Gene Expression Lecture 3

• Human globin genes


  • Expressed only in erythroid cells
  • Expressed at different stages of development and in different organs
  • Each globin gene has a specific set of regulatory elements that bind TFs that determine its pattern of expression
  • Several of the binding sites for GATA-1 overlap with those of other proteins; GATA1 appears to prevent their binding

  
• Drosophila EVEN-SKIPPED (EVE) gene

  • EVE is a 'pair-rule' gene important in embryo development
  • EVE is expressed at an early stage when the embryo is a single cell containing many nuclei
  • Transcription factors (present in the nuclei) are unevenly distributed in the embryo, giving positional information
  • Cis-elements of the EVE gene interact with the combinations of regulatory TFs present at particular locations in the embryo, i.e. they interpret the positional information, to express EVE in 7 stripes (odd-numbered parasegments)
  • The regulatory region of the EVE gene is ~20 Kbp and has modules containing cis-elements that specify a particular stripe of EVE expression
  • A specific module directs the correct spatial expression of a reporter gene in transgenic embryos
  • The 'stripe 2 module' of the EVE gene contains binding sites for 2 TFs that promote EVE transcription (Bicoid and Hunchback) and 2 that repress (Giant and Krüppel)
  • Drosophila bicoid and hunchback mutants fail to express EVE in stripe 2 whereas giant and krüppel mutants have a very broad stripe 2
  • The relative amounts of the TFs at particular locations in the embryo determine whether EVE will be transcribed
  • EVE transcription in other stripes is controlled by other elements and combinations of factors

  
• Transcription factors


  Proteins that bind to cis-elements and regulate transcription

  
• Transcription factors


  • Eukaryotic genomes encode a large number of them
  • There are numerous different types - families and sub-families - with different structures
  • Most TFs have a modular design with at least two domains: DNA binding and activation domains
  • Activation domains mediate interactions with other proteins (co-regulators or co-repressors) that stimulate or repress transcription

  
• DNA-binding domains of TFs

  • Recognise the pattern of structural features on the surface of the DNA determined by the nucleotide sequence
  • The base pairs exposed at the surface of the double helix present a distinctive chemistry
  • Only in the major groove are the surface patterns clearly different for each base pair
  • Therefore most regulatory TFs bind in the major groove

  
• Types of transcription factors

  • Helix-turn-helix proteins
  • Homeodomain proteins
  • Zn finger proteins
  • Basic zipper proteins
  • Basic helix-loop-helix proteins

  
• Helix-turn-helix DNA-binding motif


  • Two α-helices are connected by a short polypeptide chain
  • The 'recognition helix' binds the major groove
  • The proteins bind as dimers to symmetrical cis-elements
  • The 2 recognition helices are separated by one turn of the double helix

  
• Basic zipper (or leucine zipper) motif

  • Functions in both DNA binding and TF dimerisation
  • α-helices from two TF monomers interact via hydrophobic amino acids (often leucine) to form a coiled-coil
  • The inverted 'Y' structure binds the major groove
• Many TFs bind as homodimers to DNA. However, many can form heterodimers with TFs of the same family (e.g. bZIP, bHLH proteins)
• Heterodimerization


  • Increases the diversity of DNA-binding activities and also the possibilities for regulation
  • This is an example of combinatorial control

  
• Studying transcription factors


  1. Gel Shift Assays show whether a DNA fragment has protein binding activity

  2. Competition assays can be used to define the element

  3. DNase 1 footprinting defines factor-binding sequences

  4. Screening a cDNA expression library with a defined element can be used to clone proteins interacting with that element

  5. Chromatin immunoprecipitation identifies DNA sequences bound by a factor in vivo

  
• Neither gel shifts nor footprinting identify the factor that binds a DNA fragment

  
• Neither the biochemical purification nor cloning strategies show which specific factor functions in a given response in vivo
• Genetic approaches are needed to define function
• The fact that many TFs are members of large families of closely related proteins makes functional studies more difficult
• Heterodimerization


  The process where two different proteins come together to form a complex that can interact with DNA and regulate gene expression.
• Basic proteins


  A type of transcription factor that regulates gene expression by binding to DNA. They are called "basic" because they contain a high number of basic amino acids (lysine and arginine) in their DNA-binding domain.
• bZIP proteins


  A subtype of basic proteins that contain a leucine zipper motif. The leucine zipper is a structural motif that consists of a repeating pattern of leucine residues, which form a coiled-coil structure that allows the protein to dimerize with other bZIP proteins. The basic region of the protein, which is responsible for DNA binding, is located adjacent to the leucine zipper motif.