Lecture 5

• Chromatin

  The complex of DNA and protein in eukaryotic cells, plays a crucial role in regulating gene expression
• Chromatin structure

  • Can vary between an open, relaxed form that allows gene expression and a tightly packed form that inhibits gene expression
• Histone modifications

  Modifications of histones, such as methylation (Me) or acetylation (Ac), are key to the regulation of gene activity
• Histone modifications

  • H3K4me: Associated with active transcription
  • H3K9me: Linked to gene silencing and heterochromatin formation
  • H3K27me: Often involved in the inactivation of genes
• Chromatin remodeling

  A process by which the structure of chromatin is altered by enzymes, allowing transcription factors to access DNA and initiate transcription
• Chromatin remodeling is essential for gene expression and cell differentiation
• Transcriptional regulation

  Crucial for allowing cells to respond to environmental cues and for differentiating into various tissue types, plays a role in development, and its deregulation can lead to diseases such as cancer
• Mechanisms of transcription control

  • RNA Polymerase and General Transcription Factors
  • Specificity Factors
  • Activators and Repressors
• Regulatory elements and transcription factors

  Transcription is also regulated by DNA sequences called enhancers and silencers, which can be located far from the actual genes they control. These elements bind transcription factors that either increase or decrease the rate of transcription.
• Constitutive heterochromatin

  Usually found in regions that are permanently inactive, such as centromeres and telomeres
• Facultative heterochromatin

  Can switch between active and inactive states depending on the cell type or developmental stage
• Telomeres

  Repetitive DNA sequences at the ends of chromosomes that protect them from degradation
• Telomerase


  A ribonucleoprotein that extends the telomeres, thus maintaining chromosome integrity and cellular longevity
• Telomere shortening is linked to aging and cell senescence (the Hayflick limit), and in diseases like Werner syndrome, defects in components of the telomere replication machinery lead to premature aging and increased cancer risk
• RNA Polymerases in Eukaryotes

  • RNA Polymerase I (Pol I): Transcribes rRNA genes
  • RNA Polymerase II (Pol II): Handles all protein-coding genes, plus genes for small nuclear RNAs (snRNA), microRNAs (miRNA), small nucleolar RNAs (snoRNA), and small interfering RNAs (siRNA)
  • RNA Polymerase III (Pol III): Responsible for tRNA genes, 5S rRNA, some snRNA, and other small RNAs
• General Transcription Factors (GTFs)

  Essential for the initiation of transcription by RNA Polymerase II, they position the polymerase at the start sites of genes and assist in pulling apart the double helix to expose the template strand
• The Basal Transcription Apparatus

  • TATA-binding Protein (TBP): Recognizes and binds to the TATA box within the promoter
  • TFIIB: Binds to the B recognition element and helps position RNA polymerase at the start site
  • TFIID: Composed of TBP and TAFs (TBP-associated factors), it recognizes promoter elements and recruits other GTFs
  • TFIIE and TFIIF: Work together to stabilize the interaction between RNA polymerase and the DNA template
  • TFIIH: Has helicase and kinase activities; it unwinds DNA and phosphorylates the C-terminal domain (CTD) of RNA Polymerase II, marking the transition from initiation to elongation phase
• Promoter Elements

  • TATA Box: A core promoter element located about 25-30 base pairs upstream of the transcription start site, bound by TBP
  • BRE (B Recognition Elements): Located adjacent to the TATA box, these elements bind TFIIB and are crucial for precise initiation
  • Initiator Element (Inr): Encompasses the transcription start point and is recognized by TFIID
  • Upstream Control Elements: These can enhance or repress the activity of the core promoter elements depending on their nature and the context
• Transcription initiation

  Involves the coordinated assembly of the transcription machinery at the core promoter followed by the opening of the DNA helix and the start of RNA synthesis
• Transcription regulation

  Can occur through the differential binding of transcription factors to promoter elements, impacting the efficiency of assembly and initiation
• Promoter complexity varies greatly among genes, influencing their regulation. For example, promoters of genes expressed at certain developmental stages or in response to environmental changes may contain additional regulatory elements that modulate their activity.
• 1. less cassette transcription assay

  Helps researchers study transcription regulation by utilizing a promoter linked to a synthetic DNA cassette lacking guanine (G) bases, enabling the measurement of transcriptional activity by the production of RNA transcripts in a controlled setting
• Activators and Repressors

  Proteins that can enhance or inhibit transcription by interacting with specific DNA sequences or modifying chromatin structure
• DNA-Binding Domains

  • Homeodomains: Found in many developmental genes, these domains allow for highly specific DNA interactions
  • Zinc Finger Motifs: Characterized by their coordination of a zinc ion, these motifs facilitate strong and specific DNA-protein interactions
  • Leucine Zippers: These motifs facilitate dimerization (both homodimer and heterodimer) of transcription factors, broadening their regulatory potential
• Enhanceosomes

  A higher-order protein complex that assembles on certain enhancer sequences requiring tight regulation, such as those near genes activated in response to infections or other cellular stresses. They are composed of multiple activators and often recruit architectural proteins like high-mobility group (HMG) proteins and coactivators to facilitate transcription initiation by altering chromatin structure and promoting the assembly of general transcription factors.
• Combinatorial control of gene expression

  Gene expression is often controlled by the combined actions of multiple transcription factors, allowing for precise regulation of gene expression levels in response to a variety of signaling pathways
• Transcriptional misregulation and disease

  • Philadelphia Chromosome (Ph) in Chronic Myeloid Leukemia (CML): The BCR-ABL1 fusion gene is transcriptionally activated by the BCR enhancer, leading to the unregulated activity of ABL1 tyrosine kinase which drives uncontrolled cell proliferation
  • Burkitt's Lymphoma: Translocation of the MYC gene next to the immunoglobulin heavy chain locus enhancer on chromosome 14 results in the deregulated expression of MYC, promoting rapid cell division and contributing to tumor development
• Understanding the molecular mechanisms of transcriptional regulation, including the roles of specific enhancers and the formation of specific protein complexes like enhanceosomes, is crucial for developing targeted therapies. For example, drugs that inhibit the BCR-ABL1 kinase activity have been successful in treating CML.