KS

Epigenetics and How We Study It

Epigenetics: Definition & Scope

  • Epigenetic changes influence phenotype without altering DNA sequence.

  • Changes can be mitotically and in some cases meiotically heritable.

  • Represent a long-term, energy-intensive layer of gene regulation.

  • Principal mechanisms covered in this lecture:
    • DNA methylation (5-mC, 5-hmC, 5-fC, 5-caC)
    • Histone post-translational modifications & histone-tail dynamics
    • Large-scale chromatin remodeling (euchromatin ↔ heterochromatin)
    • X-chromosome inactivation as dosage compensation

  • Why study epigenetics?
    • Understand normal development & lineage programming
    • Dissect gene–environment interaction (nutrition, toxins, alcohol, etc.)
    • Clarify inheritance patterns such as imprinting
    • Identify disease mechanisms & diagnostic biomarkers (e.g., cancer, neuro-degeneration)

Cell Differentiation & Gene Regulation

  • All somatic cells originate from stem cells yet produce multiple cell types: muscle, nerve, blood, fat, skin, endothelial, pancreatic, bone, sex cells, etc.

  • As differentiation proceeds, required gene set becomes increasingly specialized; unused genes are progressively silenced.

  • Transcriptional regulation hierarchy:
    • Chromatin state → promoter accessibility
    • Cis-elements (enhancers, silencers, CpG islands)
    • Trans-acting factors (TFs, co-activators, co-repressors)
    • Post-transcriptional, translational, and post-translational layers

Classical Genetic Dosage Examples

  • Down syndrome (Trisomy 21)
    • Extra copy of chromosome 21 increases gene dose of APP (Amyloid Precursor Protein).
    • ≈50 % of individuals develop Alzheimer’s in their 50s–60s.

  • Sex-chromosome dosage
    • XX vs XY → females carry two copies of >1,000 X-linked genes; males carry one.
    • Dosage compensated via random X-inactivation (Lyonisation) producing X-linked variegation.

X-Inactivation (Chromosome-Wide Epigenetics)

  • Occurs early in embryogenesis; each precursor cell randomly inactivates one X.

  • Inactive X (Xi) forms the Barr body.

  • Features of Xi chromatin:
    • DNA hypermethylation at promoters & repeats
    • Histone macro-H2A incorporation
    • Histone hypoacetylation & H3K27me3 enrichment

Core Mechanism 1 – DNA Methylation

Chemical Basis

  • Addition of a methyl group to cytosine C5 → 5-methylcytosine (5-mC), predominantly at CpG dinucleotides.

  • CpG islands: stretches of 500–1500 bp, GC content >60\%, often overlap promoters & 5′ ends of transcripts.

Functional Consequences

  • Promoter 5-mC → transcriptional repression.

  • Represses transposable elements (genome stability).

  • Gene-body methylation may correlate with active transcription.

Writers – DNA Methyltransferases (DNMTs)

  • DNMT1: maintenance; binds hemi-methylated DNA during S-phase and copies pattern to daughter strand.

  • DNMT3a / DNMT3b: de-novo methylation; act on unmethylated DNA.

Readers & Erasers

  • MBD proteins (e.g., MeCP2) bind m5C and recruit HDACs + chromatin remodelers → compact chromatin.

  • TET1/2/3 oxidize 5-mC → 5-hmC → 5-fC → 5-caC; active demethylation proceeds via TDG-mediated base-excision repair or passive dilution during replication.

  • 5-hmC enriched in embryonic stem cells & neurons; implicated in pluripotency & alternative splicing.

Core Mechanism 2 – Histone Modifications (brief mention)

  • Acetylation (HATs/HDACs), methylation (HMTs/HDMs), phosphorylation, ubiquitination on histone tails fine-tune chromatin openness.

Transcription Primer (Revision)

  • Eukaryotic promoter contains TATA box and proximal elements; bound by basal TFs.

  • Initiation complex includes multiple TFs + RNA pol II and is large.

  • Transcription kinetics:
    • RNA pol II transcribes \approx40 nt/s.
    • Simultaneous transcription by multiple polymerases.
    • Termination: RNA pol II releases transcript 10–35 nt downstream of poly(A) signal.

Gene–Environment Interaction: Agouti (A^vy) Mouse Model

  • Mice carry an IAP retrotransposon upstream of agouti gene acting as cryptic promoter.

  • Coat-colour gradient yellow → brown correlates with methylation of the IAP.

  • Environmental effect:
    • Maternal diet enriched in methyl donors (B12, folate, choline, betaine) → higher IAP methylation → darker (pseudo-wild-type) coat, lower obesity/T2D risk.
    • Maternal alcohol exposure → hypomethylation → yellow coat, metabolic disease.

  • Demonstrates establishment of epigenetic marks early in development and transgenerational influence.

Laboratory Techniques to Study DNA Methylation

1. Restriction Fragment Length Polymorphism (RFLP)

  • Uses methylation-insensitive vs methylation-sensitive isoschizomer enzymes (e.g., Hpa II vs Msp I ; Xho I).

  • Presence/absence of digestion bands indicates methylation at specific CCGG or CTCGAG sites.

2. Methylated DNA Immunoprecipitation (MeDIP)

  • DNA is sheared, incubated with anti-5-mC antibody attached to magnetic beads.

  • Enriched methylated fraction analyzed by microarray (MeDIP-Chip) or sequencing (MeDIP-Seq).

3. Sodium Bisulfite Conversion

  • Steps: denaturation (98 °C) → bisulfite conversion (64 °C, pH 5–6) → desulphonation (alkali).

  • Unmethylated C → U, methylated 5-mC / 5-hmC remain C.

  • After PCR (polymerase reads U as T), methylation status deduced by comparing C→T changes.

  • Limitations: DNA degradation; cannot distinguish 5-mC vs 5-hmC/5-fC/5-caC.

  • Extensions: OxBS-seq, fCAB-seq add chemical/oxidative steps to discriminate modifications.

4. Primer Design & PCR for Bisulfite DNA

  • Optimal amplicon <200\text{ bp}; three primers (forward, reverse, sequencing) 18–24\text{ bp}, T_m = 62–68\,^{\circ}\text{C}.

  • Avoid hairpins, primer–dimers.

  • Each PCR cycle doubles product; after n cycles ideal yield 2^n copies.

5. Gel Electrophoresis Quality Control

  • Aim for single clean band; primer-dimers/secondary products impair sequencing.

6. Pyrosequencing

  • Sequencing-by-synthesis detects PPi release → ATP (sulfurylase) → light (luciferase).

  • Apyrase degrades residual nucleotides.

  • Quantitative: mixed C/T peak heights give % methylation at each CpG in amplicon.

  • Constraints: short reads, bisulfite DNA quality, cell-type heterogeneity.

7. Third-Generation Sequencing of Epigenetic Marks

  • SMRT (PacBio): detects kinetic delays in polymerase incorporation; identifies 5-mC & 6-mA.

  • Nanopore (Oxford Nanopore MinION/Mk1C): electrical current shift distinguishes methylated vs unmethylated bases in real time; rapid (~90 min panels) for intra-operative tumor profiling.

Epigenetics & Cancer

  • Hallmarks:
    • Promoter-specific hypermethylation → silencing of tumor-suppressor genes.
    • Global hypomethylation → genomic instability, mitotic recombination, activation of oncogenes & repetitive elements.
    • Aberrant histone modification landscape & miRNA dysregulation (onco-miRNAs, epi-miRNAs).

  • Schematic: normal cell with balanced DNA methylation/histone marks vs cancer cell displaying promoter hypermethylation, global hypomethylation & defective chromatin architecture.

Diagnostic Advances

  • HumanMethylation450K BeadChip used to profile 2,801 CNS tumor samples across 91 WHO classes → reference cohort (Capper et al., 2018 Nature).

  • Parallel testing of 1,104 new CNS tumors:
    • 76\% concordance pathology vs methylation.
    • \sim12\% re-classified after methylation feedback; 10 cases unresolved.

  • Correct methylation-based classification guides personalized therapy & prognosis.

  • Nanopore methylation panels enable same-day intra-surgical diagnosis (~90 min run).

  • DNA methylation patterns under study as biomarkers of treatment response.

Key Numbers & Facts to Memorize

  • CpG island criteria: 500–1500 bp, CG:GC > 0.6.

  • RNA pol II releases transcript 10–35 nt downstream of poly(A) signal.

  • Transcription speed: ≈40 nt/s in eukaryotes.

  • PCR doubling: after 30 cycles ≈10^9 copies of target.

  • X chromosome carries >1,000 genes.

  • Trisomy 21 increases APP gene dose → ~50 % Down-syndrome patients develop AD in mid-life.

Practical / Ethical / Philosophical Considerations

  • Epigenetic modulation provides a mechanistic link between environment and genome → raises public-health & ethical issues (e.g., prenatal nutrition, alcohol exposure).

  • Potential for transgenerational effects demands caution in exposure to epimutagens.

  • Cancer epigenome editing (e.g., DNMT inhibitors, HDAC inhibitors, CRISPR-dCas9 epigenetic editors) offers therapeutic promise but requires precise targeting to avoid off-site effects.

Take-Home Messages

  • Epigenetics adds a programmable, reversible regulation layer atop DNA that defines cellular identity and disease states.

  • DNA methylation is central; writers (DNMTs), readers (MBDs), erasers (TET/BER) coordinate dynamic patterns.

  • Nutritional and environmental factors can establish or hamper these marks, influencing phenotype and disease risk.

  • A toolbox of molecular techniques—restriction assays, MeDIP, bisulfite conversion, pyrosequencing, SMRT & Nanopore—enables locus-specific to genome-wide methylation analysis.

  • In oncology, methylation fingerprints refine diagnosis, prognosis, and therapy selection; rapid sequencing platforms are translating epigenetics into clinical practice.