Gene Expression & Regulation PDF

Gene Expression & Regulation Cellular & Molecular Biology MD105 Dr C. Michaeloudes Gene expression and regulation Dr C. Michaeloudes Cellular & Molecular Biology MD105  Lecture Objectives To understand: The basic structure and types of transcriptio...

Gene Expression & Regulation Cellular & Molecular Biology MD105 Dr C. Michaeloudes Gene expression and regulation Dr C. Michaeloudes Cellular & Molecular Biology MD105  Lecture Objectives To understand: The basic structure and types of transcription factors, and their role in transcriptional regulation The role of regulatory DNA sequences in the regulation of gene transcription The role of histone acetylation and chromatin remodelling in the regulation of gene transcription The importance and basic principles of tissue-specific regulation of gene transcription  Gene expression is controlled in time and space Every cell has the same DNA and therefore the same genes. But different genes need to be “on” and “off” in different types of cells. Therefore, gene expression must be regulated. liver embryo muscle sperm bone  Regulation of gene expression during development 10 wks 14 wks 1 day 6 mos 12 mos 18 mos At different stages of the life cycle, different genes need to be on and off.  Gene regulation DNA 1. Short-term – cells respond to Gene transcription extracellular environment (e.g growth factors, hormones) mRNA 2. Long-term –tissue-specific identity of mRNA translation cells - can be transmitted to daughter cells during cell division Protein Gene expression is regulated at different Protein activity levels: 1. Epigenetic/Chromatin remodelling 2. Transcriptional Phenotype 3. Post-transcriptional 4. Translational REMINDER: Gene Transcription  1. Transcription Initiation Transcription TF TF GENE RNA Pol II P RNA Pol II binds to a non-coding DNA sequence immediately before the target gene called the core promoter RNA Pol II cannot “read” the DNA sequence to find where to bind Guided to the core promoter by proteins called transcription factors (TF) RNA Pol II and transcription factors form the pre-initiation complex RNA Pol II phosphorylation at RBP1 initiates transcription  2. Transcription elongation Coding strand Template strand RNA Pol II is released from the promoter RNA Pol II unwinds the DNA into two separate strands– “transcription bubble” RNA Pol II moves along the DNA and uses one strand as a template to synthesise a complementary RNA sequence § Either of the strands can be a template – depends where the promoter is RNA Pol II adds nucleotides to 3’-end of the growing RNA molecule  3. Transcription termination RNA Pol II encounters a termination signal and transcription stops § Termination signal – AAUAAA hexamer in newly-formed mRNA RNA Pol and mRNA are released A new cycle of transcription begins Transcription Factors  Transcription factors Transcription factors are proteins that bind to specific DNA sequences and regulate (activate or inhibit) gene transcription Most transcription factors do not work alone Ø Recruit other proteins that activate (co-activators) or inhibit transcription (co-repressors)  Transcription factors can activate or repress transcription Transcription factors can be activators of gene transcription by: Ø Recruiting co-activators to target genes Ø Co-activators = proteins that activate or increase gene transcription by interacting with transcription factors Transcription factors can be repressors of gene transcription by recruiting co-repressors to target genes Ø Co-repressors = proteins that inhibit gene transcription by interacting with transcription factors  Transcription factors domains Structural domains: functional units shaped to allow a particular interaction (i.e with DNA or other proteins) Generally have the following structural domains: Ø DNA-binding domain (all) Recognizes specific short sequences near target gene Ø Trans-activation/trans-repression domain (all) Contains binding sites for co-activators/co-repressors Ø Ligand-binding domain (some) Some TFs require ligand binding (hormone receptors) for activation Ø Dimerisation domain (some) Some TFs need to form dimers to bind DNA  Transcription factor domains Interaction with Trans-activation/repression co-activators domain /co-repressors Binding to specific DNA-binding domain DNA sequences  DNA binding domain DNA binding domain shape allows them to bind to the major groove of DNA and interact with nucleotide bases The amino acid sequence of the DNA- binding domain determines the specific DNA sequences it can bind to Ø Determines TF specificity Recognize a range of short (6-12bp) Leucine Zipper DNA sequences called transcription DNA binding domain factor binding motifs - e.g TATA binding protein binds to TATAAAA but also TATATAT or TATATAA Transcription factors regulate transcription by binding to regulatory DNA sequences  Basic and specific transcription factors Transcription factors can be divided into two groups based on their mechanism of action during transcription: 1. Basic Transcription Factors (only 6) Ø Found in all cells and tissues Ø Recruit RNA Pol II to the gene promoter Ø Transcriptional activators 2. Specific Transcription Factors (>2000) Ø Show tissue-specificity (e.g heart-specific or lung-specific) Ø Regulate gene transcription Ø Transcriptional activators or repressors  Transcription factors bind to regulatory sequences +1 Start site Core Enhancer/Silencer Gene Enhancer/Silencer Promoter The core promoter and enhancer/silencer elements : Are regions of non-coding DNA that regulate the transcription of nearby genes Contain transcription binding motifs Recruit transcription factors (transcription activators or inhibitors) to the target gene  Core promoter RNA Pol II Basal TFs Core Enhancer/Silencer Gene Enhancer/Silencer Promoter Core promoter is immediately upstream (before) the gene – fixed position (60-120bp) Contains the transcription start site (+1) Binding sites for basal transcription factors Basal transcription factors recruit RNA Pol II to the gene Required for gene transcription  Enhancers/Silencers Specific TFs RNA Pol II Specific TFs Basal TFs Core Enhancer/Silencer Gene Enhancer/Silencer Promoter Enhancers/Silencers can be upstream (before) or downstream (after) the gene promoter Can be near (100bp) or very far (Mbp) from the gene promoter Contains binding motifs for specific transcription factors Ø More activators (enhancer)/More repressors (silencer) Regulates the activity of the core promoter (enhances or inhibits) Enhancers/silencers regulate gene expression from a distance Transcription factors bound Basal transcription complex on promoter to enhancer/silencer Enhancer/Silencer Chromatin Core promoter Cohesins Condensins looping Enhancer/Silencer Co-activators/ Co-repressors Core promoter  Enhancers/silencers regulate gene expression from a distance Enhancer Enhancer/silencers bring transcription factors and co-activators/co- repressors very close to the core promoter Core promoter Chromatin loops formed by the non-histone proteins cohesins and condensins bring the enhancer/silencer sequences in very close proximity to the promoter  Co-activators Transcription activators on enhancer elements activate gene transcription by bringing co-activators to the gene promoter These co-activators increase gene transcription by: 1. Opening chromatin (euchromatin) to allow binding of RNA Pol II 2. Recruiting RNA Pol II and basal transcription factors to the target gene promoter and stabilizing the pre-initiation complex  Co-repressors Transcription repressors on silencer elements can inhibit gene transcription by: 1. Recruiting co-repressors which induce chromatin condensation (heterochromatin) and inhibit transcription 2. Binding to DNA and directly blocking RNA Pol II recruitment to the target gene Histone modifications and chromatin remodelling  Nucleosomes prevent gene transcription Transcription complex HETEROCHROMATIN Nucleosomes prevent transcription complex NO GENE TRANSCRIPTION from accessing the gene promoter GENE TRANSCRIPTION EUCHROMATIN Transcription complex binds to the gene promoter and activates transcription  How does chromatin “open up”? 1. Nucleosome displacement 2. Chromatin unravelling ATP-dependent chromatin Histone modifications remodeling enzymes (e.g acetylation) Chromatin remodelling  Histones Histone tail + H2A + H2B H3 H4 + + - - Highly conserved proteins with positive charge – rich in lysine and arginine Globular proteins with an N-terminal tail that contains sites for covalent modifications Electrostatic interactions between positively-charged histones and negatively-charged DNA  Histone acetylation Positive charge lost Acetyl group + Histone acetyltransferases (HATs) Lysine Acetyl-CoA CoA Acetylated Lysine Acetate H2O Histone deacetylases (HDACs) Bromodomains - acetylated histone readers Acetylated lysine Lysine acetylation binding pocket Proteins containing special modules called bromodomains can bind to acetylated histones Bromodomains contain pockets where acetylated lysine residues bind Different types of proteins contain bromodomains, including chromatin remodeling complexes and transcription factors Image adapted from Brand M,, ACS Chem Biol 2015; PMID: 25549280  Histone acetylation associated with gene activation 1. Unravels chromatin 2. Creates binding sites for transcription + + + + + activators Ac Ac - - - - - GENE ON Ac Ac Transcription factors and chromatin remodelling protein contain bromodomains - - - - - Removes positive charge on histones and Binding sites for proteins with disrupts the electrostatic interactions with bromodomain DNA - chromatin unravels Recruitment of chromatin Chromatin accessible to transcription remodelling and transcription complex factors - gene activation Number of acetyl groups in the region Position of acetyl groups important important  Histone acetylation and gene regulation TRANSCRIPTION OFF Histone deacetylases (HDACs) Histone acetyltransferases (HATs) Recruited to gene promoters Recruited to gene promoters by by repressor complexes activator complexes “Condensed” chromatin “Open” chromatin/transcription Repression of gene activity complex Gene activation Ac Ac TRANSCRIPTION ON  Transcription factors regulate gene transcription by chromatin modification Chromatin remodelling Histone complex acetyltransferase RNA RNA Pol II Pol II Transcription activators can activate gene transcription by opening chromatin (euchromatin) § E.g recruitment of histone acetyltransferases and chromatin remodelling complexes  Transcription factors regulate gene transcription by chromatin modification Histone deacetylase Transcription repressors can inhibit gene transcription by inducing chromatin condensation (heterochromatin) § E.g recruitment of histone deacetylases Tissue-specific regulation of gene transcription  Tissue-specific regulation of transcription Every cell has the same DNA and therefore the same genes But different genes need to be “on” and “off” in different types of tissues and cells. Long -term, tissue-specific and cell type-specific gene expression is crucial for normal growth and development  Tissue-specific regulation of transcription Basal transcription factors are found in all tissues and cells Core promoters are usually active in all tissues and cells HOWEVER Specific transcription factors are tissue- and cell type- specific i.e found in some tissues/cells but not in others Enhancer activity shows tissue- and cell type-specificity i.e an enhancer can be active in some tissues/cells and inactive in others Cell type-specific transcription factors Differentiation of each type of lung epithelial cells requires a different set of specific transcription factors Image from Maeda, Physiol Rev 2007; PMID: 17237346  Tissue-specific enhancer activity Lung fibroblasts Histone deacetylase activity Inactive enhancer Low histone acetylation Condensed chromatin Hepatocytes Histone acetyltransferase Histone acetylation Active enhancer Open chromatin Histone modifications can activate or inactivate enhancers in different cells or tissues Ø e.g lung fibroblasts and hepatocytes  Tissue-specific enhancer/silencer activity Lung Fibroblast ON Silencer Gene X Enhancer Inactive silencer Active enhancer Hepatocyte OFF Silencer Gene X Enhancer Active silencer Inactive enhancer