OLM MCB.15 Regulation of Gene Expression - notes PDF

REGULATION OF GENE EXPRESSION Dr. David Rodda, PhD 1  Learning Objectives 1. Describe the levels at which gene expression can be regulated and discuss the advantages and disadvantages of regulation at each level. 2. Summarize the...

REGULATION OF GENE EXPRESSION Dr. David Rodda, PhD 1  Learning Objectives 1. Describe the levels at which gene expression can be regulated and discuss the advantages and disadvantages of regulation at each level. 2. Summarize the various types of transcription factors, mediator proteins, and regulatory elements that control gene expression in both prokaryotes and eukaryotes. 3. Explain how transcription factors, in concert with their ligands, can either positively or negatively regulate transcription by binding to regulatory elements. 4. Describe the regulation of the Lac operon in prokaryotes and predict how the expression of the operon will change as the availability of glucose and lactose changes. 5. Describe the epigenetic mechanisms that control chromatin conformations and gene expression in eukaryotes. 6. Analyze the results of a reporter assay to identify positive and negative regulatory elements. 7. Describe DNA-binding Domains and explain how they achieve sequence-specific DNA binding. 2  Learning Objectives 8. List the 5 major classes of steroid hormones, describe their major physiological functions and name the most import endogenous hormone in each class. 9. Describe the signal transduction mechanism for steroid hormones acting through their cognate receptors. 10. Describe the general properties of the nuclear receptor superfamily of transcription factors. 11. Contrast agonists with antagonists. 12. Summarize the mechanisms by which Iron (Fe2+) regulates gene expression post-transcriptionally. 13. Explain the role microRNAs (miRNA) play in post-transcriptional gene regulation. 3 Introduction Regulation of Prokaryotic Gene Expression Regulation of Eukaryotic Gene Expression Epigenetic Regulation of Transcription Regulatory Elements Transcription Factors Steroid Hormones and the Steroid Receptors Transcription Factors and Disease Post-Transcriptional Regulation of Gene Expression Summary INTRODUCTION 4  Cell Type-Specific Gene Expression Almost all cells carry the same genes Only a subset of these genes are expressed in any one cell at any one point in time Specific cell types express a unique profile of genes which confer the characteristics of that cell type Many genes are expressed only in one or a few specific cell types, or only at specific times during development ▪ Examples: Hemoglobin in red blood cells Opsins in retinal photoreceptor cells Nanog in the epiblast of preimplantation embryos 5  Regulation of Gene Expression Cells can also adapt to environmental and physiological stimuli by altering their gene expression profiles ▪ Inducible genes ▪ Repressible genes ▪ Examples: E. coli express enzymes to metabolize lactose only when lactose is available in the environment In humans, hypoglycemia triggers production of cortisol (a glucocorticoid steroid hormone) which signals for increased expression of enzymes that activate glycogenolysis and gluconeogenesis to release glucose into the blood Some genes are expressed in almost all cells, at relatively constitutive levels: Housekeeping genes How do cells control which genes are expressed in specific cells, at specific times during development, or in response to specific stimuli? Glycogenolysis – the breakdown of cellular glycogen to glucose. Glycogen is a polymer of glucose monosaccharaides and is used to store glucose. Gluconeogenesis – the synthesis of glucose de novo In general, the expression of Housekeeping genes is necessary to keep the cells alive. They control necessary and common cellular functions. All cells need to express these housekeeping genes, and they generally are expressed at one level at all times. 6 When Is Gene Expression Regulated? Gene expression can be (and is) regulated at each of the steps of the expression pathway 1. Initiation of transcription 2. Processing of the transcript 3. mRNA degradation 4. Translation of the transcript to protein 5. Post-translational processing of the protein 6. Protein degradation Which of these steps is the most important? Which step is the fastest? 7 Regulation of Transcriptional Initiation Initiation of Transcription ▪ The first step in gene expression ▪ The most important regulated step ▪ The human genome contains ~2600 genes encoding DNA- binding proteins Most are presumed to be transcriptional regulators >10% of all human genes are involved in regulating transcription 8  Regulation of Transcriptional Initiation Two Major Mechanisms Regulate Transcriptional Initiation 1. Interplay of trans-acting factors and cis-acting elements to control RNA polymerase recruitment All genes contain consensus sequences called ‘cis-acting elements’ which regulate the gene’s expression aka. DNA regulatory elements Bound by protein ‘trans-acting factors’ which affect recruitment of RNA- polymerase aka. Transcription factors Sequence-specific DNA-binding proteins Important in both prokaryotes and eukaryotes Jargon Elements: short discreet DNA sequences Factors: proteins Domains: regions within proteins (usually with a discreet function) Cis-acting: the regulation is conferred by a the same molecule (or part of that same molecule) that is being regulated. Ie. a cis-acting element is a DNA sequence in a chromosome that plays a role in regulating a gene on that same chromosome. These regulatory elements are on the order of 6-12 base-pairs in length and are found within larger regulatory regions such as promoters, enhancers and silencers Trans-acting: the regulation is conferred by a molecule different than that being regulated. Ie. a trans-acting factor is a protein that regulates the expression of a gene on a different molecule – the DNA strand within a chromosome. 9  Regulation of Transcriptional Initiation Two Major Mechanisms that Regulate Transcriptional Initiation 2. Alteration of chromatin structures to control accessibility of the gene to transcription factors and RNA polymerase. ie. Epigenetics Occurs only in Eukaryotes Since epigenetic regulation of gene expression involves alteration of chromatin structures, it’s not a mechanism that prokaryotes can take advantage of since they don’t have chromatin. 10  Three Classes of Transcription Factors 1. Initiation Factors Direct RNA polymerase to specific genes Initiation of transcription Eg. Sigma Factors in prokaryotes, TBP in eukaryotes 2. Repressors Impede binding of RNA polymerase to promoter Decreased rates of gene expression Negative regulation 3. Activators Promote binding of RNA polymerase to promoter Increased rates of gene expression ▪ Positive regulation The Initiation Factors described here are also sometimes called Specificity Factors, but that term is easy to confuse with the Specific Transcription Factors we will discuss later, so for our purposes we’ll call them Initiation Factors. 11 Mechanisms of Regulation by Transcription Factors 12 How to Measure Gene Expression? Quantification of mRNA ▪ Why not protein? Methods ▪ Northern Blot One probe for every gene measured Semi-quantitative ▪ RT-PCR (possibly with qPCR) Very sensitive and accurate One PCR primer set for every gene measured ▪ Gene expression microarray Can measure expression of many genes at once (high throughput) Only detects genes if a corresponding probe is on the microarray ▪ Next-generation sequencing (RNA-seq) Quantitative with sufficient sequencing depth High throughput, some bias 13 Introduction Regulation of Prokaryotic Gene Expression Regulation of Eukaryotic Gene Expression Epigenetic Regulation of Transcription Regulatory Elements Transcription Factors Steroid Hormones and the Steroid Receptors Transcription Factors and Disease Post-Transcriptional Regulation of Gene Expression Summary REGULATION OF PROKARYOTIC GENE EXPRESSION 14  Prokaryotic Promoters Promoters ▪ A regulatory region of the gene found immediately upstream (5') of the transcription start site (TSS) Prokaryotic Promoters ▪ The only type of regulatory region in prokaryotes Sequence differences can account for ~1000-fold difference in mRNA produced ▪ Promoter consensus sequences are binding sites for transcription factors ▪ Deviations from consensus sequences usually reduce promoter function TSS Consensus Sequences Binding sites for Sigma Factors It is difficult to define the size of promoters. In prokaryotes they are relatively simple, usually less than 100bp containing relatively few consensus sequences (aka. Regulatory elements / cis-acting elements). Eukaryotic promoters can be much more complex and larger, sometimes well over 1000bp, although there is no strict rule that distinguishes promoters from enhancers (see eukaryotic regulation later) Regulatory elements often have variable sequences. Consensus sequences are determined by aligning many of the same regulatory elements from different locations, and determining the most common base at each position. Usually a consensus sequence is the optimal sequence. Eg. the highest affinity binding sequence for a sigma factor (or other transcription factor) The more a specific element diverges from the consensus, the weaker it will be. So divergence from a consensus in a promoter leads to a lower rate of transcription. 15 Sigma Factors are Initiation Factors Sigma (σ) Factors Sequence specific DNA-binding proteins Components of RNA polymerase ▪ Initiation of transcription Different σ's bind different consensus sequences ▪ -10 and -35 boxes (previous slide) ▪ Specify genes that will be expressed 16  Signal Molecules (Transcription Factor Ligands) There is unique jargon in prokaryotic gene regulation Ligands that act via a Repressor 1. Co-repressors enhance repressor activity 2. Inducers inhibit repressor activity Ligands that act via an Activator 3. Co-activators enhance activator activity 4. Repressors inhibit activator activity Bacterial geneticists have come up with their own jargon which is often different compared to the eukaryotic jargon, even for molecules that act by the same mechanisms. Be careful because some of these terms are also used in eukaryotic gene regulation, but have very different meanings. 17 Identify the Ligands From The Previous Slide 18  Operons A cluster of genes encoding products involved in the same biochemical pathway ▪ Coordinated regulation Often Polycistronic Includes i. Promoter with regulatory elements ii. Structural genes (encoding products involved in the pathway) iii. May include regulatory genes (encoding regulatory proteins) Most important in prokaryotes, rare in eukaryotes Polycistronic: a single mRNA encodes multiple proteins. Transcription of the mRNA is controlled by a single promoter. Each gene within the polycistronic mRNA will have it's own Shine-Dalgarno sequence, allowing ribosomes to translate the genes independently. Polycistronic genes are most common in prokaryotes. They are rare in eukaryotes. Eukaryotic polycistronic genes do not have Shine-Dalgarno but have alternative sequences that allow ribosome binding for translation. A structural gene is a gene that codes for any RNA or protein product other than a regulatory factor (i.e. regulatory protein). It may code for a structural protein, an enzyme, or an RNA molecule not involved in regulation. 19  The Lac Operon The Lac Operon is expressed only when: 1. Lactose is available 2. Glucose is not available Lac Operon Structure i. 3 structural genes involved in Lactose catabolism ii. A regulatory region containing: ▪ Promoter (p) The binding site for RNA polymerase (Sigma Factor) ▪ Operator (o) A binding site for the repressor protein Between promoter and structural genes 20  The Lac Repressor If lactose is not available the Lac Repressor will prevent the operon from being expressed Lac Operon Structure (continued) iii. 1 Regulatory gene (i) Lac I encodes the Lac Repressor Lac Repressor ▪ Binds specifically to the Operator Operator is a negative regulatory element ▪ Blocks RNA-polymerase from transcribing the structural genes ▪ Constitutively expressed 21  The Inducer is Allolactose If lactose is available the Lac Repressor will dissociate from the Operator and transcription can proceed Lactose is converted to Allolactose by β-galactosidase Allolactose is a ligand for the Repressor ▪ When bound to Allolactose the Repressor dissociates from the Operator The RNA polymerase can bind the promoter and transcribe the structural genes Allolactose is an Inducer of the Repressor 22 Which Illustrates the Lac Repressor and the Inducer? 23  cAMP is the Co-Activator CAP (aka CRP) ▪ CAP: Catabolite Activator Protein ▪ CRP: cAMP Response Protein cAMP is a ligand for the CAP protein ▪ CAP-cAMP complex binds the CAP site (element) within the promoter The CAP-cAMP complex is a transcriptional Activator ▪ Recruits RNA polymerase to promoter cAMP is a Co-Activator of CAP 24  Glucose Inhibits cAMP Synthase cAMP is produced from If Glucose is available, it inhibits ATP by adenylyl cyclase cAMP production & Adenylyl cyclase is CAP can not activate transcription inhibited by Glucose So, in the presence of Glucose: ▪ cAMP is not synthesized Not available as a ligand for CAP ▪ CAP is unable to bind DNA ▪ RNA polymerase is not recruited to the promoter ▪ No transcription Adenylate cyclase = adenylyl cyclase = adenyl cyclase 25 Which Illustrates the CAP Protein and cAMP? 26 Lac Operon: Requirements for Transcription 1. The Inducer Allolactose (presence of Lactose) i. Lactose is converted to allolactose (the inducer) ii. The inducer binds the repressor iii. The repressor does not bind the operator iv. The promoter is available for transcription 2. The Co-Activator cAMP (absence of Glucose) i. cAMP is a ligand of the CAP activator ii. CAP-cAMP activates iii. Glucose inhibits cAMP production & prevents activation 27  Summary of Lac Operon Regulation Make sure you understand well how the lac operon is regulated under each of the four different sugar combinations shown on this slide. You should be able to explain how the regulation would change as bacteria transitions from one sugar combination to another. 28 Introduction Regulation of Prokaryotic Gene Expression Regulation of Eukaryotic Gene Expression Epigenetic Regulation of Transcription Regulatory Elements Transcription Factors Steroid Hormones and the Steroid Receptors Transcription Factors and Disease Post-Transcriptional Regulation of Gene Expression Summary REGULATION OF EUKARYOTIC GENE EXPRESSION 29  Regulation of Eukaryotic Gene Expression A hypothetical order of events Contrast with Prokaryotic Gene which may occur in different orders on different genes Regulation ▪ Increased complexity i. Chromatin structure plays a central role ii. A variety of regulatory regions containing many more regulatory elements iii. A larger number of transcription factors Basal / General Specific iv. Multiple transcription factors regulate most genes May act synergistically Combinatorial effects determine levels of gene expression 30 Introduction Regulation of Prokaryotic Gene Expression Regulation of Eukaryotic Gene Expression Epigenetic Regulation of Transcription Regulatory Elements Transcription Factors Steroid Hormones and the Steroid Receptors Transcription Factors and Disease Post-Transcriptional Regulation of Gene Expression Summary EPIGENETIC REGULATION OF TRANSCRIPTION 31 Regulation of Chromatin Structures (Epigenetics) 1. DNA Methylation ▪ In vertebrates, only C's within 5'-CG-3' (aka CpG) sequences are methylated ▪ Associated with heterochromatin formation and transcriptional silencing ▪ Permanent After a gene is silenced by DNA methylation it generally will stay silent for the rest of the lifetime of that cell, and also in daughter cells Exceptions: germ cells and zygotes Important role in differentiation and determination of cell-specificity ▪ CpG Islands A region of DNA sequence rich in CpG dinucleotides. Often found upstream of genes Hypermethylation within CpG islands is very strongly silencing 32  Regulation of Chromatin Structures (Epigenetics) 2. Histone Modification Histone code hypothesis: histone modifications partially determine chromatin structure Post-translational modifications of Histone N-terminal tails (aka "marks") i. Acetylation Activating Histone Acetyltransferases (HATs) Add acetyl groups - Coactivators Histone Deacetylases (HDACs) Remove acetyl groups-Corepressors ii. Methylation H3 Variable effects iii. Phosphorylation H3 Variable effects Roles in other processes H3 DNA replication DNA repair H3 As part of the histone code hypothesis, each specific histone modification will have a determining effect on the chromatin structure. The effects of some individual marks are now known, but what effects combinations of marks will have is largely unknown. In addition to Acetylation, Methylation and Phosphorylation, many other post- translational modifications also occur to histone tails. We are only beginning to research how these all control chromatin structure. 33 Regulation of Chromatin Structures (Epigenetics) 3. Chromatin Remodeling ▪ ATP-dependent chromatin remodeling ▪ Eg. Swi/SNF family ▪ Nucleosome remodeling, removal & replacement 34 Introduction Regulation of Prokaryotic Gene Expression Regulation of Eukaryotic Gene Expression Epigenetic Regulation of Transcription Regulatory Elements Transcription Factors Steroid Hormones and the Steroid Receptors Transcription Factors and Disease Post-Transcriptional Regulation of Gene Expression Summary REGULATORY ELEMENTS 35  Regulatory Regions in Eukaryotic Genes 1. Promoters ▪ Larger and more complex than prokaryotic promoters ▪ Contain multiple regulatory elements (ie. consensus sites) Initiation of Transcription TATA box, Inr sequence Binding of General / Basal transcription factors Usually found at consistent locations Other regulatory / response elements Specificity (tissue and/or temporal) and response to stimuli Binding of Specific transcription factors Can be found at different locations Only about 50% of human genes have a TATA box, others may have an Inr sequence which directs transcription initiation instead of the TATA box 36 Regulatory Regions in Eukaryotic Genes 2. Enhancers and Silencers ▪ Enhancers activate Enhancers ▪ Silencers repress ▪ Located upstream, downstream, or within introns of genes ▪ rarely if ever in exons ▪ Usually location and orientation non-specific Can be > 106 bp from the gene ▪ Regulatory / Response elements Specificity (tissue and temporal) and response to stimuli ▪ DNA-folding brings the region into closer proximity of the promoter Regulate transcription initiation 37  Eukaryotic Regulatory Elements Regulatory Elements ▪ Discrete DNA sequences of 6-12 bp ▪ Binding sites for transcription factors ▪ Found within promoters, enhancers or silencers Response Elements ▪ A regulatory element that mediates a response to a stimuli ▪ Eg. steroid response elements, estrogen response elements, cAMP response elements Insulator Elements ▪ Prevent enhancers or silencers from regulating the inappropriate genes Barrier Sequences ▪ Prevent heterochromatin from spreading inappropriately More on barrier sequences: Within a chromosome, regions of heterochromatin can vary in size from cell to cell, even among cells of the same type. This can result in genes nearby the heterochromatin region being silenced inappropriately in some cells if the heterochromatin region has expanded. So cells of the same type may either express or not express these genes. This is a phenomenon called "position-effect variegation". It is relatively rare in humans (thanks largely to barrier sequences) but does occur in some animals and plants. Eg. fruit-fly eyes. 38  Identifying Regulatory Elements Reporter Assays ▪ Mutational analysis of a regulatory region (eg. a promoter) to identify regulatory elements ▪ The regulatory region is cloned upstream of a reporter gene plasmid plasmid an interesting promoter reporter gene ▪ Mutations are introduced into the regulatory region plasmid plasmid an interesting promoter luciferase gene luciferase gene luciferase gene luciferase gene luciferase gene ▪ Transcription is measured by assaying the expression of the reporter gene Reporter genes Not found endogenously in the organisms in which the assay is being carried out So endogenous activity does not confound results Sensitive assays are available Eg. Luciferase, Green Fluorescent Protein Luciferase is the enzyme which catalyzes the reaction that produces the light in fireflies. It is not found in mammals so is often used as a reporter gene in human (or other mammalian) systems. The assay produces light and very sensitive assays are available using photodetectors. Green Fluorescent Protein is a jellyfish protein that naturally fluoresces under UV light. It is not expressed in mammals and can also be easily assayed using fluorescence detectors. 39  Reporter Assay Analysis In this promoter, are elements A, B and C activating or repressing elements? Wild-type C B A luc Deletion Mutants B A luc A luc luc Point Mutants X B A luc C X A luc C B X luc 0 50 100 150 Relative Luciferase Activity (%) 40 Introduction Regulation of Prokaryotic Gene Expression Regulation of Eukaryotic Gene Expression Epigenetic Regulation of Transcription Regulatory Elements Transcription Factors Steroid Hormones and the Steroid Receptors Transcription Factors and Disease Post-Transcriptional Regulation of Gene Expression Summary TRANSCRIPTION FACTORS 41  General Transcription Factors Sequence-specific DNA binding proteins ▪ Bind regulatory elements ▪ Control rates of transcriptional initiation Directly or indirectly Basal/General Transcription Factors ▪ Assembly around transcriptional initiation site Eg. TATA box, Inr sequence ▪ Directly recruit the RNA polymerase TFII factors recruit RNA polymerase II ▪ Necessary for transcription of all genes ▪ TBP and TFIID are the most important to know about specific transcription factors specific transcription factors In the figure on the right, the names of the transcription factors have been abbreviated to remove the TF from their names (e.g. IID in the figure is TFIID) General transcription factors are by convention named TF + number of RNA polymerase they regulate + letter represented order of discovery So any factor beginning with TFI regulates RNA polymerase I (ie. rRNA genes) Factors beginning with TFIII regulate RNA polymerase III (ie. tRNA and the 5S rRNA genes) Factors beginning with TFII regulate RNA polymerase II (ie. protein coding (mRNA) genes) – these are the most interesting. 42  Specific Transcription Factors Specific Transcription Factors ▪ Thousands of factors that bind regulatory elements and control: i. Strength of expression ii. Specificity of expression both cell type and temporal specificity iii. Response to stimuli ▪ Only regulate specific gene(s) ▪ May regulate many different genes, sometimes having drastically different effects 43  Mediator Proteins Specific transcription factors affect transcriptional initiation indirectly They make contacts with either: i. General transcription factors, or ii. Mediator proteins Act as "bridges" with the general transcription factors Do not bind to specific DNA sequences May have enzymatic activity Eg. HAT, HDAC, other histone modifications, chromatin remodeling Coactivators Corepressors As a general rule specific transcription factors don't make direct contacts with the RNA polymerase, and so don't affect transcriptional initiation directly. 44  Transcription Factor Synergy Most genes are regulated by multiple transcription factors Transcriptional activators may act synergistically ▪ Combined effect is greater than the additive effect of the factors acting individually 45  DNA-Binding Domains Transcription factors bind DNA through their DNA-binding domains (DBDs) ▪ Make sequence-specific bonds with the DNA ▪ Most types use an alpha-helix binding within the major groove There are a limited number of types of DBDs shared among all transcription factors Families of transcription factors share the same type of DBD Zinc finger domains are the most important to remember basic bHLH Nuclear Receptors c-jun, c-fos Homeodomains (Hox) Myo-D, Myf5 Octamer factors (Oct1 and 4) E. HMG box Sox factors (Sox2) SRY 46  Sequence Specific DNA-Binding Within DNA-binding domains, amino acid side-chains make contact with: ▪ Bases Hydrogen Bonds / Hydrophobic Interactions ▪ Sugar-phosphate backbone Ionic bonds / Salt bridges ▪ Which contacts would provide sequence specificity? 47 Introduction Regulation of Prokaryotic Gene Expression Regulation of Eukaryotic Gene Expression Epigenetic Regulation of Transcription Regulatory Elements Transcription Factors Steroid Hormones and the Steroid Receptors Transcription Factors and Disease Post-Transcriptional Regulation of Gene Expression Summary STEROID HORMONES AND THE STEROID RECEPTORS 48  Steroid Hormones Important endocrine signaling molecules ▪ Secreted by a gland into blood circulation ▪ Have effects on tissues far from the gland of origin Endogenous steroids regulate metabolism, inflammation, immune functions, salt and water balance, sexual development etc. Synthetic steroids are used as pharmaceuticals with many different indications 49  Classes of Steroid Hormones There are 5 classes of steroid hormone which can be divided into 2 broad groups based on the glands which secrete them: ▪ Corticosteroids produced in the adrenal cortex I. Glucocorticoids II. Mineralocorticoids ▪ Sex steroids produced in the gonads (testes or ovaries) or placenta I. Androgens II. Estrogens III. Progestogens 50  Corticosteroids Glucocorticoids ▪ Eg. Cortisol, corticosterone ▪ Signal chronic stress ▪ Regulate energy metabolism, increase glucose production ▪ Inhibit inflammation Mineralocorticoids ▪ Eg. Aldosterone ▪ Regulates salt metabolism and blood pressure 51  Sex Steroids Androgens ▪ Eg. Testosterone, dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), androstenodione ▪ Regulate male sexual development Estrogens ▪ Eg. Estradiol, estriol, estrone ▪ Regulate female sexual development Progestogens ▪ Eg. Progesterone ▪ Regulate female reproductive cycles 52  Steroid Hormone Signaling Steroid Hormones Circulate with plasma steroid-binding proteins (SBP) Diffuse through the plasma membrane Bind their cognate receptor in the cytosol Steroid Hormone Receptors Are maintained in the cytosol in a ligand- receptive conformation by chaperones ▪ Eg. Hsp90 & Hsp70 Upon hormone binding to receptor ▪ Chaperones are released Receptor-Steroid complexes: i. Form homodimers ii. Translocate to nucleus iii. Bind response-elements iv. Activate (or sometimes repress) transcription Steroid hormones are derived from cholesterol and are largely hydrophobic molecules. This allows them to pass through the plasma membrane by diffusion. Homodimer – two identical molecules interacting. Contrast with: Heterodimer – two different molecules interacting The binding of the receptor to the chaperones masks a nuclear localization signal. After steroid binding and release of the chaperones, the nuclear localization signal is exposed and the homodimers rapidly translocate into the nucleus. 53  The Steroid Hormone Receptor Family Steroid hormones act only through their cognate receptors to regulate transcription The Steroid Receptors i. Glucocorticoid receptor ii. Mineralocorticoid receptor iii. Progesterone receptor iv. Androgen receptor v. Estrogen receptor The elements they bind are These 3 are nuclear variously called: receptors but ▪ Hormone Response Elements not steroid receptors (HREs) ▪ Steroid Response Elements (SREs) ▪ Specific Steroid Response Elements Eg. Glucocorticoid Response Elements, Estrogen Response Elements etc. There are five classical steroid receptors, although variant isoforms exist. The steroid hormone listed under each is the primary physiological steroid hormone that each responds to, although for each type there are other physiological and pharmaceutical steroids that can also signal through these receptors. 54  The Nuclear Receptor Superfamily The Nuclear Receptor Superfamily ▪ In humans a superfamily of 48 different receptor proteins ▪ Ligand-binding transcription factors ▪ Zn-finger DNA-binding domains ▪ Classes: I. Steroid Hormone Receptors II. Thyroid Hormone, Vitamin D, Retinoic Acid Receptors III. Orphan Receptors ▪ All family members have the same general domain structure Orphan receptors have no known ligands. Either they are not regulated by a ligand, or the ligand has not been discovered yet. 55 Binding of the Glucocorticoid Receptor to a GRE Note there are both: i. Protein-DNA interactions between the Zn-fingers and the glucocorticoid response elements Red highlights ii. Protein-Protein interactions between the two monomers of the homodimer Green highlights 56  Steroid Agonists vs. Antagonists Agonists ▪ Bind to a receptor and stimulate gene expression Examples: Most endogenous steroids Dexamethasone - a synthetic glucocorticoid agonist Antagonists ▪ Block the activity of the endogenous steroid by competitive binding to the receptor ▪ Repress gene expression and/or block activation Examples: Mifepristone (RU486) – a glucocorticoid and progesterone antagonist Indications: termination of pregnancy, Cushing's syndrome 57 Treatment of Breast Cancer by Tamoxifen Growth of some breast cancers depends on estrogen signaling Tamoxifen ▪ An estrogen antagonist in the breast ▪ Estrogen Receptor bound to Tamoxifen recruits a corepressor instead of a coactivator ▪ Cell division stops ▪ An estrogen agonist in the endometrium ▪ Estrogen Receptor bound to tamoxifen recruits a coactivator ▪ Increased growth, risk of developing endometrial cancer 58  Coordinated Gene Expression A single transcription factor can coordinate the expression of many genes Example Left: multiple genes bound by different specific transcription factors ▪ Insufficient for activation Right: upon glucocorticoid signaling, the glucocorticoid receptor synergizes with the other factors to activate all of the genes A mechanism for coordinated expression of many genes Also a mechanism for cell-type specificity ▪ The glucocorticoid receptor may activate different sets of genes in different cell types, depending on what other transcription factors are expressed in that cell type 59 Introduction Regulation of Prokaryotic Gene Expression Regulation of Eukaryotic Gene Expression Epigenetic Regulation of Transcription Regulatory Elements Transcription Factors Steroid Hormones and the Steroid Receptors Transcription Factors and Disease Post-Transcriptional Regulation of Gene Expression Summary TRANSCRIPTION FACTORS AND DISEASE 60  Transcription Factors and Disease Mutations in transcription factors are associated with many diseases Some examples: Cancer Autoimmune ▪ Many proto-oncogenes are transcription factors ▪ Polyendocrinopathy c-myc, c-jun, c-fos Syndrome AIRE Developmental ▪ Nail-patella syndrome LMX1B ▪ Cleidocranial dysplasia Diabetes RUNX2 ▪ Maturity Onset Diabetes ▪ Bicuspid aortic valve GATA4 of the Young (MODY) ▪ Synpolydactyly HNF1α, HNF1β, HNF4α, HOXD13 PDX1, NEUROD1 You will need to know several of these disease in the future, but you don't need to memorize them for the purposes of this lecture. Syndactyly – fusion of digits Polydactyly – extra digits 61 Transcription Factor Mutations Transcription factors regulating development ▪ Regulation of multiple genes through many stages of development ▪ Mutations often result in pleiotropy 62 Introduction Regulation of Prokaryotic Gene Expression Regulation of Eukaryotic Gene Expression Epigenetic Regulation of Transcription Regulatory Elements Transcription Factors Steroid Hormones and the Steroid Receptors Transcription Factors and Disease Post-Transcriptional Regulation of Gene Expression Summary POST-TRANSCRIPTIONAL REGULATION OF GENE EXPRESSION 63  Post-Transcriptional Regulation While the initiation of transcription is the most important regulatory point for expression of most genes, some genes are also regulated post transcriptionally Regulation at the following levels can also occur: 1. mRNA stability, processing, transport or editing 2. Translation initiation or elongation 3. Post-translational processing or modifications To illustrate post-transcriptional regulation, we will look at examples of post-transcriptional regulation by Iron (Fe2+) 1. Translation of Globin 2. Translation of Ferritin 3. mRNA stability of Transferrin Receptor 64  Translational Regulation of Globin In reticulocytes α- and β-globin must bind heme and Fe2+ for functional hemoglobin Globins are only synthesized when heme and Fe2+ are available Heme Kinase eIF2 eIF2 P Stable Unstable eIF2 stability is controlled by phosphorylation Heme inhibits the kinase If Fe2+ and heme are available ▪ Kinase is inhibited and translation of globin proteins proceeds If Fe2+ and heme are not available ▪ Kinase is active, eIF2 is unstable, and translation stops 65  Regulation of Ferritin Translation Iron Response Element Binding Protein (IRE-BP) Regulates expression of Ferritin and the Transferin Receptor post-transcriptionally RNA-binding protein Dissociates from RNA when bound by Fe2+ Ferritin ▪ Cellular Fe2+ storage protein ▪ Expressed when cellular Fe2+ levels are high ▪ An IRE is found at the 5’ end of the Ferritin mRNA At Low Fe2+ ▪ IRE-BP binds the IRE and blocks the ribosome, blocking translation At High Fe2+ ▪ IRE-BP dissociates from the mRNA, the ribosome can bind, and translation proceeds 66  Regulation of Stability of Transferrin Receptor mRNA Transferrin Receptor ▪ Cellular Fe2+ import protein ▪ Expressed when cellular Fe2+ levels are low ▪ An IRE is found at the 3’ end of the Transferrin Receptor mRNA At Low Fe2+ ▪ IRE-BP binds the IRE and masks a 3’ degradation signal in the mRNA At High Fe2+ ▪ IRE-BP dissociates from the mRNA, the degradation signal is revealed, and the mRNA is degraded 67  microRNAs microRNA (miRNA) Short RNA molecules ▪ ~22-23 nucleotides Pre-miRNA ▪ Processed by Dicer to produce mature miRNA Functions with RISC complex to inhibit expression of homologous mRNA ▪ High homology triggers mRNA degradation ▪ Less homology inhibition of translation 68  mRNA Editing mRNA Editing ▪ Enzymatic modification of mRNA sequence Eg. Substitution, insertion, deletion ▪ Example: Editing of the APOB gene in enterocytes of the small intestine Deamination of a specific cytosine in the ApoB mRNA Generates a premature stop codon No nonsense mediated decay! Instead, a truncated protein is generated The APOB gene encodes apolipoprotein B (ApoB) a protein component of Chylomicrons, VLDL and LDL lipoproteins VLDL and LDL, produced in the liver, carry ApoB100 (the protein is of 100% size) Chylomicrons, produced in the intestine, carry ApoB48 (the protein is 48% the size of the liver protein) 69 Introduction Regulation of Prokaryotic Gene Expression Regulation of Eukaryotic Gene Expression Epigenetic Regulation of Transcription Regulatory Elements Transcription Factors Steroid Hormones and the Steroid Receptors Transcription Factors and Disease Post-Transcriptional Regulation of Gene Expression Summary SUMMARY 70  Summary 1. Gene expression is regulated at all steps of the expression pathway ▪ Most important: transcriptional initiation ▪ Fastest: post-translational modification of proteins 2. Regulatory Regions of Genes ▪ Promoters (both Prokaryotes and Eukaryotes) ▪ Enhancers, Silencers (only Eukaryotes) ▪ Contain cis-acting elements 3. Cis-acting elements ▪ Regulatory elements / consensus sequences ▪ Binding sites for transcription factors 4. Trans-acting Factors ▪ Transcription factors ▪ Activators – positive regulation ▪ Repressors – negative regulation ▪ Activity may be controlled by ligands eg. DNA-binding 5. Measuring Gene Expression ▪ Quantifying mRNA levels ▪ Northern, RT-PCR, Microarray, Next-gen sequencing 71  Summary 6. Prokaryotic Regulation ▪ Promoters are the only regulatory region ▪ Relatively few regulatory elements / consensus sequences ▪ Sigma factors – specificity and transcriptional initiation ▪ Co-repressors & Inducers: ligands that act via a repressor ▪ Co-Activators & Repressors: ligands that act via an Activator 7. Operons ▪ Polycistronic clusters of genes regulated by a single promoter. ▪ Coordinated regulation 8. Lac Operon ▪ Active when Lactose is available and Glucose is not ▪ Allolactose is the inducer Causes Repressor to dissociate from operator ▪ cAMP is the Co-Activator Causes CAP protein to bind CAP site 9. Eukaryotic Regulation: Chromatin Structure ▪ DNA Methylation is silencing, permanent ▪ Histone Acetylation is activating, dynamic ▪ Histone Methylation is variable, dynamic ▪ ATP-Dependent Chromatin Remodeling (SWI/SNF) removes/modifies nucleosomes 72  Summary 10. Regulatory Regions ▪ Promoters Immediately 5' of every gene Initiate transcription via TATA Box, Inr Sequence Bind Basal/General Transcription Factors Control specificity via other regulatory elements Bind Specific Transcription Factors ▪ Enhancers, Silencer Located upstream, downstream, in introns of some genes Control specificity via other regulatory elements Bind Specific Transcription Factors Enhancers activate, Silencers repress 11. Regulatory Elements ▪ Response elements mediate a response to a stimuli 12. Reporter Assays ▪ Be able to identify positive and negative regulatory elements in a reporter assay 13. Transcription Factors ▪ General: Assemble around a TATA or Inr Sequence on ALL genes, directly recruit RNA polymerase ▪ Specific: Bind at other regulatory elements only on specific genes, indirectly recruit RNA polymerase ▪ May act synergistically ▪ Contact DNA through DNA-binding domains. Sequence-specific bonds with bases. 73  Summary 14. Mediator Proteins ▪ "Bridges", may have enzymatic activity ▪ Coactivators or Corepressors 15. Nuclear Receptors ▪ Superfamily of ligand-activated transcription factors ▪ Conserved domain structure 16. Steroid Receptors ▪ Activated by steroid hormones ▪ Bind steroid in cytoplasm, translocate to nucleus, activate transcription ▪ Agonists – ligands that activate ▪ Antagonists – ligands that repress 17. Post-translational regulation of expression by Iron ▪ Globin translation is regulated by phosphorylation of eIF2 ▪ Ferritin translation is regulated by IRE-BP ▪ Transferrin receptor mRNA stability is regulated by IRE-BP 18. Other mechanisms ▪ microRNAs inhibit translation or trigger mRNA degradation ▪ RNA editing modifies the sequence of the mRNA eg. APOB 74