Regulation Of Gene Expression & Modification 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 m...

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 signals (for example consumption of nutrients, stress, infection…etc).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 element–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 β apoprotein (apoβ) 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