Regulation of Gene Expression PDF

20 REGULATION OF GENE EXPRESSION & ITS MODIFICATION ILOs By the end of this lecture, students will be able to 1. Deduce different mechanisms of regulation of gene expression 2. Correlate how intracellular signaling molecules aff...

20 REGULATION OF GENE EXPRESSION & ITS MODIFICATION ILOs By the end of this lecture, students will be able to 1. Deduce different mechanisms of regulation of gene expression 2. Correlate how intracellular signaling molecules affect gene expression 3. Outline how drugs can be used to modify gene expression & nuclear signaling What is meant by regulation of gene expression? It is the control on the amount of protein that is being expressed (transcription and translation) from the genetic DNA. Types of gene expression 1- Constitutive gene expression: It is unvarying expression of a gene (i.e. expressed at all times). It is responsible for expression of House Keeping genes, which are needed to maintain viability of cell (e.g. genes expressing glycolysis enzymes). 2- Regulated gene expression: It is the expression of genes whose products (proteins) level changes in response to molecular ]Pvo~(}˘uo}vu]}v}(v]vUU]v(]}vYX They can be inducible genes or repressible genes. Inducible genes: They are the genes whose products increase in concentration under particular molecular circumstances, i.e.; positive regulation. Repressible genes: They are the genes whose products decrease in concentration in response to molecular signals. Regulation of gene expression If you remember the sequence of central dogma of life, you can deduce that regulation of gene expression can occur at different levels: I) At the level of chromatin (Chromatin remodeling) In eukaryotes, DNA is found complexed with histone and nonhistone proteins to form chromatin. Transcriptionally active, decondensed chromatin (euchromatin) differs from the more condensed, inactive form (heterochromatin) in a number of ways: 1- Active chromatin contains histone proteins that have been covalently modified at their amino terminal ends by reversible acetylation, or phosphorylation. Such modifications increase the negative charge of histones, thereby decreasing the strength of their association with negatively charged DNA. This relaxes the nucleosome, allowing transcription factors access to specific regions on the DNA and hence more protein expression. The opposite is true. Page 1 of 5 2-Methylation can occur on cytosine bases in CG-rich regions (CpG islands) in the promoter region of many genes. Transcriptionally active genes are less methylated (hypomethylated) than their inactive counterparts, suggesting that DNA hypermethylation silences gene expression. Clinical implication Some drug can affect gene expression by inducing DNA methylation or histone modification as the use of histone deacetylase inhibitors (HDACi); to kill cancer cells by inducing cell cycle II) At the level of DNA (genes) 1) Gene copy number: An increase or decrease in the number of copies of a gene can affect the amount of gene product produced. An increase in copy number (gene amplification) can lead to increased gene expression. An example of this is amplification of the gene coding for the enzyme dihydrofolate reductase (DHFR) (required for the synthesis of thymidine triphosphate (TTP), which is essential for DNA synthesis), leading to increased production of the enzyme. Another example is gene deletion, with the famous example of RBCs. During development of RBCs, immature erythroblasts contain nuclei that produce mRNA for synthesis of the globin chain of hemoglobin. As the cells maturate, the nuclei are extruded, so that the fully mature red blood cells have no genes, so they can no longer produce mRNA and proteins. Clinical implication Gene amplification is the mechanism by which many tumour cells develop resistance to anticancer drugs. E.g., The anticancer drug, methotrexate, acts by inhibiting the enzyme dihydrofolate reductase. Amplification of DHFR genes by cancer cells makes them less responsive (resistant) to methotrexate. 2. Re-arrangement of DNA: A single gene of immunoglobulins (Antibodies) can produce from 10 9>1011 different immunoglobulins, providing the diversity needed for the recognition of an enormous number of antigens. This diversity is due to the process of DNA rearrangement. The chains of Igs contain segments called constant (C ), variable (V), diversity (D), and joining (J). Each time an IG is produced, re-arrangement of different segments (Except the constant segment) occur to produce a different Ig. (Figure 1) Figure 1.Gene re-arrangement to produce different Igs Page 2 of 5 3. Mobile DNA elements (Transposons (Tn): These are mobile segments of DNA that move in a random manner from one site to another on the same or a different chromosome. Movement is mediated by transposase, an enzyme encoded by the Tn itself. Transposition has contributed to structural variation in the genome and the potential to alter gene expression and even to cause disease. III) At the level of transcription For transcription factors to work, they have to interact in harmony with DNA sequences acting as regulatory regions as follows: 1- Basal expression elements: (Figure 2) It consists of the proximal element of TATA box that direct RNA polymerase II to the correct start site(+1) and the upstream element e.g. CAAT box or GC box that specify the frequency of initiation. Figure 2.Basal expression elements 2-Regulated expression elements: (Cis-acting elements): They are specific DNA sequences that are present on the same gene, so termed Cis-elements, and are responsible for regulation of expression. They can exert their effect on transcription even when separated by thousands of base pairs from a promoter. They include Enhancers and Silencers (Refer to gene expression 1: transcription lecture Regulatory proteins Activators and inhibitors (called trans- factors as they are produced by other genes than this being transcribed (Refer to gene expression 1: transcription lecture )(Figure 3) Figure 3.Interaction between basal and regulated expression elements Page 3 of 5 3- Response elements They are sequences on DNA to which signaling molecules bind, causing change in gene expression. a. Signaling molecules binding to intracellular receptors Members of the nuclear receptor superfamily include the steroid hormone (glucocorticoids, mineralocorticoids, androgens, and estrogens), vitamin D, retinoic acid, and thyroid hormone receptors. These molecules, in addition to some metals such as iron, have intracellular receptors (either cytoplasmic or nuclear) When such molecules diffuse inside the cell, they bind to their receptors. The receptor-ligand complex translocates to DNA to bind to its specific response element, which is a pre-defined sequence on DNA. (for e.g hormone response element (HRE), or metal response element). This binding can alter rate of expression of certain genes (for example, binding of steroid to steroid response element, causes increased expression of gluconeogenesis enzymes, binding of vitamin D to its response element causes increased expression of calcium binding protein) b. Signaling molecules binding to cell surface receptors These receptors include those for insulin, epinephrine, and glucagon. This extracellular signal is then transduced to intracellular 2nd messengers to end by phosphorylation and activation of different kinases. One of these is cAMP response elementt binding [CREB] protein that can bind to a specific responsive element sequence on DNA and result in transcription of some target genes of metabolism or of growth. Clinical implications: Pharmacological modulation of certain gene transcription is a common mechanism of action of many classes of drugs either: A) Directly by targeting nuclear receptor superfamily (that are ligand activated transcriptional factors), where the drug receptor complex, itself induces activation or suppression of gene transcription. For e.g., all steroid hormones, as Glucocorticoids or related steroid drugs; dexamethasone, are used in treatment of many inflammatory and autoimmune disorders by suppressing the transcription of inflammatory mediators and cytokines. B) Indirectly by targeting cell surface receptors to affect their downstream signalling cascades to finally affect their kinases involved in activation e or inhibit of transcriptional factors. For e.g., Insulin (targeting tyrosine kinase receptors) used in treatment of diabetes mellitus by affecting the genes involved in metabolism. C) Indirectly by interacting with enzymes that activate or inhibit transcriptional factors. For e.g., Calcineurin inhibitors, cyclosporine, inhibit calcineurin to inhibit transcription factors involved in transcription of a cytokine mediator (IL-2) so can be used to suppress increased immune activity during treatment of many autoimmune disorders and in graft rejection. (Refer to cytokines lecture) Page 4 of 5 III-Post-Transcriptional Regulation: Regulation can occur during processing of the primary transcript (hn RNA) and during the transport of mRNA from nucleus to the cytoplasm. 1- Alternative splicing and polyadenylation sites In certain cases, the use of alternative splicing and polyadenylation sites causes different proteins to be produced from the same gene. For example, in parafollicular cells of the thyroid gland, the calcitonin gene produces mRNA that codes for calcitonin. In the brain, the transcript of this gene undergoes alternative splicing and polyadenylation to produce a different protein called calcitonin gene-related protein (CGRP). 2-RNA editing It is change in a single nucleotide on mRNA after it has been transcribed. The nucleotide is changed either by insertion, deletion or substitution. An example of RNA editing occurs in the production of t}}]v~}t that is synthesized in liver and intestinal cells and serves as a component of the lipoproteins (carriers of lipid in circulation). Although these apoproteins are encoded by the same gene, the version of the protein made in the liver (B-100) contains 4563 amino acid residues, while the (B-48) made in intestinal cells has only 2152 amino acid. This is due to RNA editing in intestinal cells results in a change of a single nucleotide causing the creation of a stop codon earlier than usual and hence a shorter protein. 3-Stability of mRNA Developmental or environmental stimuli like nutrient levels, cytokines, hormones and temperature shifts as well as environmental stresses like hypoxia, hypocalcemia, viral infection, and tissue injury affect stability of mRNA and hence amount of protein produced In addition, a group of RNAs called microRNA act as post-transcriptional regulators of their mRNA causing mRNA degradation and/or translational repression. IV- Regulation at the level of Translation: Most eukaryotic translational controls affect the initiation of protein synthesis. The initiation factors for translation, particularly eukaryotic initiation factor 2 (eIF2), are the focus of these regulatory mechanisms. The action of eIF2 can be inhibited by phosphorylation. V- Post-Translational Regulation: After proteins are synthesized, their lifespan is regulated by proteolytic degradation. Proteins have different half-lives, some last for hours or days, others last for months or years. Some proteins are degraded by lysosomal enzymes, other proteins are degraded by proteases in the cytoplasm. Clinical Implications: Post translationally, many drugs can affect certain protein level. For e.g., Protease inhibitors; antivirals that inhibit post-translational processing of precursor viral proteins and is used in treatment of HIV and Hepatitis C infection. Page 5 of 5  21 Autophagy, Lysosomes, Peroxisomes & cell inclusions ILOs By the end of this lecture, students will be able to 1. Explain the role of autophagy as a cellular sink 2. Describe the origin of lysosomal enzymes and their function in health and disease. 3. Predict the role especially of autolysosome and phagolysosome in physiological conditions 4. Connect the structure of proteasome to its degrative function. 5. Predict the role of peroxisomes in cell adaptation to patterns of stress. 6. Correlate the types of cytoplasmic inclusions to patterns of cell activity 7. Justify the impact of its derangement on cellular health. 1. Lysosomes Lysosomes are membrane bounded cell organelles that have an acidic pH and contain hydrolytic enzymes. It contains at least 40 different types of acid hydrolases, such as sulfatases, proteases, nucleases, lipases that are active in acidic pH. These enzymes are manufactured in the same steps of protein synthesis following the same steps in rER, packed in Golgi complex and released in vesicles from trans Golgi network. Lysosomes receive contents to be digested from late endosomes. Lysosomes aid in digesting phagocytosed microorganisms, cellular debris, and cells but also excess or senescent organelles, such as mitochondria and RER. The various enzymes digest the engulfed material into small, soluble end products that are transported by carrier proteins in the lysosomal membrane from the lysosomes into the cytosol andare either reused by the cell or exported from the cell into the extracellular space. Transport of Substances into Lysosomes Substances destined for degradation within lysosomes reach these organelles in one of three ways: through phagosomes, pinocytotic vesicles, or autophagosomes. 1- Phagosomes: Phagocytosed material, contained within phagosomes, moves toward the interior of the cell. The phagosome joins either a lysosome or a late endosome. The hydrolytic enzymes digest most of the contents of the phagosome, especially the protein and carbohydrate components. Lipids, however, are more resistant to complete digestion, and they remain enclosed within the spent lysosome, now referred to as a residual body. (Fig. 1) 1  Fg 1. Pathways of intracellular digestion by lysosomes 2- Autophagy: The term autophagy is derived from the Greek word meaning 'self- devouring'. Senescent organelles such as mitochondria or the RER, need to be degraded. The organelles in question become surrounded by elements of the endoplasmic reticulum and are enclosed in vesicles called autophagosomes. Fate of autophagosomes: These structures fuse either with late endosomes or with lysosomes and share the same subsequent fate as the phagosome. (Fig. 1) Autophagy is a self-digesting mechanism responsible for removal of long-lived proteins, damaged organelles, and malformed proteins during biosynthesis by lysosome. Significance of autophagy Regulation of diverse cellular functions including growth, differentiation, response to nutrient deficit and oxidative stress, cell death, and macromolecule and organelle turnover. Mechanism A}ZP}}u(}u]}v]Po˙}v}(^}ZP˙ -oPv_oo Atgs. Mutation leads to formation of a double-membrane vesicle, which encapsulates cytoplasm, malformed proteins, long-lived proteins, and organelles and then fuses with lysosomes for degradation. 2  Autophagy Regulation Autophagy is activated in response to diverse stress and physiological conditions. For example, food deprivation, hyperthermia, and hypoxia, which are known as major environmental modulators of ageing, are also conditions that induce autophagy. Figure 2 - Stages of autophagy Autophagy and Diseases Autophagy is important in normal development and responds to changing environmental stimuli. On starvation, autophagy is greatly increased, allowing the cell to degrade proteins and organelles and thus obtain a source of macromolecular precursors, such as amino acids, fatty acids, and nucleotides, which would not be available otherwise. Autophagy roles in cancer are a topic of intense debate. In one hand, autophagy has an anticancer role. On the other hand, when tumor cells are starved due to limited angiogenesis, autophagy stops them from dying. Autophagy is important in numerous diseases, including bacterial and viral infections, neurodegenerative disorders, several myopathies, and cardiovascular diseases. 3 Autophagy and weight loss A type of intermittent fasting is used to stimulate autophagy and to 'trick' one's metabolism into working longer hours and burning more fat. Notably, pharmacological stimulation of autophagy can reduce both weight gain and obesity-associated alterations upon hypercaloric regimens usage. Proteasomes Proteasomes are small organelles composed of protein complexes (proteases) that are responsible for proteolysis (protein breakdown) of malformed and ubiquitin-tagged proteins. Proteasomes monitor the protein content of the cell to ensure degradation of unwanted proteins, such as excess enzymes and other proteins that become unnecessary to the cell after they perform their normal functions, and malformed proteins. Protein encoded by virus should also be destroyed. The process of cytosolic proteolysis is carefully controlled by the cell, and it requires that the protein be recognized as a potential candidate for degradation. This recognition involves ubiquination, a process whereby several ubiquitin molecules (a 76-amino acid long polypeptide chain) are attached to the candidate protein using ATP. Once a protein has been marked, it is degraded by proteasomes. (Fig 2) During proteolysis, the ubiquitin molecules are released and re-enter the cytosolic pool to be re used. Fig. 3. The structure and function of the proteasome 4 Protein degradation by proteasomes in health and disease Proteins destined for degradation are labeled with ubiquitin through covalent attachment to a lysine side chain. The amino acid composition at the amino terminus determines how quickly the protein will be ubiquinated and thus the half-life of the protein. Some proteins have very long half-lives, such as the crystallins in the lens of the eye; these proteins do not turn over significantly during the human life span. Because they were synthesized largely in utero, about half the crystallins in the adult lens are older than the person. Other proteins have half-lives of 4 months (proteins such as hemoglobin that last as long as the red blood cell), or the half-life can be very short, such as for ornithine decarboxylase, which has a half-life of 11 minutes. The half-lives of proteins is influenced by the amino (N)-terminal residue, the so- called N-end rule. Destabilizing N-terminal amino acids (causing short half-life) include arginine and acetylated alanine. In contrast, serine is a stabilizing amino acid. Additionally, proteins rich in sequences containing proline, glutamate, serine, and threonine (called PEST sequences) are rapidly ubiquinated and degraded and, therefore, have short half-lives Poly-ubiquination, which increases the rate of turnover/degradation of a protein, occurs by successive addition of free ubiquitin to that which is already bound to the protein. Failure of degradation of misfolded proteins by proteasomes, can lead to accumulation of abnormal proteins and development of certain diseases such as AoZ]u[]vC(o t Jakob disease (Mad-cow disease). Peroxisomes Peroxisomes are small membrane bounded, self-replicating organelles. They contain more than 40 oxidative enzymes, especially urate oxidase, and D- amino acid oxidase that contain oxidative enzymes. Peroxisomes function in the catabolism of long-chained fatty acids (beta oxidation), forming acetyl coenzyme A (CoA) as well as hydrogen peroxide (H2O2) by combining hydrogen from the fatty acid with molecular oxygen. Similar to mitochondria, peroxisomes increase in size and undergo fission to form new peroxisomes; however, they possess no genetic material of their own. Inclusions Inclusions are non-living components of the cell that do not possess metabolic activity and are not bounded by membranes. The most common inclusions are glycogen, lipid droplets, pigments, and crystals. 5  1. Glycogen Glycogen is the most common storage form of glucose in human and is especially abundant in cells of muscle and liver. It appears in electron micrographs as clusters, or }U }( t ]o ~v oP r ]o ]v Z o] Z semble ribosomes, located in the vicinity of the SER. On demand, enzymes responsible for glycogenolysis degrade glycogen into individual molecules of glucose. 2. Lipids Lipids, triglycerides in storage form, not only are stored in specialized cells (adipocytes) but also are located as individual droplets in various cell types, especially hepatocytes. Lipids are considered as potential source of energy within the cells. 3. Pigments It could be natural pigments as haemoglobin of red blood cells, melanin in the skin and hair and a yellow-to-brown pigment, lipofuscin in the long-lived cells, such as neurons and cardiac muscle. Tattoos is the injection of ink intracellular that could be phagocytosed by macrophages leading to its permanent effect. Fig. 4 Types of inclusions A. TEM of glycogen ganules in rosettes B. Lipid droplets in fat cell Clinical hint: abnormal accumulations I. Lipids 1-Steatosis (Fatty Change) Means excessive, abnormal accumulations of triglycerides within parenchymal cells due to alcohol abuse, diabetes mellitus, obesity, toxins, protein malnutrition, and anoxia 2- Cholesterol and Cholesterol Esters as in atherosclerosis. 6  I. Proteins as inAlzheimer disease. II. Glycogenas in Diabetes mellitus and Glycogen storage diseases. III. Pigments; Exogenous Carbon (coal dust),The most common air pollutant in urban areas. Its accumulation could lead to Anthracosis occurs in heavy smokers, and coal mines workers with accumulation of carbon pigment within lungs and regional lymph nodes. Endogenous Pigments Lipofuscin Patients with severe malnutrition& Cancer cachexia. Melanin: Hyperpigmentation generalized due to excessive sun exposure or localized as in benign (nevus) and malignant cutaneous tumors. Hypopigmentation Generalized as in albinism or localized as in vitiligo (autoimmune disorder). 7