11  Regulation of Gene Expression

11.1 Learning Objectives

By the end of this chapter, you should be able to:

  1. Explain why gene regulation is essential for cellular function and development
  2. Compare and contrast gene regulation mechanisms in prokaryotes and eukaryotes
  3. Describe transcriptional regulation through promoters, enhancers, and transcription factors
  4. Explain post-transcriptional regulation including RNA processing, stability, and translation control
  5. Describe epigenetic mechanisms of gene regulation including DNA methylation and histone modifications
  6. Analyze how regulatory networks coordinate gene expression programs
  7. Explain how misregulation of gene expression leads to disease
  8. Apply understanding of gene regulation to biotechnology and therapeutic strategies

11.2 Introduction

Gene expression must be precisely regulated to ensure that the right genes are expressed at the right time, in the right cells, and in the right amounts. This chapter explores the sophisticated mechanisms that cells use to control gene expression, from transcriptional initiation to protein degradation. We will examine how regulatory systems implement the information processing principles introduced earlier, enabling cells to respond to signals, differentiate into specialized types, and maintain homeostasis. Understanding gene regulation is key to comprehending development, cellular adaptation, and diseases like cancer.

11.3 Principles of Gene Regulation

11.3.1 Why Regulate Gene Expression?

Cellular economy: Protein synthesis is energetically expensive

  • Cost: ~4 ATP equivalents per peptide bond
  • Strategy: Express genes only when needed

Cellular differentiation: Different cell types express different genes

  • Example: Hemoglobin in red blood cells, insulin in pancreatic β-cells

Environmental response: Adapt to changing conditions - Examples: Heat shock response, nutrient availability

Development: Temporal and spatial control of gene expression

11.3.2 Levels of Regulation

  1. Transcriptional: Control of when and how often gene is transcribed
  2. Post-transcriptional: RNA processing, stability, localization
  3. Translational: Control of translation initiation and efficiency
  4. Post-translational: Protein modification, localization, degradation

Prokaryotes: Mainly transcriptional regulation

Eukaryotes: Regulation at all levels, with greater complexity

11.3.3 Regulatory Elements

cis-acting elements: DNA sequences affecting nearby genes

  • Promoters: Initiate transcription
  • Enhancers: Increase transcription (can be distant)
  • Silencers: Decrease transcription
  • Insulators: Block enhancer-promoter interactions

trans-acting factors: Proteins that bind regulatory elements

  • Transcription factors: Bind DNA, regulate transcription
  • Coactivators/repressors: Don’t bind DNA directly, modulate transcription

11.4 Prokaryotic Gene Regulation

11.4.1 The Operon Model

Operon: Cluster of genes with related functions transcribed as single mRNA

Components:

  • Structural genes: Code for enzymes
  • Promoter: RNA polymerase binding site
  • Operator: Repressor binding site
  • Regulatory gene: Codes for repressor (often elsewhere)

11.4.2 Lac Operon (Inducible System)

Function: Lactose metabolism

Genes: lacZ (β-galactosidase), lacY (permease), lacA (transacetylase)

Regulation:

  • Lactose absent: Repressor bound to operator, no transcription
  • Lactose present: Allolactose binds repressor, inactivates it, transcription occurs
  • Glucose effect: Catabolite repression via cAMP-CAP complex

Dual control:

  1. Negative control: Repressor prevents transcription
  2. Positive control: CAP activates transcription when glucose low

11.4.3 Trp Operon (Repressible System)

Function: Tryptophan synthesis

Regulation:

  • Tryptophan absent: Repressor inactive, transcription occurs
  • Tryptophan present: Tryptophan binds repressor (corepressor), activates it, transcription blocked

Attenuation: Additional regulation via leader peptide and transcription termination

  • High trp: Ribosome moves quickly, termination structure forms
  • Low trp: Ribosome stalls, antitermination structure forms

11.4.4 Two-Component Systems

Common in: Bacteria, archaea, plants, fungi

Components:

  1. Sensor kinase: Membrane protein, autophosphorylates when signal detected
  2. Response regulator: Receives phosphate, activates transcription

Example: Chemotaxis in E. coli

11.5 Eukaryotic Transcriptional Regulation

11.5.1 Complexity of Eukaryotic Regulation

Challenges:

  1. DNA packaged in chromatin
  2. Nucleus separates transcription and translation
  3. Larger genomes with more genes
  4. Multicellularity requires cell-type specific expression

11.5.2 Chromatin Structure and Regulation

Nucleosomes: DNA wrapped around histone octamers

  • Core histones: H2A, H2B, H3, H4 (2 each)
  • Linker histone: H1

Chromatin remodeling complexes: Use ATP to slide, evict, or restructure nucleosomes

  • Examples: SWI/SNF, ISWI, CHD families

Histone modifications: Chemical groups added to histone tails

  • Acetylation: Generally activates transcription (neutralizes positive charge)
  • Methylation: Can activate or repress depending on position
  • Phosphorylation: Often activates
  • Ubiquitination: Variable effects

Histone code hypothesis: Combinations of modifications specify chromatin states

11.5.3 DNA Methylation

Occurs at: CpG dinucleotides (cytosine followed by guanine)

Enzyme: DNA methyltransferases (DNMTs)

Effect: Generally represses transcription

  • Mechanisms: Blocks transcription factor binding, recruits repressive proteins
  • Patterns: Tissue-specific, developmental regulation

Genomic imprinting: Parent-specific gene expression via differential methylation X-inactivation: One X chromosome silenced in female mammals

11.5.4 Transcription Factors

General transcription factors (GTFs): Required for all Pol II transcription

  • TFIID: Contains TBP (TATA-binding protein)
  • TFIIH: Helicase and kinase activities

Specific transcription factors: Regulate specific genes

  • DNA-binding domains: Zinc fingers, helix-turn-helix, leucine zippers, helix-loop-helix
  • Activation domains: Interact with coactivators and basal machinery

Combinatorial control: Combinations of factors determine expression

11.5.5 Enhancers and Promoters

Enhancers:

  • Can be upstream, downstream, or within genes
  • Can be far from promoter (50+ kb)
  • Looping brings enhancer to promoter
  • Mediator complex: Bridges transcription factors and Pol II

Promoter-proximal elements: Near transcription start site

  • TATA box: ~-30 in many genes
  • CAAT box: ~-80
  • GC box: ~-100

11.5.6 RNA Polymerases and Their Regulation

RNA Pol I: rRNA genes (except 5S)

RNA Pol II: mRNA, snRNA, miRNA

RNA Pol III: tRNA, 5S rRNA, other small RNAs

Initiation complexes: 50+ proteins at Pol II promoters

11.6 Post-Transcriptional Regulation

11.6.1 RNA Processing

5’ capping: 7-methylguanosine added co-transcriptionally

  • Functions: Protection, translation initiation, RNA export

3’ polyadenylation: Poly-A tail added (100-250 adenines)

  • Signal: AAUAAA in mRNA
  • Functions: Stability, translation, export

Splicing: Removal of introns, joining of exons

  • Spliceosome: snRNPs (U1, U2, U4, U5, U6) + proteins
  • Mechanism: Two transesterification reactions
  • Regulation: Alternative splicing produces multiple mRNAs from one gene

RNA editing: Post-transcriptional alteration of RNA sequence

  • Examples: A→I (adenosine deamination), C→U (cytidine deamination)
  • APOBEC1: Edits apolipoprotein B mRNA (creates stop codon)

11.6.2 RNA Stability and Degradation

mRNA half-lives: Minutes to days

Stability elements:

  • 5’ cap: Protects from 5’→3’ exonucleases
  • 3’ poly-A tail: Protects from 3’→5’ exonucleases

Degradation pathways:

  • Deadenylation-dependent: Poly-A shortening then decapping and degradation
  • Endonucleolytic cleavage: Specific cleavage sites

AU-rich elements (AREs): In 3’ UTR, target mRNAs for rapid degradation

11.6.3 RNA Localization

Localized translation: mRNA transported to specific cellular locations

  • Examples: Ash1 mRNA in yeast (bud tip), β-actin mRNA (leading edge)

Mechanisms: Motor proteins, localization sequences in 3’ UTR

11.6.4 Translational Regulation

Initiation control:

  • eIF2 phosphorylation: Inhibits general translation (stress response)
  • eIF4E availability: Rate-limiting for cap-dependent translation
  • IRES elements: Internal ribosome entry sites (cap-independent)

mRNA-specific regulation:

  • 5’ UTR secondary structure: Can block ribosome scanning
  • Upstream ORFs: Small open reading frames before main coding sequence
  • MicroRNAs: Bind 3’ UTR, inhibit translation or cause degradation

Global regulation:

  • mTOR pathway: Integrates nutrient and growth factor signals
  • Integrated stress response: Phosphorylation of eIF2α

11.7 RNA-Based Regulation

11.7.1 MicroRNAs (miRNAs)

Biogenesis:

  1. Transcription as pri-miRNA
  2. Processing by Drosha to pre-miRNA
  3. Export to cytoplasm
  4. Processing by Dicer to miRNA duplex
  5. Loading into RISC (RNA-induced silencing complex)

Mechanism: Partial complementarity to 3’ UTR of target mRNAs

  • Effects: Translational repression and/or mRNA degradation
  • Specificity: One miRNA can regulate hundreds of mRNAs

Functions: Development, differentiation, homeostasis, stress response

11.7.2 Small Interfering RNAs (siRNAs)

Origin: Long double-stranded RNA (viral, transposon, or experimental)

Processing: Dicer cleavage to 21-23 nt fragments

Mechanism: Perfect complementarity leads to mRNA cleavage

Functions: Antiviral defense, transposon silencing, experimental gene knockdown

11.7.3 Long Non-Coding RNAs (lncRNAs)

Definition: RNAs >200 nt without protein-coding potential

Functions diverse:

  • Xist: X-chromosome inactivation
  • HOTAIR: Represses HOX genes (chromatin regulation)
  • NEAT1: Paraspeckle formation
  • MALAT1: Alternative splicing regulation

Mechanisms: Scaffold, guide, decoy, signal

11.7.4 CRISPR-Cas Systems

Natural function: Adaptive immunity in bacteria and archaea

Components:

  • CRISPR array: Stores viral DNA sequences

  • Cas proteins: Nucleases that cleave foreign DNA/RNA

Applications: Genome editing (CRISPR-Cas9)

11.8 Epigenetic Regulation

11.8.1 Definition and Characteristics

Epigenetics: Heritable changes in gene expression without DNA sequence change

Key features:

  • Stable: Maintained through cell divisions
  • Reversible: Can be erased and reestablished
  • Influenced by environment: Diet, stress, toxins

11.8.2 Mechanisms

DNA methylation: Covalent modification of cytosine (5-methylcytosine)

  • Maintenance methylation: After replication
  • Demethylation: Active (TET enzymes) or passive (lack of maintenance)

Histone modifications: Over 60 different modifications identified

  • Readers: Proteins that recognize specific modifications
  • Writers: Enzymes that add modifications
  • Erasers: Enzymes that remove modifications

Chromatin remodeling: ATP-dependent alteration of nucleosome position

Non-coding RNAs: Especially in transcriptional silencing

11.8.3 Epigenetic Inheritance

Cellular inheritance: Through cell division

Transgenerational inheritance: Through germline (evidence in animals, plants)

Examples:

  • Genomic imprinting: Parent-specific expression
  • X-inactivation: Random in early embryo, clonal thereafter
  • Position effect variegation: Gene silencing near heterochromatin

11.8.4 Epigenetics in Development and Disease

Development: Cell fate decisions, tissue-specific expression

Cancer: Global hypomethylation, local hypermethylation of tumor suppressors

Neurological disorders: Rett syndrome (MeCP2 mutation), fragile X syndrome

Metabolic diseases: Influenced by parental diet

11.9 Regulatory Networks

11.9.1 Gene Regulatory Networks (GRNs)

Definition: Set of genes and their regulatory interactions

Properties:

  • Modularity: Functional units
  • Hierarchy: Master regulators control sub-networks
  • Robustness: Buffer against perturbations
  • Evolution: Conservation with modification

11.9.2 Network Motifs

Recurrent patterns in regulatory networks:

  • Feed-forward loops: Common in development
  • Single-input modules: One regulator controls multiple targets
  • Dense overlapping regulons: Multiple regulators control multiple targets

11.9.3 Systems Biology Approaches

High-throughput methods:

  • ChIP-seq: Protein-DNA interactions
  • RNA-seq: Transcriptome analysis
  • ATAC-seq: Chromatin accessibility
  • Hi-C: Chromosome conformation

Computational modeling: Boolean networks, ordinary differential equations

11.9.4 Development and Differentiation

Morphogen gradients: Concentration-dependent gene activation

  • Example: Bicoid in Drosophila embryos

Combinatorial code: Combinations of transcription factors specify cell fate

  • Example: HOX genes in body patterning

Stem cell regulation: Balance between self-renewal and differentiation

11.10 Disease and Therapeutics

11.10.1 Cancer

Oncogenes: Mutated forms of normal regulatory genes

  • Examples: Myc (transcription factor), Ras (signal transduction)

Tumor suppressors: Normally inhibit growth

  • Examples: p53 (transcription factor), Rb (cell cycle regulator)

Epigenetic alterations: DNA methylation changes, histone modification alterations

11.10.2 Genetic Disorders

Regulatory mutations: Affect expression levels rather than protein sequence

  • Examples: β-thalassemia (promoter mutations), Fragile X syndrome (CGG expansion)

Imprinting disorders:

  • Prader-Willi syndrome: Paternal deletion/maternal UPD chromosome 15
  • Angelman syndrome: Maternal deletion/paternal UPD chromosome 15

11.10.3 Therapeutic Strategies

Small molecule inhibitors: Target specific regulatory proteins

  • Example: Imatinib (BCR-ABL tyrosine kinase inhibitor)

Epigenetic drugs:

  • DNMT inhibitors: 5-azacytidine (for myelodysplastic syndrome)
  • HDAC inhibitors: Vorinostat (for cutaneous T-cell lymphoma)

Gene therapy: Replace or correct defective regulatory elements

RNA therapeutics: Antisense oligonucleotides, RNA interference

11.11 Chapter Summary

11.11.1 Key Concepts

  1. Gene regulation occurs at multiple levels: transcriptional, post-transcriptional, translational, post-translational
  2. Prokaryotes use operons for coordinate regulation of related genes
  3. Eukaryotes have complex regulatory systems involving chromatin, transcription factors, and RNA processing
  4. Epigenetic mechanisms provide stable, reversible regulation without DNA sequence change
  5. Regulatory networks coordinate gene expression programs for development and homeostasis
  6. Misregulation of gene expression underlies many diseases including cancer
  7. Understanding regulation enables therapeutic interventions

11.11.2 Comparison: Prokaryotic vs. Eukaryotic Regulation

Aspect Prokaryotes Eukaryotes
Primary level Transcriptional All levels, emphasis on transcriptional
DNA packaging Minimal Chromatin (nucleosomes)
Gene organization Operons (polycistronic) Individual genes (monocistronic)
Transcription/translation Coupled Separated (nuclear/cytoplasmic)
RNA processing Minimal Extensive (capping, polyA, splicing)
Regulatory complexity Relatively simple Highly complex

11.11.3 Major Regulatory Mechanisms

Level Key Mechanisms Examples
Transcriptional Promoters, enhancers, TFs, chromatin remodeling Lac operon, histone acetylation
Post-transcriptional Splicing, editing, stability, localization Alternative splicing, miRNA regulation
Translational Initiation control, miRNA, RNA-binding proteins eIF2 phosphorylation, microRNAs
Post-translational Phosphorylation, ubiquitination, localization Protein degradation via proteasome

11.11.4 Epigenetic Marks

Mark Enzyme Typical Effect
DNA methylation DNMTs Repression
H3K4me3 SET1/COMPASS Activation
H3K27me3 PRC2 Repression
H3K9me3 SUV39H Heterochromatin
H3K27ac p300/CBP Activation
H3S10ph Aurora B Mitosis, activation

11.11.5 Network Properties

  1. Modularity: Functional units
  2. Hierarchy: Master regulators
  3. Redundancy: Multiple paths to same outcome
  4. Robustness: Resists perturbations
  5. Evolution: Conservation with innovation

11.12 Review Questions

11.12.1 Level 1: Recall and Understanding

  1. Why is gene regulation important for cellular function?
  2. Compare inducible and repressible operons in bacteria.
  3. What are the main differences between prokaryotic and eukaryotic gene regulation?
  4. List three types of histone modifications and their general effects.
  5. How do microRNAs regulate gene expression?

11.12.2 Level 2: Application and Analysis

  1. Explain how the lac operon is regulated when both lactose and glucose are present.
  2. How does chromatin structure affect gene expression, and how can it be modified?
  3. A gene has alternative splice forms. How might this increase proteomic diversity?
  4. Why might mutations in regulatory regions sometimes have more severe effects than coding mutations?
  5. Design an experiment to identify transcription factor binding sites in a genome.

11.12.3 Level 3: Synthesis and Evaluation

  1. Evaluate the statement: “Most phenotypic variation between individuals is due to differences in gene regulation rather than protein-coding sequences.”
  2. How does epigenetic regulation provide a mechanism for environmental influences on phenotype?
  3. Compare the evolutionary advantages of simple vs. complex regulatory systems.
  4. Propose how understanding gene regulatory networks could lead to new cancer therapies.

11.13 Key Terms

  • Operon: Cluster of prokaryotic genes with related functions transcribed together
  • Promoter: DNA sequence where RNA polymerase binds to initiate transcription
  • Enhancer: DNA sequence that increases transcription, can be distant from gene
  • Transcription factor: Protein that binds DNA and regulates transcription
  • Chromatin remodeling: ATP-dependent alteration of nucleosome position
  • Histone modification: Chemical changes to histone tails affecting chromatin structure
  • DNA methylation: Addition of methyl group to cytosine, usually represses transcription
  • Alternative splicing: Different combinations of exons from same gene
  • MicroRNA (miRNA): Small RNA that regulates gene expression post-transcriptionally
  • Epigenetics: Heritable changes in gene expression without DNA sequence change
  • Gene regulatory network: Set of genes and their regulatory interactions
  • Morphogen: Substance whose concentration gradient determines cell fate

11.14 Further Reading

11.14.1 Books

  1. Ptashne, M., & Gann, A. (2002). Genes and Signals. Cold Spring Harbor Laboratory Press.
  2. Allis, C. D., et al. (Eds.). (2015). Epigenetics (2nd ed.). Cold Spring Harbor Laboratory Press.
  3. Lewin, B., et al. (2018). Genes XII. Jones & Bartlett.

11.14.2 Scientific Articles

  1. Jacob, F., & Monod, J. (1961). Genetic regulatory mechanisms in the synthesis of proteins. Journal of Molecular Biology, 3, 318-356.
  2. Bird, A. (2007). Perceptions of epigenetics. Nature, 447(7143), 396-398.
  3. Lee, R. C., et al. (1993). The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell, 75(5), 843-854.

11.14.3 Online Resources

  1. ENCODE Project: https://www.encodeproject.org
  2. RegulonDB: http://regulondb.ccg.unam.mx
  3. Epigenomics: https://www.roadmapepigenomics.org

11.15 Quantitative Problems

  1. Operon Regulation: In E. coli, the lac operon has a basal transcription rate of 1 unit. With CAP activated (glucose low), rate increases 50-fold. With repressor removed (lactose present), rate increases another 10-fold.
    1. Calculate transcription rate under: (i) glucose high, lactose absent; (ii) glucose high, lactose present; (iii) glucose low, lactose present.
    2. If a mutation reduces CAP binding 10-fold, what are the new rates?
  2. miRNA Regulation: A miRNA reduces target mRNA stability. Normally, mRNA half-life is 4 hours. With miRNA, it becomes 1 hour.
    1. What is the steady-state mRNA level with miRNA relative to without?
    2. If translation rate is proportional to mRNA level, what is the effect on protein production?
    3. If miRNA also reduces translation efficiency by 50%, what is the total effect?
  3. Epigenetic Inheritance: DNA methylation is maintained with 95% fidelity per cell division.
    1. After 10 cell divisions, what percentage of methylation marks remain?
    2. If a stem cell divides 100 times during a lifetime, what percentage remains?
    3. How does this explain epigenetic stability over many cell divisions?

11.16 Case Study: p53 Regulation Network

Background: p53 is a tumor suppressor transcription factor activated by DNA damage.

Questions:

  1. How is p53 normally kept at low levels in cells?
  2. What signals activate p53, and how does activation occur?
  3. What genes does p53 activate, and what are their functions?
  4. Why is p53 mutation so common in cancers?
  5. How might understanding p53 regulation lead to cancer therapies?

Data for analysis:

  • p53 half-life: Normally 20 minutes, stabilized by phosphorylation
  • MDM2: E3 ubiquitin ligase that targets p53 for degradation
  • Activation: Phosphorylation by ATM/ATR kinases after DNA damage
  • Targets: p21 (cell cycle arrest), Bax (apoptosis), GADD45 (DNA repair)
  • Mutation frequency: >50% of human cancers

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