telomeres, depends on RNAi machinery.
  - Involvement of chromatin regulators (e.g., Clr4 & Swi6) and RNAi components (e.g., Ago1, Dcr1).
  - Suppresses gene expression and recombination and is crucial for mating-type switching and chromosome segregation.

Methyltransferase Targets and Drug Development

Histone methyltransferases:
  - Large group of enzymes that methylate specific lysine residues, using S-adenosylmethionine (SAM) as a cofactor and methyl-donor.
  - Specific PRMT proteins methylate arginine residues on histones.
  - Many have been implicated in cancer, becoming targets for drug development efforts.
Key modifications include: H3K9, H3K27, H3K4, H4K20, and H3K36.

Mechanisms of HP1 Proteins and Chromodomain Function

HP1 proteins:
- Bind to H3K9me2/3 methylated histones and recruit client proteins via PxVxL motifs.
- Comprised of a "chromodomain-linker-chromoshadow domain" architecture allowing high specificity binding.
Chromodomains:
- Readers of di- and tri-methylated lysines (e.g., H3K9, H3K27, H4K20).
- Recognize methylated lysines using an aromatic cage.

Epigenetic Memory and Feedback Loops

Mechanism for heterochromatin maintenance:
  - HP1 binds to H3K9me and recruits the Su(var)3-9 H3K9 methyltransferase, which reinforces positive feedback loops.
  - This creates heritable components of chromatin structure, linking readers and writers to stabilize signals.
  - Reference: Schotta G et al. (2003). SU(VAR)3-9 is a conserved key function in heterochromatic gene silencing. Genetica 117, 149-58. DOI.

Summary

The histone code hypothesis suggests post-translational modifications on histones regulate genome function and phenotype.
Each modification has specific writer, reader, and eraser proteins.
Histone variants and DNA methylation serve as components of chromatin signaling, marking specific genomic regions.
Histone methyltransferases and demethylases create lysine methylation patterns, while acetylation patterns are formed by HATs and HDACs.
H3K9 di- and tri-methylation exhibit strong epigenetic features due to powerful feedback loops stabilizing repressive chromatin.
Caveat: The dynamic nature of many histone marks complicates proving their epigenetic properties.