Unit 6: Regulation of Gene Expression and Epigenetics

I. Orientation: The Logic of Gene Regulation

Gene expression is controlled so that a cell synthesises the right products at the right time and level; regulation predominantly acts at the point of transcription initiation because it is the most economical step to control (mid-20th century onward, from Jacob and Monod, 1961).

• Central problem: every cell carries the same genome, so phenotype differences and responses to signals arise from differential expression, not differential DNA content.
• Cis-acting elements: DNA sequences (promoters, operators, enhancers) that influence only the molecule they sit on.
• Trans-acting factors: diffusible molecules (repressors, activators, RNAs) that act on any target bearing the cognate sequence.
• Positive vs negative control: activators raise transcription above a low baseline; repressors lower it from a high baseline.
• Levels of control: transcriptional (main focus here) > processing > stability/translation > post-translation.
• Epigenetic layer: heritable changes in expression without DNA sequence change, encoded in histone marks, DNA methylation and chromatin state.

II. Regulation of Transcription in Prokaryotes

Fast, mostly transcription-initiation control clustered around operons.

Prokaryotes group functionally related genes into operons under a single promoter, giving coordinate, ligand-responsive control.

A. Operon models

An operon is a transcription unit of a promoter, operator and structural genes producing one polycistronic mRNA.

1. The lac operon (inducible, negative + positive):
   • Structural genes: lacZ (β-galactosidase), lacY (permease), lacA (transacetylase).
   • Negative control: LacI repressor binds the operator; allolactose (inducer) binds LacI, releasing it → transcription ON.
   • Positive control: low glucose → high cAMP → CAP–cAMP complex binds and recruits RNA polymerase.
   • Logic: maximal expression needs lactose present and glucose absent (catabolite repression).
2. The trp operon (repressible + attenuation):
   • Repression: tryptophan acts as corepressor, activating TrpR to block transcription when Trp is abundant.
   • Attenuation: a leader region forms alternative RNA hairpins; abundant charged tRNA^Trp lets the ribosome force a terminator (3-4) hairpin, aborting transcription; scarcity favours the antiterminator (2-3) hairpin.

B. Riboswitches

Riboswitches are structured mRNA segments in the 5′ UTR that bind a metabolite directly, without protein, to control their own gene.

• Architecture: an aptamer domain (ligand-binding) coupled to an expression platform.
• Mechanism: ligand binding switches RNA folding to either form a terminator/anti-terminator or occlude the Shine–Dalgarno sequence.
• Examples: the TPP (thiamine pyrophosphate) riboswitch; the FMN and guanine riboswitches — all sense their pathway's end-product for feedback control.
• Significance: an ancient, protein-free regulatory mode consistent with an RNA-world origin.

C. Bacteriophage lambda transcriptional switch

The lambda switch is a bistable genetic circuit choosing between lysis and lysogeny in E. coli.

• Key regulators: cI (lambda repressor) favours lysogeny; Cro favours lysis.
• Operator region: O_R with three sites (O_R1, O_R2, O_R3) between divergent promoters P_R and P_RM.
• Lysogenic state: cI binds O_R1 and O_R2 cooperatively → blocks P_R (Cro off), stimulates P_RM (own synthesis) → stable maintenance.
• Induction: DNA damage activates RecA, promoting cI autocleavage → Cro dominates, cI drops → switch to lysis.
• Design principle: cooperative binding plus autoregulation produces a robust, near-irreversible switch.

III. Regulation of Transcription in Eukaryotes

Control layered onto chromatin, so accessibility of DNA is itself regulated.

Eukaryotic DNA is packaged with histones into nucleosomes; transcription requires opening this chromatin, so covalent marks, remodelling and methylation gate access.

A. Covalent histone modifications

Reversible chemical marks on histone tails constitute a "histone code" read by regulatory proteins.

• Acetylation: HATs add acetyl groups to lysines, neutralising charge and loosening DNA → activation; HDACs remove them → repression.
• Methylation: context-dependent — H3K4me3 (active promoters) vs H3K9me3 and H3K27me3 (repressive/heterochromatin).
• Other marks: phosphorylation (e.g., H3S10 in mitosis), ubiquitination (H2A/H2B).
• Readers, writers, erasers: bromodomains read acetyl marks; chromodomains read methyl marks; enzymes deposit or remove them.

B. Nucleosome remodelling

ATP-dependent complexes physically reposition or evict nucleosomes to expose or hide regulatory DNA.

• Energy source: ATP hydrolysis by SNF2-family ATPases.
• Complexes: SWI/SNF (sliding/ejection to expose promoters), ISWI (spacing/assembly), INO80 (histone-variant exchange).
• Outcomes: sliding along DNA, complete eviction, or exchange of canonical H2A for variant H2A.Z.
• Coupling: remodellers are recruited by, and cooperate with, histone-modifying enzymes and transcription factors.

C. DNA methylation and gene regulation

Addition of a methyl group to cytosine, chiefly at CpG sites, generally silences transcription and is heritable.

• Reaction: DNMTs transfer a methyl to the 5-carbon of cytosine → 5-methylcytosine.
• Enzymes: DNMT3a/3b establish de novo methylation; DNMT1 copies the pattern at replication (maintenance).
• CpG islands: clustered CpGs at promoters, normally unmethylated in active genes; methylation blocks factor binding and recruits MeCP2/methyl-binding proteins that pull in HDACs.
• Roles: genomic imprinting, X-chromosome inactivation, transposon silencing.

IV. Mechanism of Gene Silencing

Sequence-specific and chromatin-based routes to switching genes off.

Cells silence genes both through small-RNA guides that target complementary transcripts and through condensed chromatin that occludes whole regions.

A. RNA interference — RISC-mediated silencing

RNAi is post-transcriptional silencing directed by small RNAs loaded into an effector complex.

• RISC: the RNA-Induced Silencing Complex, whose catalytic core is an Argonaute protein.
• Guide loading: one strand of a small duplex is retained as the guide; the passenger strand is discarded.
• Target recognition: base-pairing of the guide to mRNA directs the outcome.
  • Perfect complementarity: Argonaute ("slicer") cleaves the mRNA.
  • Partial complementarity (seed match): translational repression and mRNA destabilisation.

B. Mechanisms of RNA interference

RNAi originates from double-stranded RNA processed into defined small RNA classes.

dsRNA / pre-miRNA
      │  Dicer (RNase III) cleavage
      ▼
~21–23 nt duplex (2-nt 3′ overhangs)
      │  loading into Argonaute
      ▼
RISC → mRNA cleavage or translational repression

• siRNA pathway: exogenous/viral dsRNA → Dicer → siRNA → perfect-match cleavage.
• miRNA pathway: genome-encoded hairpins → Drosha (nucleus) → export by Exportin-5 → Dicer (cytoplasm) → miRNA → usually partial-match repression via 3′ UTR seed sites.
• Amplification: in plants/worms, RdRP enzymes generate secondary siRNAs.
• Symbols: Dicer, Drosha = RNase III enzymes; seed = guide nucleotides 2–8.

C. Role of heterochromatin in gene silencing

Heterochromatin is condensed, transcriptionally inert chromatin that silences genes over broad domains.

• Constitutive vs facultative:
  1. Constitutive: permanently silent, gene-poor regions (centromeres, telomeres).
  2. Facultative: silenced in a context-dependent way (e.g., inactive X, Barr body).
• Molecular marks: H3K9me3 recruits HP1, which spreads and compacts chromatin.
• RNAi link: in fission yeast, siRNAs guide the RITS complex to nascent transcripts, directing H3K9 methylation and heterochromatin formation.
• Position-effect variegation: a gene relocated near heterochromatin is stochastically silenced, revealing spreading of the silent state.

V. Epigenetic Regulation

Heritable expression states shaped by environment and disrupted in disease.

Epigenetic marks are stable yet reversible, letting cells record past signals and, when miswired, drive pathology.

A. Epigenetics and the environment

External conditions leave lasting marks on the epigenome that alter expression without changing sequence.

• Diet and metabolism: folate/methyl-donor availability changes DNA methylation; the classic agouti mouse shows coat colour set by maternal methyl-donor intake.
• Developmental programming: prenatal stress and nutrition (e.g., Dutch Hunger Winter cohort) associate with altered methylation and later metabolic risk.
• Environmental exposures: toxins, smoking and stress modify methylation and histone marks.
• Transgenerational effects: some marks escape reprogramming and persist across generations.

B. Epigenetics and cancer

Cancer arises partly from epigenetic disruption alongside genetic mutation, altering which genes are silenced or activated.

• Global hypomethylation: genome-wide loss of methylation → chromosomal instability and reactivation of transposons.
• Promoter hypermethylation: localised methylation silences tumour-suppressor genes (e.g., BRCA1, MLH1, CDKN2A/p16).
• Histone/remodeller defects: mutations in HATs, HDACs and SWI/SNF subunits (e.g., SMARCB1) perturb chromatin.
• miRNA dysregulation: loss of tumour-suppressor miRNAs or gain of oncogenic miRNAs (oncomiRs).
• Therapeutic angle: reversibility makes marks druggable — DNMT inhibitors (azacitidine) and HDAC inhibitors (vorinostat) can restore silenced genes.