Class 16: Control of Gene Expression II

Misplicing & β-Thalassemia

• Core idea: A single‐nucleotide change inside an intron can create a cryptic 3′ splice site, tricking the spliceosome and dragging intronic sequence into the mature mRNA.
  • Canonical 3′ splice motif to remember: $\text{NCAGG}$ (in the β-globin intron the N = T, i.e., $\text{TCAGG}$).
  • Point mutation: $\text{TC\underline{G}GG}\;\rightarrow\;\text{TC\underline{A}GG}$ creates a second, upstream 3′ splice site.
  • Spliceosome (snRNPs) now pairs the authentic 5′ site with the new mutant 3′ site → extra intronic stretch becomes part of the mRNA (a "cryptic exon").
  • Within that intronic fragment lie premature stop codons (e.g., $\text{TAG}$ → $\text{UAG}$ in RNA).
    • Stop codon inside an exon halts translation early → truncated β-globin chain incapable of efficient O₂ transport.
• Consequences to compare on exams:
  • Mutant message = longer mRNA ➔ shorter protein (translation stops early).
  • Wild type message = shorter mRNA ➔ longer, functional protein.
  • Students often reverse this relationship—don’t!
• Physiological outcome: Hb tetramer ($\alpha\alpha\beta\beta$ + heme) can’t bind O₂ efficiently → clinical β-thalassemia.

RNA Editing – ApoB100 vs ApoB48

• Post-transcriptional regulation after splicing; involves RNA editing, not splice choice.
• Same APOB mRNA length in liver & intestine; protein size differs.
  • Liver lacks APOBEC1 ➔ codon remains $\text{CAA}$ ➔ full-length $\text{ApoB}_{100}$.
  • Intestine expresses APOBEC1 (C→U deaminase) ➔ $\text{CAA}\rightarrow\text{UAA}$ (stop) ➔ shorter $\text{ApoB}_{48}$.
• Tissue specificity driven by transcription factors that activate APOBEC1 only in intestinal cells.
• Exam trap: mRNAs are identical in size; proteins differ.

Telomeres & Telomerase

• Chromosome ends = repetitive, non-coding $\text{TTAGGG}$ sequences called telomeres.
  • Protect coding DNA from attrition each S-phase.
  • Without maintenance cells reach the Hayflick limit (replicative senescence).
• Telomerase (ribonucleoprotein RT) extends telomeres by adding $\text{TTAGGG}$ repeats.
  • Active in: germ cells, embryonic stem cells, adult stem cells, many cancers.
  • Absent/low in most somatic tissue → progressive shortening.

Stem Cell Fundamentals

Potency Spectrum

• Totipotent – can generate embryo + extra-embryonic tissues; only the zygote.
• Pluripotent – form any embryonic germ-layer derivative, not placenta; e.g., inner-cell-mass ESCs.
• Multipotent – multiple lineages within one family; e.g., hematopoietic stem cells (HSCs).
• Unipotent – one lineage; e.g., basal epidermal stem cells → keratinocytes.

Major Stem-Cell Categories

1. Embryonic stem cells (ESC).
2. Somatic/Adult stem cells.
3. Induced pluripotent stem cells (iPSC).
4. Umbilical-cord/cord-blood stem cells.

Adult Stem-Cell Niche & Division Modes

• Stem cells reside in a specialized micro-environment – the niche.
• Division patterns:
  • Symmetric self-renewal: $\text{SC}\to2\,\text{SC}$ (one may return to niche).
  • Asymmetric: $\text{SC}\to\text{SC}+\text{progenitor}$.
  • Symmetric differentiation: $\text{SC}\to2\,\text{progenitors}$.
    • Neighboring SC may compensate by symmetric self-renewal to keep stem-cell pool constant.
• Example turnover times:
  • Small-intestinal epithelium ≈ 5 days
  • Epidermis ≈ 30 days
  • Hair follicles ≈ 4 years
• Niche signalling ligands: WNT, EGF, Notch, etc. awaken quiescent ($G_0$) stem cells.

Induced Pluripotent Stem Cells (iPSC)

• 2006 – Shinya Yamanaka reprograms somatic fibroblasts to ESC-like state.
  • Requires four transcription factors ("Yamanaka factors": Oct4, Sox2, Klf4, c-Myc).
  • Reactivates telomerase & embryonic gene networks.
• Validation of potency: Replace ESCs in a blastocyst with iPSCs → viable mice born (proved functional pluripotency).
• Ethical advantage: sidesteps embryo destruction; huge for regenerative medicine.
• Clinical progress:
  • Lab-grown skin with follicles, glands, fat layers for burn patients – reduced graft rejection (autologous source).
  • Ongoing trials for retinal, cardiac, and neural repair.

Gene Therapy for Hemoglobinopathies (CTX001)

Hemoglobin Review

• Adult tetramer: $\alpha<em>2\beta</em>2$ (HbA); Fetal: $\alpha<em>2\gamma</em>2$ (HbF).
• HbF has higher O₂ affinity → curve left-shifted relative to HbA; beneficial in utero.
• Developmental switch:
  • At birth: gradual $\gamma\downarrow,\;\beta\uparrow$; by ~6 months HbF gone → symptoms of β-thalassemia or sickle-cell become evident.

Molecular Switch Control

• Transcription factor BCL11A ("B cell 11A") = repressor of γ-globin gene.
  • Erythroid expression of BCL11A depends on enhancer bound by KLF1.
  • $\text{High KLF1}\Rightarrow\text{BCL11A on}\Rightarrow\gamma\text{-globin off}$.

CRISPR/Cas9 Strategy (CTX001)

1. Harvest autologous HSCs from patient’s bone marrow.
2. Ex vivo CRISPR/Cas9 mutates the erythroid enhancer of BCL11A.
   • Disrupts KLF1 binding site → BCL11A transcription drops specifically in erythroid cells.
3. Edited HSCs now lack γ-globin repression → HbF re-expressed.
4. Myeloablation + reinfusion of edited cells → engraftment.
5. Result: new erythrocytes carry high HbF, ameliorating sickling or β-chain deficit.

Drug Concept

• CTX001 isn’t a chemical; it is a population of CRISPR-edited HSCs.
• Represents next-generation “living” therapeutics; patient becomes own drug factory.

Broader Implications & Ethics

• Precision editing avoids global knockout of BCL11A (vital in other tissues).
• Autologous approach avoids graft-versus-host disease.
• Ongoing questions: Long-term genotoxicity, cost, equitable access.

Quick Exam Reminders

• TAG (DNA) ↔ UAG (RNA) ↔ one of three stops ($\text{UAA},\;\text{UAG},\;\text{UGA}$).
• Longer mRNA ≠ longer protein if a premature stop is introduced.
• Post-transcriptional regulation covers splicing, editing, capping, poly-A—not transcription initiation.
• Yamanaka factors are transcription factors—they re-write epigenetic programs.
• Telomerase active in $\text{germ cells} + \text{embryonic SC} + \text{adult SC} + \text{cancers}$.
• Stem-cell potency: \text{Totipotent}>\text{Pluripotent}>\text{Multipotent}>\text{Unipotent}.