DMD5025/CHS5042 Regulation of Gene Expression PDF

DMD5025/CHS5042 Regulation of Gene Expression Q. Quinn Li, PhD Office: DOC108 Office phone: 469-8523 [email protected] Objectives Define constitutive vs inducible gene expression patterns Describe major steps of gene expression regulation Defi...

DMD5025/CHS5042 Regulation of Gene Expression Q. Quinn Li, PhD Office: DOC108 Office phone: 469-8523 [email protected] Objectives Define constitutive vs inducible gene expression patterns Describe major steps of gene expression regulation Define epigenetic regulation of gene expression Describe chromatin modifications associated with hetero- and euchromatin Relate the importance of genome imprinting in genetics Recognize major structural classes and functions of transcription factors Relate the impact of RNA processing in gene expression Describe mechanisms to regulate mRNA stability and rate of translation including lncRNA and miRNA, and RNA binding proteins Relate processes of gene regulation to cell signaling pathways required for tooth development Flow of genetic information -- CENTRAL DOGMA MORPHOLOGY DNA RNA PROTEIN PHYSIOLOGY DEVELOPMENT Non-coding RNA DNA: deoxyribonucleic acid RNA: ribonucleic acid Messenger RNA - mRNA Transfer RNA - tRNA Ribosomal RNA - rRNA Small non-coding RNA Long non-coding RNA  Gene Expression Regulation Expression of almost all genes are regulated at different levels, but in relative terms, there are differences. Constitutively expressed genes: Genes that are generally continuously expressed (housekeeping genes), e.g. genes for ribosomal proteins Regulated expressed genes: Expression is controlled by the needs of the cell, e.g. responding to the environment. More dramatically up or down regulated than constitutive genes. Example of controlled gene expression: Comparison of synthesis of different globin chains at given stages of human development  Multi level gene expression controls in eukaryotes 1 2 Nucleus The Big Picture Cytoplasm 3 5 4 6 Protein modifications 7 Not all steps applicable to all genes/proteins  Most Eukaryotic Genes are “Silenced” Eukaryotic RNA polymerases have very low affinity for promoters without help from DNA binding proteins Usually several different proteins must bind before a gene is activated Positive regulation makes more sense in highly differentiated cell systems (most genes are “off” unless needed); allows fine control for huge genomes like ours  Transcriptional control in Eukaryotes The key is to control RNA polymerase activities: similar to prokaryote in this sense 1. Accessibility to DNA: Epigenetics Histone modifications Chromatin remodeling DNA methylation 2. Promoters, enhancers, and transcriptional factors Trans-acting factors: protein transcriptional factors Cis-elements: DNA sequences recognized by transcriptional factors and other proteins  Two Types of Chromatin Heterochromatin is not transcribed (silent) DNA is hypermethylated at CpG dinucleotides Histones are deacetylated Euchromatin is transcribed (active) DNA is hypomethylated Histones are acetylated Chromatin relaxes, resulting in hypersensitivity to DNase treatment  What Is Epigenetics? Epi: over or above; Epigenetics: inheritance of variation above and beyond differences in DNA sequences. Epi-genetics: Phenotypes and and processes that are transmitted through cell division and sometimes to future generations, but are not the results of differences in DNA sequence. Changes in chromatin structure, or DNA methylation Changes in patterns of DNA methylation Chemical modification of histone proteins RNA molecules that affect chromatin structure and gene expression Epigenetic regulation: Chromosome Changes Histones are general repressors of transcription Histone modifications: Addition or removal of phosphate groups, methyl groups, or acetyl groups etc. Histone code, patterns of histone modification that may encode information affecting how genes are expressed. Chromatin remodeling: Some proteins alter chromatin structure through protein interactions, without modifying histone proteins.  Epigenetic Modifications Histone 3, #9 amino acid Lys Activating Marks Silencing Marks Histone methylation Histone methylation H3: K4, K36, K79 H3: K27, K9 H4: K20 Histone acetylation Histone deacetylation H3 and H4 H3 and H4 Most prominent Histone phosphorylation H3S10 Histone ubiquitylation Histone ubiquitylation H2BK123 H2BK119 Unmethylated CpG Methylated CpG Epigenetic regulation: Histone modifications Acetylation generally stimulates transcription Acetyl group (CH3CO-) added Histone acetyltransferases (HATs) Histone deacetylase Closed form Open form  DNA modification DNA methylation: addition of methyl groups to nucleotide bases; mostly 5- methylcytosine Methylated DNA generally become transcriptionally inactive Heterochromatin are normally highly methylated Chromosome maintain certain patterns of methylation, particularly CpG islands (C-p-G sequence).  Parent DNA Maintenance of DNA methylation through replication Methylated DNA may be transmitted to next generations Methyltranferase emzyme recognizes and Replicated DNA makes the new strand methylated May be de-methylated or de novo methylated according to epigenetic regulations One daughter DNA Methylcytosines attract “repressor” proteins Co-repressors MeC Binding Protein Chromatin Modifiers MBP: methyl binding protein Trends in Biochem Sci 31:89-97, 2006. TF: transcription factor  Epigenetic Processes Produce a Diverse Set of Effects Behavioral epigenetics: life experiences, especially early in life, have long-lasting effects on behavior: stresses, food tastes Epigenetic changes induced by maternal behavior: transgenerational effects Epigenetic effects by environment Folate deficiency during development increases risk for neural tube defects Hyperhomocysteinuria increases risk for cardiovascular disease Cigarette smoke causes global epigenetic changes in multiple tissues hypomethylation of oncogenes hypermethylation of tumor suppressor genes  Genomic Imprinting Gene Silencing as Dosage Compensation Genome Imprinting: differential expression of maternal and paternal genes in offspring Mostly DNA methylation differences for purposes of dosage compensation/gene silencing Affects the expression of certain disease phenotypes Examples: Many embryogenesis related genes X-chromosome inactivation May be maintained by siRNA  X Chromosome inactivation PRC2: polycomb repressive complex 2 lncRNA: long non-coding RNA Genomic Imprinting Prader-Willi Syndrome vs Angelman Syndrome 1st identified as deletion in region 15q11-13 That region contains genes that are differentially silenced in male vs female gametes  if mutant chromosome 15 is inherited from father, only maternal genes from that region are expressed and vice versa  only paternal genes expressed = Angelman (mutant maternal chromosome 15)  only maternal genes expressed = Prader-Willi (mutant paternal chromosome 15) Genomic Imprinting Chromosome 15q11-13 has imprinted regions: a hypothetical example Maternal Paternal Maternal Paternal 1 1 1 1 Deleted in 2 2 2 2 Deleted in Angelman’s 3 3 3 3 Prader Wili 4 4 4 4 5 5 5 5 Prader-Willi vs Angelman Syndrome Prader-Willi Angelman mental retardation mental retardation obesity hypotonia hypogonadism absence of speech small hands & feet large mandible itchy skin tongue thrusting voracious appetite epilepsy No gender differences  Expression of only maternal genes (mutant region of paternal chromosome 15) causes Prader-Willi Expression of only paternal genes (mutant region of maternal chromosome 15) causes Angelman Syndrome Changes in DNA methylation and chromatin structure affect development E.g. In vitro cell culture studies from induced pluripotent stem cells (iPSCs) Q u e s t i o n s  Transcriptional Initiation: Two basic classes of genes 1. Constitutive: essential for cell survival relatively “unregulated” gene expression simple promoters E.g.: genes that encode enzymes of glycolysis, the TCA cycle, and respiratory chain and many structural proteins (actin, tubulin) 2. Inducible/repressible: highly regulated respond to cell signals complex promoters E.g.: genes that encode hormone responsive proteins, inflammatory proteins, etc. Transcriptional control: Typical Eukaryotic Promoters for Pol II Upstream elements, bound by activators: vary, or combination of common motifs; control specificity and/or efficiency Coordinating Transcription Regulation in Eukaryotes Few operon structures (lower organisms). Coordinately regulated genes usually are dispersed. Cis-acting sequences (or cis-elements) core promoter and promoter-proximal elements enhancers Silencers Insolator: a DNA cis-element that blocks the action of a promoter and/or enhancer. Trans-acting control numerous transcription factors Some are DNA biding proteins  Classes of Transcription Factors Basal Factors: position RNA polymerase on the core promoter: TFIID,A,B,E,F & H Activators: bind to enhancer elements; recruit chromatin modifiers and increase rate of assembly of transcriptional machinery Co-Activators or mediator: adapter molecules that connect activators (& repressors) to basal factors Repressors: bind to silencer elements; interfere with activators to slow transcription or induce heterochromatin formation Chromatin modifiers: HATs, HDACs, HMG proteins; promotes and stabilizes euchromatin formation to allow core binding proteins access to promoter Combination of a few different transcriptional factors may control different arrays of gene expression  Structural Motifs of DNA Binding Proteins homeodomain (include TFs) proteins (Hox, Msx-1, Barx-1) steroid involved in hormone protein/protein receptors (ER, interactions e.g. PR, AR, GCR) AP-1, Nrf2 Eukaryotic Transcriptional Activation CTD: C-terminal domain of RNA Pol II HMG: High-mobility group (HMG) proteins Inr: initiator element UAS: upstream activation sequence TBP: TATA binding protein Transcriptional Repression RNA processing control of gene expression Alternative splicing: Splice introns differently in different tissues or response to different signals Produce different mature mRNA >> proteins Alternative polyadenylation: Use of different poly(A) sites of a pre-mRNA molecule, produce different mature mRNA>> proteins Affect the stability of mRNA Could suppress translation Increase transcriptome and proteome diversity One gene generates many version of mature mRNA More mRNA diversity >> proteome diversity Example: Alternative splicing and alternative polyadenylation of human calcitonin gene AAA Selective outcome of exons 4 and 5 result in different proteins produced RNA processing control Alternative polyadenylation regulates oncogene expressions Poly(A) site 1 Poly(A) site 1 Use of different poly(A) sites leads to inclusion or exclusion of cis-elements (miRNAs) These cis-elements are targets of other regulations e.g. microRNA (miR-15), stability factor (ARE: AU-repeat element) Poly(A) site in introns or exons are also found. Impact? Cyclin D1 regulates cell cycle Mayr and Bartel, 2009, Cell 138, 673–684 RNA stability control mRNA Degradation Control mRNA turnover very quickly once in cytoplasm The rate of turnover is a gene expression control point Cis-elements reside in 3’-UTR, like UUUUUAU Other factors affect this process Two pathways in yeast: De-adenylation dependent: poly(A) degradation first De-adenylation independent: no relying on poly(A) degradation Endonuclease mediated decay RNA stability control mRNA Stability Stability time span: From minutes to days, differ in genes and conditions. RNA stability control RNA Interference: a post-transcriptional gene expression regulation RNAi: Gene expression regulation mechanism by small RNA molecules. AKA: Gene silencing, or post-transcriptional gene silencing Nobel prize: Andrew Fire and Craig Mello, 2006 Small interfering RNAs and microRNAs (20-30 nts) regulate gene expression through at least four distinct mechanisms: (1) cleavage of mRNA, (2) inhibition of translation (3) transcriptional silencing (4) degradation of mRNA. Micro RNA and small silencing RNA (Chpt 14) RNAi: originated as a defense mechanism General cellular RNA are single stranded Origin Viral RNA some time are double stranded Originally found in plants or RISC: RNA-induced silencing complex  Processing Cellular origin: Internal External  Action function Or RNA degradation  RNA induced transcriptional silencing  lncRNA: long non-coding RNA Long (>200 bases) RNA that is transcribed but not translated into a protein product Most are transcribed by RNA polymerase II Most have 5’ cap and 3’ polyA tails Introns are spliced but not as efficiently as in mRNA Some remain in nucleus; some transported to cytoplasm Functions: Modulate chromatin function Alter stability and translation of cytoplasmic mRNAs Interfere with signaling pathways Translational control Example immune response Enhancement of translation Translational control (conti.) Cell proliferation  Protein Control - Degradation Protein degradation rate determines its level A short-lived mRNA may make a very stable protein A long-lived mRNA may make a unstable protein Protein stability varies Lens proteins, lift-time Steroid receptors, minutes Through co-factor ubiquitin and degradesome Protein Control - activation and inactivation Protein amount may not change, but activity changes Phosphorylation of protein mostly activates a protein Dephosphorylation mostly inactivates a protein Co-factor protein binding to activate a protein Other modifications (e.g. acetylation, glycosylation) Application: Tooth Development BMP BMP BMP BMP Dental lamina = band of epithelial tissue FGF FGF FGF FGF Dental papilla = condensation of PDGF PDGF SHH SHH SHH SHH ectomesenchymal cells that lie below enamel WNT WNT organ TGF TGF WNT WNT ACTIVIN BMP BMP BMP FGF FGF Barx 1 Barx 1 WNT Gli1-3 Cbfa1 Barx 1 Msx1 Gli1-3 Cbfa1 Msx2 Msx1 Gli1-3 Pax9 Pax9 Msx1 Pax9 Transcriptional regulation of tooth development Inhibition, no expression p19ink4d Activation, expression Transcriptional factor Msx1 functions as an inhibitor of the expression of gene p19ink4d (a cell cycle regulator act on cyclin D-dependent kinase (CDK) )  Transcriptional regulation of tooth development p19ink4d Promoter p19ink4d gene Two Msx1 binding sites on the promoter of p19ink4d. It works with many other transcriptional factors to reach final goal of regulating p19ink4d expression.  Cell Signaling & Incisor/Molar Determination in mice noggin BMP-4 Molar Growth factor FGF-8: Barx-1 >>>>> molars Growth factor BMP-4: Msx-1 & Barx-1 >>>>> incisors If block BMP-4 with an inhibitor protein (noggin), influence of Barx-1 extends medially; can convert future incisors into molars.