Gene Expression Regulation in Eukaryotes (Part 2) Lecture Notes PDF

GENE EXPRESSION REGULATION OF GENE EXPRESSION IN EUKARYOTES (PART2) Dr. HAYTHAM MOHAMED Lecture ACADEMIC YEAR 2024/2025 Previously ▪ CRISPR Cas ▪ RNA Mediated Repression Through Ribozymes ▪ Alternative sigma factors ▪...

GENE EXPRESSION REGULATION OF GENE EXPRESSION IN EUKARYOTES (PART2) Dr. HAYTHAM MOHAMED Lecture ACADEMIC YEAR 2024/2025 Previously ▪ CRISPR Cas ▪ RNA Mediated Repression Through Ribozymes ▪ Alternative sigma factors ▪ Control of Transcription in Viruses ▪ Regulation of gene expression eukaryotes Objectives ▪ Gene Regulation at Transcription level ▪ Transcription Factors ▪ Promoters ▪ Examples for gene regulation ▪ Within core promoters are numerous DNA elements (short nucleotide sequences) that are bound by transcription factors. ▪ While it is not yet clear how dispersed promoters specify multiple transcriptional start sites, much more is known about the structure of focused promoters ▪ All the elements in core or proximal promoters are considered as Cis-acting DNA element. ▪ Different transcription factors bind to each Cis- acting DNA element, so each promoter responds to a unique combination of transcription factors. ▪ Glucocorticoid hormones (like cortisol) stimulate many genes that encode proteins involved in several different cellular processes, including the synthesis of glucose, the breakdown of proteins, and the mobilization of fats. Although the genes are not physically adjacent to each other, but all of them have GRE (Glucocorticoid response element) DNA sequce. ▪ When a transcription factor binds to the GC box in the cell, which promoter will generate the maximum amount of RNA? ▪ When a transcription factor binds to the CAAT box in the cell, what amount of RNA will be generated from the three promoters? Cis-acting DNA element (Control DNA elements) could be: ▪ Response Elements: Response elements are cis-acting sequences that allow genes to be regulated in response to specific signals or factors. (Gene expression in certain times during development or in response to certain external or internal signals). ▪ Tissue-Specific Elements: These elements are found in the promoters and enhancers of genes that are expressed in specific tissues or cell types. They bind tissue- specific transcription factors to regulate gene expression in a cell-type-specific manner. ▪ In plants, the dehydration response involves specific elements and transcription factors that activate gene expression to help the plant cope with water stress. ▪ DRE, Dehydration response element (DNA sequence present in the promoter of many genes in plants) 3- Distal promoters (Enhancers) Enhancer A D N A sequence that enhances transcription and the expression of genes. Enhancers can act over a distance of thousands or millions of base pairs and can be located upstream, downstream, or internal to the gene. enhancers often confer time- and tissue-specific gene expression. 1. Whereas c ore or pr ox imal promoters must be immediately upstream of the genes they regulate, the position of an enhancer is not critical; it will function the same whether it is upstream, downstream, or within a gene. 2. Enhancer can be inverted, relative to the gene it regulates. A single gene can have one enhancer or several. As described in more detail later, a single enhancer may have multiple binding sites for different transcription factors ▪ Bending of the DNA by a protein enables enhancers to influence a promoter hundreds or even thousands of nucleotides away. ▪ Specific transcription factors (activators) bind to the enhancer DNA sequences and then to a group of mediator proteins. These in turn bind to general transcription factors and then RNA polymerase II, thus assembling the transcription initiation complex. ▪ These protein-protein interactions lead to correct positioning of the complex on the promoter and the initiation of RNA synthesis. Only one enhancer is shown here, but a gene may have several enhancers that act at different times or in different cell types. Enhancers often regulate genes in a cell type specific manner ▪ Both liver cells and lens cells have the genes for making the proteins albumin and crystallin, but only liver cells make albumin (a blood protein) and only lens cells make crystallin (the main protein of the lens of the eye). ▪ Specific transcription factors made in a cell determine which genes are expressed ▪ Transcription factors required for high-level expression of the albumin gene are present in liver cells only (left), whereas the factors needed for expression of the crystallin gene are present in lens cells only ▪ Another example of an enhancer located downstream of the gene it regulates is the β globin g e n e enhancer. ▪ An example of an enhancer located within the g e n e it regulates is the immunoglobulin heavy-chain g e n e enhancer, which is located in an intron within the gene sequence. ▪ In chickens, an enhancer located between the β-globin g e n e and the ϵ-globin g e n e works in one direction to control transcription of the ϵ-globin during embryonic development and in the opposite direction to regulate expression of the β-globin gene during adult life. Mechanisms of Transcriptional Activation in Eukaryotes Basal factors (General transcription factors) Basal factors assist the binding of RNA polymerase II to the promoter. The key component of the basal factor complex that forms on most promoters is the TATA box– binding protein, or TBP. As its name implies, this transcription factor interacts directly with the TATA box at the promoter. TBP recruits other proteins called TBP-associated factors, or TAFs, to the promoter. Once the basal factor complex has formed, RNA polymerase II can initiate a low level of transcription (basal transcription). ▪ In the recruitment model, DNA loops serve to recruit activators and GTFs to the promoter region. (Enhanceosomes). ▪ The Mediator complex acts as a bridge or mediator between transcription factors, RNA polymerase, and the promoter region of genes. Its primary function is to facilitate the initiation of transcription by promoting the assembly of the pre-initiation complex (PIC) In the chromatin alterations model, transcription factors physically interact with chromatin modifiers such as histone acetyl transferases (HATs) to promote an “open” chromatin conformation In the nuclear relocation model, D N A looping may relocate a target gene to a nuclear region that is favorable or inhibitory to transcription regions of the nucleus that contain high or low concentrations of RNAP II and transcription regulatory factors.  Mechanisms of Transcriptional Repression in Eukaryotes Most eukaryotic repressors do not directly block RNA polymerase like in bacteria Three main mechanisms: ▪ Compete with activators for binding sites Repressor binding prevents activator from binding ▪ Bind near activators and interfere to blocks activator from recruiting transcription machinery ▪ Directly interfere with assembly of basal transcription complex (polymerase/factors)  Silencers ▪ Silencers is DNA sequence that reduces or blocks the transcription and the expression of genes. Silencers can act over a distance of thousands of base pairs and can be located upstream, downstream, or internal to the gene they affect. Super-enhancers ▪ Have High occupancy of transcription factors which Stimulate higher transcription levels than Regular enhancers Enhancer RNAs and Super-Enhancers ▪ Many enhancers are transcribed into enhancer RNA (eRNAs) ▪ eRNA is produced bidirectionally which means Transcription copies DNA in both directions ▪ eRNAs do not encode proteins May play a role in enhancer function What stops an enhancer from coregulating genes on the same chromosome that are transcribed at different times or in different cell types? Insulator : a DNA sequence that serves as a boundary element. Insulators are located between an enhancer and the promoter of a non-target gene to prevent the enhancer from influencing the transcription of the non target gene. Example : Insulators positioned upstream of the β globin gene and downstream of the ϵ globin gene ensure that this particular β enhancer does not influence the expression of a gene located outside its region. Insulator must lie between an enhancer (Enhancer I) and a promoter (Promoter of Gene B), to block the action of the enhancer, but if an insulator lies outside the region between the two, it has no effect  Mechanisms of Action ▪ Insulators bind proteins that allow chromatin to form topologically associated domains (TADs) ▪ CTCF Insulators which is found bound to DNA at the base of TADs and, with the help of cohesion protein, is thought to be involved in the formation of chromatin loops ▪ which create “neighborhoods” of regulatory elements and genes that are able to physically interact but are insulated from regulatory elements in other neighborhoods. Correct enhancer/promoter interactions require specific TAD boundaries ▪ A gene called EPHA4 is normally transcribed during limb development; EPHA4 protein is required for innervation of the limb. ▪ An adjacent gene called IHH, which encodes a regulator of developmental patterning, is not normally transcribed in the limb. ▪ A chromosomal deletion that removes the normal TAD boundary between EPHA4 and IHH results in activation of gene expression in both genes. ▪ Ectopic transcription of IHH in the developing limb causes polydactyly. Regulation of Transcriptional Stalling and Elongation ▪ A protein called negative elongation factor (NELF), which binds to RNA polymerase and causes it to stall after initiation. (Abortive elongation) ▪ Another protein, called positive transcription elongation factor (PTEFb), relieves stalling and promotes elongation by phosphorylating NELF and RNA polymerase, perhaps by causing NELF to dissociate from the polymerase. (Productive elongation) ▪ For example, stalling is observed at genes that encode heat-shock proteins in Drosophila— proteins that help prevent damage by stressors such as extreme heat. Common ways to modulate the function of regulatory transcription factors. (a) The binding of an effector molecule (ligand) such as a hormone may influence the ability of a transcription factor to bind to the DNA. (b) Protein-protein interactions among transcription factor proteins may influence their functions. (c) Covalent modifications such as phosphorylation may alter transcription factor function. ▪ Transcription factors have both ligand binding domain and dimerization domain. Coordinately Controlled Genes in Eukaryotes ▪ The binding of an effector molecule (growth factor) to transcription factor activate gene expression ▪ For example, the cAMP response element (CRE) is involved in gene regulation in response to cyclic AMP levels by CREB Transcription factor CREB (cAMP Response Element Binding protein) This system is important in many biological processes, including: Circadian rhythms Cellular metabolism ▪ The binding of an effector molecule (hormone) to transcription factor activate gene expression Steroid Hormones Exert Their Effects by Binding to a Regulatory Transcription Factor ▪ Glucocorticoids are steroid hormones produced in the adrenal cortex ▪ They diffuse into the cell and bind to glucocorticoid receptors ▪ Glucocorticoid receptors initially complexed with HSP90 proteins ▪ Hormone binding causes release of HSP90 and exposure of the nuclear localization signal (NLS)- allostric interactions ▪ NLS- function is to direct protein to the nucleus ▪ Receptors dimerize and enter the nucleus through the nuclear pore ▪ In the nucleus, the receptor-hormone complex binds GRE response element and regulates gene transcription ▪ This influences activation of many genes expression to produce many proteins involved in a wide range of physiological responses, including metabolism, immune response, and stress adaptation.  Examples for gene regulation The Human Metallothionein 2 A Gene: Multiple Cis- Acting Elements and Transcription Factors  ▪ TATA box and Inr, Bound by general transcription factors and RNAP II to initiate ▪ The product of the M T 2 A g e n e is a transcription (low rates of transcription protein that binds to heavy metals such produced). Repressor protein PZ120 can also as zinc an d cadmium, thereby bound to stop transcription protecting cells from the toxic effects of h i g h levels of these metals. The protein ▪ The presence of heavy metals stimulates the is also implicated in protecting cells from binding of transcription factor MTF-1 to the MRE, the effects of oxidative stress. which elevates the rate of transcription of the metallothionein gene. Because there are multiple ▪ The MT2A g en e is ex p res s ed at a copies of the MRE, high rates of transcription are low or basal level in all cells but is induced by metals. transcribed at h i g h levels when cells are exposed to heavy metals or stress hormones such as glucocorticoids. ▪ A second response element, called GRE, upstream of the metallothionein gene stimulates transcription in response to Glucocorticoids hormones. Gene Regulation in a Model Organism: Transcription of the G A L Genes of Yeast The G A L g e n e system in yeast served as the initial model system for studying gene regulation in eukaryotes. This system comprises four structural genes (GAL1, GAL10, GAL2, and GAL7) and three regulatory ge nes (GAL4, GAL80, and GAL3). Transcription of the G A L structural genes is inducible. In the absence of galactose, the G A L structural g e ne s are not transcribed. But in the presence of galactose, transcription begins immediately and the mRNA concentrations for G A L structural proteins increase a thousand-fold.  The Gal4 protein (Gal4p) is required for transcription of the G A L structural genes. Gal4p GAL4 is a transcription factor consists of a homodimer that includes a DNA- that binds upstream activating binding domain (DBD) that recognizes and binds sequences (UAS) specific D N A sequences and an activation domain (AD) that activates transcription. GAL4 binds UAS, it recruits transcription machinery and In the absence of galactose, G a l 80 p binds to activates genes involved in Ga l 4p and hides or masks the Gal 4p AD. This galactose metabolism These association inhibits Gal4’s ability to activate genes encode enzymes that transcription of the G A L structural genes. break down galactose such as (GAL 1 and GAL 10) In the presence of galactose, Gal3p binds directly to galactose and undergoes a conformational change that allows it to bind to Gal80p. This interaction relieves Gal4p inhibition leading to the activation of the G A L structural genes.  In the presence of glucose, Mig1p, a zinc finger repressor protein, binds to cis acting silencers to stop genes expression in the G A L system, such as G A L 1 and GAL4. Summary of the Tryptophan Biosynthesis Pathway The overall pathway can be summarized as follows: 1.Chorismate → Anthranilate (via trpE) 2.Anthranilate → Phosphoribosyl Anthranilate (via trpD) 3.Phosphoribosyl Anthranilate → Indole-3-Glycerol Phosphate (via trpC) 4.Indole-3-Glycerol Phosphate → Indole (via trpB) 5.Indole + Serine → Tryptophan (via trpA) For your information only Summary of Main Concepts Next Lecture POSTTRANSCRIPTIONAL REGULATION IN EUKARYOTES END OF LECTURE 6