KS

Epigenetics and Chromatin Regulation Lecture

Epigenetics – Definition & Scope

  • Core Idea: Epigenetics studies heritable changes in gene expression that do not involve alterations in the DNA nucleotide sequence.

  • Primary Molecular Layers

    • \text{DNA methylation} (mainly at CpG dinucleotides).

    • Histone tail modifications (acetylation, methylation, phosphorylation, etc.).

    • Chromatin‐structure remodeling (ATP-dependent changes in nucleosome position or composition).

  • Why We Study It

    • Understand gene regulation in both health & disease.

    • Grasp mechanisms of development, differentiation, and cellular re-programming.

    • Explore gene–environment interactions and how external cues leave molecular “marks.”

    • Explain non-Mendelian inheritance patterns such as imprinting.

Cell Differentiation & Epigenetics

  • Stem-cell potential is shaped by epigenetic programs that give rise to specialized lineages:

    • Bone, blood, muscle, fat, skin, nerve, endothelial, pancreatic, sex cells, etc.

    • Cancer cells represent aberrant epigenetic programming.

  • Key Take-home: The same genome → diverse cell fates because epigenetic marks selectively open/close chromatin regions.

DNA Methylation

  • Classic Mark: 5\text{-methylcytosine} (5mC) added to the 5-carbon of cytosine in CpG dinucleotides.

  • Functional Consequence:

    • Promoter CpG methylation → recruitment of MBD (Methyl-CpG Binding Domain) proteins → HDACs → chromatin condensation → transcriptional silencing.

    • Picture: \text{CpG}_{\text{methylated}} \xrightarrow{\text{MBD}} \xrightarrow{\text{HDAC}} \text{Inactive Gene}

  • DNA Demethylation Pathway

    • Ten-eleven translocation (TET) enzymes oxidize 5mC → \text{5-hydroxymethyl-C (hm5C)} \to \text{5-formyl-C (5fC)} \to \text{5-carboxyl-C (5caC)}.

    • Final reconversion to cytosine via TDG-mediated base-excision repair or passive replication dilution.

  • Biological Relevance

    • \text{hm5C} enriched in embryonic stem cells & neurons; implicated in pluripotency maintenance & alternative splicing.

Additional DNA Base Modifications

  • 6-methyladenine (6mA) prominent in prokaryotes; now documented at low frequency in eukaryotes.

  • Detection platforms: SMRT sequencing (integration time changes) & Oxford Nanopore (ionic current shifts for methylated vs. unmethylated bases).

Chromosome & Chromatin Architecture

  • Hierarchy (Annunziato et al.)

    • 2\,\text{nm} DNA double helix.

    • \approx 11\,\text{nm} nucleosome (core eight histones + DNA wrapped 1.65 turns).

    • Chromatosome = nucleosome + H1 linker histone.

    • 30\,\text{nm} fiber (helically coiled).

    • 300\,\text{nm} looped domains → 700\,\text{nm} chromatid.

  • Euchromatin vs. Heterochromatin

    • “Beads-on-a-string” open euchromatin.

    • Compacted 30-nm fiber heterochromatin.

  • Key Principle: Transcription requires chromatin opening; histone modifications govern accessibility.

Histone Tail Modifications

  • Residues Available: Lysines, arginines, serines, threonines on H2A, H2B, H3, H4.

  • Acetylation

    • Enzymes: HATs/KATs → neutralize lysine + charge → loosen DNA-histone interaction → transcriptional activation.

    • Opposed by HDACs; associated with repressors.

  • Methylation

    • Context-dependent: \text{H3K4me}^3 (active), \text{H3K9me}^3 or \text{H3K27me}^3 (repressive).

  • Phosphorylation

    • Linked to transcription, DNA repair, chromosome condensation, cell-cycle progression.

  • Crosstalk

    • Remodeling complexes ↔ acetyltransferase complexes.

    • Histone marks recruit additional chromatin modifiers, facilitating transcription elongation (Li et al., 2007 Cell).

Chromatin Remodeling Complexes

  • ATP-dependent movers can:

    • Slide nucleosomes.

    • Evict nucleosomes.

    • Exchange histone variants (e.g., H2A.Z).

  • Recruitment: Sequence-specific activators bind promoters → recruit remodeling machines.

  • Energy Demand: Hydrolysis of \text{ATP} powers nucleosome reconfiguration.

Experimental Techniques

  • Chromatin Immunoprecipitation (ChIP)

    • Cross-link proteins ↔ DNA, shear DNA, immunoprecipitate with target-specific antibody on magnetic beads, purify DNA.

    • Downstreams: ChIP-chip, ChIP-Seq → genome-wide maps of histone marks or transcription-factor binding.

  • Sequencing-Based Methylation Mapping

    • Bisulfite sequencing, SMRT, Nanopore.

Bioinformatics Resource – ENCODE @ UCSC

  • ENCODE browser integrates:

    • RNA-seq expression across 9 cell lines.

    • ChIP-seq tracks (161 factors).

    • Histone mark layers (e.g., H3K27ac).

    • DNaseI hypersensitivity clusters.

    • Comparative genomics & SNP annotations.

  • Example locus displayed: \text{chr21 (q22.11)} surrounding SOD1 gene.

Genomic Imprinting & Disease

  • Definition: Parent-of-origin-specific gene silencing by epigenetic marks.

  • Dynamic Cycle

    • Imprint establishment during gametogenesis.

    • Maintenance through fertilization & development.

    • Erasure in primordial germ cells → reset for next generation.

Angelman Syndrome (UBE3A)

  • Molecular Basis: Loss of maternal UBE3A (15q11-q13); paternal allele imprinted in brain.

  • Phenotype: Severe speech impairment, microcephaly, fair complexion, ataxia, epilepsy, happy demeanor.

  • Mechanism Recap:

    • Maternal deletion \Rightarrow no active UBE3A \Rightarrow absent ubiquitin-protein ligase E3A.

Prader–Willi Syndrome (SNRPN, others)

  • Molecular Basis: Paternal deletion of same 15q11-q13 region; maternal alleles imprinted.

  • Phenotype: Neonatal hypotonia, feeding difficulty → later hyperphagia & obesity, mild–moderate ID, hypogonadism, infertility.

  • Key Contrast: Maternal deletion of region does not cause PWS because paternal allele is active.

Epigenetics & Cancer

  • Global phenomena

    • Hypomethylation → genomic instability.

    • Promoter hypermethylation of tumor suppressors.

    • Histone-modification pattern shifts.

  • Therapeutic Implications: HDAC inhibitors, DNMT inhibitors.

Integrative Summary

  • Active/Open Chromatin

    • \text{Ac–H3/H4 tails}, low DNA CpG methylation → transcriptionally competent.

  • Inactive/Condensed Chromatin

    • \text{Me–H3 (specific Lys)} and \text{CpG–Me} → repressed genes.

  • Cell-type Identity = unique combinatorial “epigenetic barcode.”

Further Reading & Study Tools

  • Lewin’s Genes XII, Ch. 26 – Eukaryotic Transcription Regulation.

  • Jorde, Carey, Bamshad – Medical Genetics (Imprinting, Epigenetics).

  • “Epigenetics in Cancer,” Ch. 11.

  • Student Consult digital resources.

Potential Exam Questions (Self-Check)

  • Explain how 5mC \to hm5C conversion contributes to active demethylation.

  • Compare the roles of HATs and HDACs in transcription regulation.

  • Outline the ChIP-Seq workflow and its application in mapping histone marks.

  • Distinguish Angelman from Prader–Willi syndromes mechanistically.

Key Numbers & Equations Recap

  • DNA wraps 1.65 turns (≈147 bp) around a histone octamer.

  • 30\,\text{nm} fiber diameter for heterochromatin.

  • \approx 70\% of Angelman or PWS cases = deletion events.

  • 161 transcription-factor ChIP datasets in the highlighted ENCODE track.

Ethical, Philosophical, Practical Implications

  • Inheritance of epigenetic marks challenges classical genetics; prompts debate on trans-generational environmental effects.

  • Clinical Translation: Epigenetic drugs necessitate careful off-target monitoring due to genome-wide effects.

  • Societal Impact: Understanding gene–environment interplay fuels public policy discussions on exposures (nutrition, toxins).

End-of-Lecture Prompt

  • “Any questions?” – Engage with ChIP, TET pathways, or imprinting case studies to reinforce learning.