L3 Eukaryotic Transcription Factors.pdf

L3 Eukaryotic Transcription Factors Learning outcomes: Definition of a transcription factor and the distinction between general and specific transcription factors Experimental methods that are used to investigate transcription factors and their binding sites Four structura...

L3 Eukaryotic Transcription Factors Learning outcomes: Definition of a transcription factor and the distinction between general and specific transcription factors Experimental methods that are used to investigate transcription factors and their binding sites Four structural motifs that are used to classify transcription factors: examples of factors that contain these motifs Examples of mechanisms by which transcription factors activate and repress transcription Examples of mechanisms by which regulated expression/activity of transcription factors achieve tissue specific or inducible gene expression Definition of a transcription factor and the distinction between general and specific transcription factors, general principles of TF binding Transcription factor: Any protein (other than RNA polymerase) required to initiate or regulate transcription General (basal) factors participate in formation of the basal transcription complex near the start site (e.g. TFIID etc.) Specific factors stimulate (or repress) transcription of particular genes by binding to their regulatory sequences which leads to non-ubiquitous protein synthesis. Transcription factors bind through specific amino acid side chain: base interactions. There are likely 1639 human TFs identified Each individual interaction is weak and it takes around 10-20 interactions for a stable DNA-protein interface. Generally, it is difficult for transcription factors to access DNA as it is tightly wrapped around nucleosomes. TFs have evolved to establish high specificity: o In each binding site multiple weak interactions work in combination. o Chromatin remodellers that move nucleosomes around and affect their binding strength which allows TFs to bind. o Multiple TF binding sites are used in combination to activate a gene. TFs often bind as dimers in order to modulate the binding frequency. In solution TF is found mostly as monomer, on DNA it will bind as dimer. Cis-regulatory sequences: enhancers and silencers In the chromatin context, transcription initiation requires additional activators Cis-regulatory Sequences: Enhancers/Silencers; distant from promoter/transcription start site Insulators; prevent interference to neighbouring transcription units Housekeeping genes Found in all cells Promoters contain binding sites for ubiquitous transcription factors which are present in all tissues. Increase efficiency of transcription: action of each TF is additive in a non-linear way – if A and B each boost transcription two and ten-fold respectively, together they boost it 100-fold. Tissue specific genes Contain binding sites for ubiquitous and tissue- specific transcription factors E.g. Globin genes contain binding sites for ubiquitous factors, sp1 and cp1 , and also for tissue-specific factors such as gata- 1. Combination of binding sites for general ubiquitous TFs + specialized TFs will determine when a specific gene is expressed the same binding sites can bind ubiquitous or tissue specific factors – depends on context. Combinatorial control determines the final gene expression outcome The action of multiple TFs is integrated at the PIC and determines if transcription will occur. Structural motifs are used to classify to TFs into families, e.g. o Helix-turn-helix o Zinc fingers o Leucine zippers o Helix loop helix Four structural motifs that are used to classify transcription factors: examples of factors that contain these motifs Motif generally sit on the major groove. Helix turn helix DNA-binding motif found in many prokaryotic and eukaryotic TFs.  2 α helices connected by an amino acid “turn” and held in fixed position. C terminal “recognition” helix makes contact with major groove of DNA. Usually act as dimers to increase sequence- specificity. Dimerisation will occur through separate domain. They recognise 6-8 nucleotides. Homeodomain proteins (extended HTH) Form 3 alpha helices including the helix turn helix motif Amino acid sequence of helix 3, the recognition helix, determines DNA bindinf specificity. Examples include the regulators of hox genes which must be tightly regulated to ensure correct embryonic patterning. Zinc fingers Diverse transcriptional activators Zn finger structures maintained by Zn2+ ions. Zn2+ connected to C2H2. More than 30 variations known. Finger loop of 12 amino acids, formed by 2 Cys, 2 His (or 4 Cys), binding a single zinc atom Alpha Helix binds along major groove, often occurs as arrays and so can bind along helix. Various Zn finger motifs can be combined in one protein Example includes nuclear receptors which have Cys2/Cys2 fingers Ligand binding to steroid receptors causes them to transloacate into the nucleus. Leucine zippers 3 classes known. Leu (hydrophobic) every 7th amino acid for at least three repeats. Forms aliphatic helix (on every second turn on same side of α helix) Hydrophobic interactions allow protein dimerization (coiled coil) and DNA positioning. Protein dimerization allows positioning of N-terminal, basic stretch (23 aa) to bind into major groove as !- helix. Substitution of leucine abolishes dimerisation and DNA binding. They are Homodimers or Heterodimers, therefore allowing greater combinatorial specificity. Helix loop helix Dimerisation domain, often with adjacent basic DNA-binding domain Many bHLH factors involved in muscle development e.g. MyoD, myogenin, and in neurogenesis e.g.neurogenin Binding of bHLH factors to DNA regulated by dimerisation with ubiquitous bHLH TFs e.g. E12 or with inhibitors e.g. Id (lack basic DNA-binding region). Summary Basic DNA binding motifs are used as modules/domains in many Transcription factors These domains can be combined leading to a vast array of binding specificities Combinatorial power forms the basis of gene-expression regulation both on the single cell, as well as level of a whole organism TFs diversification (radiatin) during evolution mirrors cell-type complexity Examples of mechanisms by which transcription factors activate and repress transcription Transcriptional activators or repressors mediate contact to effectors. Broadly this can be distinguished into three different activities o 1) Recruitment of General TFs or Pol II itself o 2) Altering the structure of histones through recruitment of chromatin modifying enzymes  o 3) Affecting the processivity and elongation speed of RNA polymerase II. Recruiting trans activators of pol II Binding of TFs promotes binding of additional regulators and recruits RNA polymerase to the promoter. Certain transcription factors can prevent nucleosomes from binding and keep nucleosomes open. Affecting chromatin structure Certain transcriptional regulators have histone acetylase (HAT) or histone deacetylase (HDAC) activity: Histone acetylases: CBP (transcriptional co-activator that regulates genes in response to cAMP). Has HAT activity and stimulates basal transcription apparatus. Acetylates histones at n-terminal end and stimulates basal transcription apparatus. Histone deacetylase: thyroid hormone receptor co-repressor. Affecting Pol II transcription Transcription factors can affect how efficiently genes are transcribed Transcriptional repression Some repressors act indirectly: a) Bind DNA and block activator binding b) Bind DNA near activator and block its activation domain e.g. mammalian repressor WT1 (proposed mechanism) Examples of mechanisms by which regulated expression/activity of transcription factors achieve tissue specific or inducible gene expression Spatio-temporal control of TF expression allows diversification of gene-expression patterns in different cell-types. Mis-regulation of this expression control can lead to disease and malformations Experimental methods that are used to investigate transcription factors and their binding sites Protein purification o Transcription factors can be 1:Identifying TFs using footprinting techniques purified by affinity chromatography, using their property of binding specific DNA sequences Low through put: Transcription factor binding to DNA can be detected by “footprinting” techniques o E.g. fluorophores coupled to 5’ ends. (see figure) Reporter gene assay  ChiP or CUT and RUN ** Knock-down assays and subsequent sequencing o Observe altered transcriptional profile after genomic deletion of putative TF 4.5 structure-homology motif predition and binding site prediction using machine learning and AI approaches o Detect if motifs are similar to known DNA binding regions and therefore make predictions about its role.