BioA Ch-18-JYCHEN-2017 Ch 18 Regulation of (Control of Gene Expression) 陳正繹

國防醫學院生物及解剖學科

Cover: Cross-section of Drosophila eye imaginal discs stained for Elav (red), which is expressed in the nuclei of all photoreceptor cells, and Bar (green), which is expressed in the nuclei of undifferentiated cells. Note, the nuclei of undifferentiated cells are basal to those of photoreceptor cells. R1 and R6 photoreceptors express both Elav and Bar (yellow). The surface of eye disc cells is labelled by the membrane marker Discs-large (blue). 18.0 Beauty in the Eye of the Beholder * Prokaryotes and eukaryotes precisely regulate gene expression in response to their changing environment * In multicellular eukaryotes, gene expression regulates development and is responsible for differences in cell types * RNA molecules play many roles in regulating gene expression in eukaryotes

How can this fish’s eyes see equally well in both air and water 2 18.1: Bacteria often respond to environmental change by regulating Genes that encode enzymes 1, 2, and 3

* Natural selection has favored bacteria that produce only the products needed by that cell * A cell can regulate the production of enzymes by feedback inhibition回饋抑制 or by gene regulation * Gene expression in bacteria is controlled by the model操縱子模式

Regulation of a metabolic pathway 3 A. : The Basic Concept * A cluster of functionally related genes can be under coordinated control by a single on-off “switch” * An operon 操縱子is the entire stretch of DNA that includes the operator操 縱子, the promoter啟動子, and the genes that they control * Operator :the regulatory “switch”, is a segment of DNA within the promoter * Repressor抑制劑is the product of a separate regulatory gene調節 基因, can switched off the operon by binding to the operator and blocking RNA polymerase * The repressor can be in an active or inactive form, depending on the presence of other molecules.

4 * A corepressor輔抑制劑is a molecule that cooperates with a repressor protein to switch an operon off. * When tryptophan is present, it binds to the trp repressor protein, which turns the operon off * The repressor is active only in the presence of its corepressor tryptophan; thus the trp operon is turned off (repressed) if tryptophan levels are high.

5 B. Repressible and Inducible Operons: Two Types of Negative Gene Regulation * A repressible operon is one that is usually on; binding of a repressor to the operator shuts off transcription * The trp operon is a repressible operon * An inducible operon is one that is usually off; a molecule called an inducer誘 導劑 inactivates the repressor and turns on transcription * The is an inducible operon and contains genes that code for enzymes used in the hydrolysis and metabolism of lactose * By itself, the lac repressor is active and switches the lac operon off

The lac operon in E. coli: regulated synthesis of inducible enzymes.

6 * A molecule called an inducer誘導劑 inactivates the repressor to turn the lac operon on * Inducible enzymes usually function in catabolic pathways分解路徑; their synthesis is induced by a chemical signal * Repressible enzymes usually function in anabolic pathways合成路徑; their synthesis is repressed by high levels of the end product * Regulation of the trp and lac operons involves negative control 負向調控of genes because operons are switched off by the active form of the repressor

7 C. Positive Gene Regulation * Some operons are also subject to positive control through a stimulatory protein, such as catabolite activator protein (CAP), an activator 活化劑of transcription * When glucose (a preferred food source of E. coli) is scarce, CAP is activated by binding with cyclic AMP * Activated CAP attaches to the promoter of the lac operon and increases the affinity of RNA polymerase, thus accelerating transcription * When glucose levels increase, CAP detaches from the lac operon, and transcription returns to a normal rate * CAP helps regulate other operons that encode enzymes used in catabolic pathways 8 18.2: Eukaryotic gene expression can be regulated at many stages

* All organisms must regulate which genes are expressed at any given time * In multicellular organisms gene expression is essential for cell specialization A. Differential Gene Expression * Almost all the cells in an organism are genetically identical * Differences between cell types result from differential gene expression, the expression of different genes by cells with the same genome * Abnormalities in gene expression can lead to diseases including cancer * Gene expression is regulated at many stages

9 B. Regulation of Chromatin Structure

* The structural organization of chromatin helps regulate gene expression in several way * Genes within highly packed heterochromatin are usually not expressed * Chemical modifications to histones組蛋白 and DNA of chromatin influence both chromatin structure and gene expression

10 1. Histone Modifications  In histone acetylation組蛋白乙醯化, acetyl groups are attached to positively charged lysines in histone tails  This loosens chromatin structure, thereby promoting the initiation of transcription  The addition of methyl groups (methylation甲基化) in histone tails can condense chromatin; the addition of phosphate groups (phosphorylation磷 酸化) next to a methylated amino acid can loosen chromatin.

A simple model of histone tails and the effect of histone acetylation. 11 * DNA methylation, the addition of methyl groups to certain bases in DNA, is associated with reduced transcription in some species. Ex. tortoiseshell cat * DNA methylation can cause long-term inactivation of genes in cellular differentiation * In 基因印痕, methylation regulates expression of either the maternal or paternal of certain genes at the start of development

2. Epigenetic Inheritance

* Although the chromatin modifications just discussed do not alter DNA sequence, they may be passed to future generations of cells * The inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called epigenetic inheritance外(表)基因遺傳

12 C. Regulation of Transcription Initiation * Chromatin-modifying enzymes provide initial control of gene expression by making a region of DNA either more or less able to bind the transcription machinery 1. Organization of a Typical Eukaryotic Gene * Associated with most eukaryotic genes are multiple control elements調控元 件, segments of noncoding DNA that serve as binding sites for transcription factors thathelp regulate transcription by binding certain * Control elements and the proteins they bind are critical to the precise regulation of gene expression in different cell types

13 A eukaryotic gene and its transcript 2. The Roles of Transcription Factors *To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors轉錄因子 a. General Transcription Factors at the Promoter * General transcription factors一般 轉錄因子 are essential for the transcription of all protein-coding genes * A few bind to the TATA box within the promoter * Many bind to proteins, including other transcription factors and RNA polymerase II * Only when the complete initiation complex has assembled can the RNA polymerase begin to move along the template strand of the DNA

14 b. Enhancers and Specific Transcription Factors

* Proximal control elements 近端調控元件are located close to the promoter * Distal control elements遠端調控元件, groups of which are called enhancers 增強子, may be far away from a gene or even located in an intron * An activator活化劑 is a protein that binds to an and stimulates transcription of a gene * Activators have two domains, one that binds DNA and a second that activates transcription * Bound activators facilitate a sequence of protein-protein interactions that result in transcription of a given gene

* The mediator proteins interact with general transcription factors at the promoter * Some transcription factors function as repressors抑制劑, inhibiting expression of a particular gene * Some activators and repressors act indirectly by influencing chromatin structure to promote or silence transcription The structure of MyoD, an activator 15 A model for the action of enhancers and transcription activators

16 Cell-type specific transcription

c. Combinatorial Control of Gene Activation * A particular combination of control elements can activate transcription only when the appropriate activator proteins are present

17 Eukaryotic genes are regulated by combinations of proteins

18 3. Coordinately Controlled Genes in Eukaryotes * Co-expressed eukaryotic genes are not organized in operons (with a few minor exceptions) * These genes can be scattered分散over different chromosomes, but each has the same combination of control elements * Copies of the activators recognize specific control elements and promote simultaneous transcription of the genes

19 4. Nuclear Architecture and Gene Expression

• Chromosome conformation capture techniques allow identification of regions of chromosomes that interact with each other • Loops from different chromosomes may congregate at particular sites, some of which are rich in transcription factors and RNA polymerases • These may be areas (transcription factories轉錄工 廠) specialized for a common function

Chromosomal interactions in the interphase nucleus. D. Mechanisms of Post-Transcriptional Regulation轉錄後調控 *Transcription alone does not account for gene expression *Regulatory mechanisms can operate at various stages after transcription *Such mechanisms allow a cell to fine-tune微調gene expression rapidly in response to environmental changes 1. RNA Processing * In alternative RNA splicing交 互RNA剪接, different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons外顯子 and which as introns內接子 * More than 90% of the human protein-coding genes undergo alternative splicing

21 2. Initiation of Translation and mRNA Degradation * The initiation of translation of selected mRNAs can be blocked by regulatory proteins that bind to sequences or structures of the mRNA * Alternatively, translation of all mRNAs in a cell may be regulated simultaneously * For example, translation initiation factors are simultaneously activated in an egg following fertilization

Model of CPEB-dependent masking and activation of mRNA translation in . Typically, maternal mRNAs initially carry a short poly(A) tail and their CPE motif is bound by CPEB. CPEB forms a complex with maskin, which in turn interacts with eIF4E. This configuration is thought to represent a translationally inactive or “masked” state of the mRNA. The signal for maturation leads to a phosphorylation of CPEB and a stimulation of binding between CPEB and CPSF. Through further association of CPSF with poly(A)-polymerase, this leads to elongation of the poly(A) tail (indicated by the line with star). The binding between maskin22 and eIF4E is reduced, possibly as a consequence of polyadenylation. *The life span of mRNA molecules in the cytoplasm is a key to determining protein synthesis * Eukaryotic mRNA is more long lived than prokaryotic mRNA * The mRNA life span is determined in part by sequences in the leader and trailer regions

23 3. Protein Processing and Degradation * After translation, various types of protein processing, including cleavage and chemical modifications, are subject to control * The length of time each protein function is regulated by selective degradation * Cells mark proteins for degradation by attaching ubiquitin泛素 to them * This mark is recognized by proteasomes蛋白質解體, which recognize and degrade the proteins * Proteasomes are giant protein complexes that bind protein molecules and degrade them

24 ★Eukaryotic gene expression can be regulated at any stage

25 18.3: Noncoding RNAs play multiple roles in controlling gene expression *Only a small fraction of DNA codes for proteins, rRNA, and tRNA * A significant amount of the genome may be transcribed into noncoding RNAs (ncRNAs)非編碼RNA * Noncoding RNAs非編碼RNA regulate gene expression at two points: mRNA translation and chromatin configuration染色體構形 A. Effects on mRNAs by MicroRNAs and Small Interfering RNAs * MicroRNAs (miRNAs) 微型RNA are small single-stranded RNA molecules that can bind to mRNA * These can degrade mRNA or block its translation * It is estimated that expression of at least half of all human genes may be regulated by miRNAs * Small interfering RNAs (siRNAs)小型干擾RNA are similar to miRNAs in size and function * The blocking of gene expression by siRNAs is called RNA interference (RNAi) RNA干擾 * RNAi is used in the laboratory as a means of disabling genes to investigate their function 26 Regulation of gene expression by miRNA

27 B. Chromatin Remodeling and Silencing of Transcription by Small RNAs * Some ncRNAs act to bring about remodeling of chromatin structure. In some yeasts siRNAs play a role in heterochromatin formation and can block large regions of the chromosome * In some yeasts siRNAs re-form heterochromatin at centromeres

28 Condensation of chromatin at the centromeres B. Chromatin Remodeling and Silencing of Transcription by Small RNAs

* Some ncRNAs act to bring about remodeling of chromatin structure. In some yeasts siRNAs play a role in heterochromatin formation and can block large regions of the chromosome * In some yeasts siRNAs re-form heterochromatin at centromeres * Small ncRNAs called piwi-interaction RNAs (piRNAs) induce heterochromatin, blocking the expression of parasitic DNA elements in the genome, known as transposons轉座子 * piRNAs help to reestablish appropriate methylation patterns during gamete formation in many animal species

*Long noncoding RNAs (lncRNAs) range from 200 to hundreds of thousands of nucleotides in length *One type of lncRNA is responsible for X chromosome inactivation *RNA-based regulation of chromatin structure plays an important role in gene regulation

30 C. The Evolutionary Significance of Small ncRNAs • Small ncRNAs can regulate gene expression at multiple steps • An increase in the number of miRNAs in a species may have allowed morphological complexity to increase over evolutionary time • siRNAs may have evolved first, followed by miRNAs and later piRNAs

Noncoding RNAs play multiple roles in controlling gene expression

31 18.4: A program of differential gene expression leads to the different cell types in a multicellular organism * During embryonic development, a fertilized egg gives rise to many different cell types * Cell types are organized successively into tissues, organs, organ systems, and the whole organism * Gene expression orchestrates the developmental programs of animals A. A Genetic Program for Embryonic Development * The transformation from zygote to adult results from , cell differentiation, and * Cell differentiation細胞分化is the process by which cells become specialized in structure and function * The physical processes that give an organism its shape constitute morphogenesis形態發生 * Differential gene expression results from genes being regulated differently in each cell type * Materials in the egg can set up gene regulation that is carried out as cells divide 32 B. Cytoplasmic Determinants and Inductive Signals

* An egg’s cytoplasm contains RNA, proteins, and other substances that are distributed unevenly in the unfertilized egg * Cytoplasmic determinants 胞 質決定因素are maternal substances in the egg that influence early development * As the zygote divides by mitosis, cells contain different cytoplasmic determinants, which lead to different gene expression

33 * The other important source of developmental information is the environment around the cell, especially signals from nearby embryonic cells * In the process called induction誘導, signal molecules from embryonic cells cause transcriptional changes in nearby target cells * Thus, interactions between cells induce differentiation of specialized cell types

34 C. Sequential Regulation of Gene Expression During Cellular Differentiation * Determination命定commits a cell to its final fate * Determination precedes differentiation * Cell differentiation is marked by the production of tissue-specific proteins

* Myoblasts肌母細胞 are cells determined to produce muscle cells and begin producing muscle- specific proteins * MyoD is a “master regulatory gene” 主控基 因encodes a that commits the cell to becoming skeletal muscle * The MyoD protein can turn some kinds of differentiated cells—fat cells and liver cells—into muscle cells 35 D. : Setting Up the * Pattern formation樣式形成is the development of a spatial organization空間組成 of tissues and organs * In animals, pattern formation begins with the establishment of the major axes * Positional information位置訊 息, the molecular cues that control pattern formation, tells a cell its location relative to the body axes and to neighboring cells * Pattern formation has been extensively studied in the fruit fly * Combining anatomical, genetic, and biochemical approaches, researchers have discovered developmental principles common to many other species, including 36 humans. 1. The Life Cycle of Drosophila * In Drosophila, cytoplasmic determinants in the unfertilized egg determine the axes before fertilization * After fertilization, the embryo develops into a segmented larva with three larval stages * The larva then forms a pupa, which undergoes metamorphosis into the adult fly

37 2. Genetic Analysis of Early Development: Scientific Inquiry * Edward B. Lewis, Christiane Nüsslein-Volhard, and Eric Wieschaus won a Nobel 1995 Prize for decoding pattern formation in Drosophila * Lewis demonstrated that homeotic genes同源性基因 which control pattern formation in late embryo, larva, and adult stages * Nüsslein-Volhard and Wieschaus studied segment formation體節生成 * They created mutants, conducted breeding experiments, and looked for corresponding genes * Breeding experiments were complicated by embryonic lethals胚胎致死, embryos with lethal mutations * They found 120 genes essential for normal

38 3. Axis Establishment體軸建立 * Maternal effect genes母效基因 encode for cytoplasmic determinants that initially establish the axes of the body of Drosophila * These maternal effect genes are also called egg-polarity genes卵向基因 because they control orientation of the egg and consequently the fly

39 http://flymove.uni-muenster.de/  Bicoid: A Determining Head Structure http://www.ncbi.nlm.nih.gov/books/NBK10039/ * One maternal effect gene, the bicoid gene雙尾基因, affects the front half of the body * An embryo whose mother has a mutant bicoid gene lacks the front half of its body and has duplicate posterior structures at both ends * This suggests that the product of the mother’s bicoid gene is concentrated at the future anterior end

40 卵極性基因(egg polarity gene)  gap genes配對法則基因(pair rule gene) 體節 * gradient hypothesis 性基因(segmentation gene) : 同源性基因 形態成形素梯度假說, in which ( ) gradients of substances called morphogens形態成形素establish an embryo’s axes and other features * The bicoid 雙尾research is important for three reasons: i. It identified a specific protein required for some early steps in pattern formation ii. It increased understanding of the mother’s role in embryo development iii. It demonstrated a key developmental principle that a gradient of molecules can determine polarity and position in the embryo

41 4. Evolutionary (“Evo-Devo”)  The fly with legs emerging from its head in is the result of a single mutation in one gene  Some scientists considered whether these types of mutations could contribute to by generating novel body shapes  This line of inquiry gave rise to the field of evolutionary developmental biology, “evo-devo”演化發育學

42 18.5: Cancer results from genetic changes that affect cell cycle control *The gene regulation systems that go wrong during cancer are the very same systems involved in embryonic development A. Types of Genes Associated with Cancer * Cancer can be caused by mutations to genes that regulate cell growth and division * Mutations in these genes can be caused by spontaneous mutation or environmental influences such as chemicals, radiation, and some viruses 1. Oncogenes致癌基因 and Proto-Oncogenes原致癌基因 * Oncogenes are cancer-causing genes in some types of viruses * Proto-oncogenes are the corresponding normal cellular genes that are responsible for normal cell growth and division * Conversion of a proto-oncogene to an oncogene can lead to abnormal stimulation of the cell cycle

43 * Proto-oncogenes can be converted to oncogenes by i. Movement of DNA within the genome: if it ends up near an active promoter, transcription may increase ii. Amplification of a proto-oncogene: increases the number of copies of the gene iii. Point mutations in the proto-oncogene or its control elements: causes an increase in gene expression

44 2. Tumor-Suppressor Genes腫瘤抑制基因 * Tumor-suppressor genes help prevent uncontrolled cell growth * Mutations that decrease protein products of tumor-suppressor genes may contribute to cancer onset * Tumor-suppressor proteins i. Repair damaged DNA ii. Control cell adhesion iii. Act in cell-signaling pathways that inhibit the cell cycle pathway

45 B. Interference with Normal Cell-Signaling Pathways * Mutations in the ras proto-oncogene and p53 tumor-suppressor gene are common in human cancers * Mutations in the ras gene can lead to production of a hyperactive Ras protein and increased cell division

46 * Suppression of the cell cycle can be important in the case of damage to a cell’s DNA; p53 prevents a cell from passing on mutations due to DNA damage * Mutations in the p53 gene prevent suppression of the cell cycle

47 C. The Multistep Model of Cancer Development

* Multiple mutations are generally needed for full-fledged cancer; thus the incidence increases with age * At the DNA level, a cancerous cell is usually characterized by at least one active oncogene and the mutation of several tumor-suppressor genes * Routine screening for some cancers, such as colorectal cancer大腸直腸癌, is recommended * In such cases, any suspicious polyps瘜肉 may be removed before cancer progresses

48 49 D. Inherited Predisposition傾向 and Other Factors Contributing to Cancer  Individuals can inherit oncogenes or mutant alleles of tumor-suppressor genes  Inherited mutations in the tumor-suppressor gene adenomatous polyposis coli(APC)大腸腺瘤性瘜肉基因are common in individuals with colorectal cancer大腸直腸癌  Mutations in the BRCA1 (60% before the age of 50) or BRCA2 gene are found in at least half of inherited breast cancers

E. The Role of Viruses in Cancer  A number of tumor viruses can also cause cancer in humans and animals  Viruses can interfere with normal gene regulation in several ways if they integrate into the DNA of a cell  Viruses are powerful biological agents

50 You should now be able to: * Explain the concept of an operon and the function of the operator, repressor, and corepressor * Explain the adaptive advantage of grouping bacterial genes into an operon * Explain how repressible and inducible operons differ and how those differences reflect differences in the pathways they control * Explain how DNA methylation and histone acetylation affect chromatin structure and the regulation of transcription * Define control elements and explain how they influence transcription * Explain the role of promoters, enhancers, activators, and repressors in transcription control * Explain how eukaryotic genes can be coordinately expressed * Describe the roles played by small RNAs on gene expression * Explain why determination precedes differentiation * Describe two sources of information that instruct a cell to express genes at the appropriate time * Explain how maternal effect genes affect polarity and development in Drosophila embryos * Explain how mutations in tumor-suppressor genes can contribute to cancer * Describe the effects of mutations to the p53 and ras genes 51