LEC1 - 10/14/11 Evaluation for Lisheng Wang Bch 4122 
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Unformatted text preview: 10/14/11 Evaluation for Lisheng Wang Bch 4122 
 Macromolecules:
 The regulation of transcription by chromatin structure during skeletal muscle development" Ilona Skerjanc (Sker/yants)" October 17 – November 21, 2011" 1 10/14/11 BCH 4122 - MACROMOLECULES - ACADEMIC YEAR 2011 - 2012 FALL SEMESTER 2011 COORDINATOR: DR. ILONA SKERJANC LECTURE 1: MONDAY FROM 13:00 - 14:30 P.M. - MACDONALD HALL - ROOM 146 LECTURE 2: WEDNESDAY FROM 11:30 A.M - 13:00 P.M. - MACDONALD HALL - ROOM 146 DATE PROFESSOR TOPICS Wednesday, September 7 Lisheng Wang Introduction of stem cells (Part I) Monday, September 12 Lisheng Wang Introduction of stem cells (Part II) Wednesday, September 14 Lisheng Wang Surrogate markers to characterize adult stem cells Monday, September 19 Lisheng Wang Adult stem cells - HSC Wednesday, September 21 Lisheng Wang Adult stem cells & applications Monday, September 26 Lisheng Wang Human embyryonic stem cells Wednesday, September 28 Lisheng Wang Epigenetic reprogramming (Part I) Monday, October 03 Lisheng Wang Epigenetic reprogramming (Part II) Wednesday, October 05 Lisheng Wang Differentiation of pluripotent stem cells & class summary Monday, October 10 THANKSGIVING DAY NO COURSE Wednesday, October 12 Midterm Bch 4122 Calendar" Monday, October 17 Wednesday, October 19 Monday, October 24 Wednesday, October 26 Monday, October 31 Wednesday, November 02 Monday, November 07 Wednesday, November 09 Monday, November 14 Wednesday, November 15 Vendredi, 18 novembre Monday, November 21 Wednesday, November 23 Monday, November 28 Wednesday, November 30 Monday, December 5 ** Wednesday, December 7 Ilona Skerjanc Ilona Skerjanc STUDY WEEK STUDY WEEK Ilona Skerjanc Ilona Skerjanc Ilona Skerjanc Ilona Skerjanc Ilona Skerjanc Ilona Skerjanc LAST DAY TO WITHDRAW FROM THE COURSE Ilona Skerjanc Robin Parks Robin Parks Valerie Wallace Valerie Wallace Revision-Ilona Skerjanc The regulation of transcription during skeletal myogenesis by chromatin structure NO COURSE NO COURSE Manipulating the genome through gene therapy Hedgehog signaling pathway: signal transduction, development, and disease Marking Scheme" Midterm = 39% of total course marks " = all questions from Dr. Wang (9 lectures)" Final exam = 61% of total course marks" "" "= 70% of the final exam from Dr. Skerjanc " " "(10 lectures)" "" "=15% of the final exam from Dr. Parks " " "(2 lectures)" "" "=15% of the final exam from Dr. Wallace " " "(2 lectures) 2 10/14/11 Bch 4125 Lectures: Ilona Skerjanc Regulation of Muscle-specific Transcription during Skeletal and Cardiac Muscle Development" Skeletal Muscle Development Papers:" •  Paper #1: Regulation of skeletal myogenesis by association of the MEF2 transcription factor with Class II histone deacetylases, Lu, J., McKinsey, T.A., Zhang, C., and Olson, E.N., Molecular Cell, 6:233-244, 2000" •  Paper #2: Signal-dependent nuclear export of a histone deacetylase regulates muscle differentiation, T.A. McKinsey, C. Zhang, J. Lu, and E.N. Olson, Nature, 408: 106-111, 2000." •  Paper #3: Skeletal muscle specification by myogenin and Mef2D via the SWI/SNF ATPase Brg1, Yasuyuki Ohkawa, Concetta GA Marfella and Anthony N Imbalzano*, The EMBO Journal (2006) 25, 490–501. " • Paper #4: Pax7 activates myogenic genes by recruitment of a histone methyltransferase complex, I.W. McKinnel, J. Ishibashi, F. Le Grand, V.G. J. Punch, G.C. Addicks, J.F. Greenblatt, F.J. Dillworth, and M.A. Rudnicki, Nature Cell Biology (2008) 10, 77-84." 3 10/14/11 My Exam " Short answer questions based on the material provided in class:" 1) You should understand the four papers presented and be able to answer questions about the methods used, the assumptions, results, and conclusions from the data. " 2) You should be able to describe cell therapy approaches " 3) You should be able to extrapolate your knowledge of the papers to new situations" *(no specific questions asked on intro material; lecture #1). Overview of Lecture #1" 1. Why study muscle development?
 2. Overview of Early Human Development
 3. Basis of transcriptional control 
 4. Introduction to Genes Regulating Skeletal "Muscle Development
 5. Where are these genes expressed in the " "developing embryo?" 4 10/14/11 Skeletal Muscle diseases" •  Genetic diseases (dystrophies) involving mutation in the dystrophin glycoprotein complex or contraction apparatus" •  Sarcopenia - muscle wasting due " to aging processes" •  Fibromyalgia and viral myositis" • From: Brennan et al., Eur J Surg Onc 30:589, 2004 " A Lot is Known about Muscle Biology " Many genes involved in muscle contraction/relaxation have been cloned and purified and studied for years" From Bioessays 27:809, 2005 5 10/14/11 A Lot is Known about Muscle Development" Many genes regulating muscle development have been cloned and purified and studied for years" Pax7 Why do we not yet have a cure?" Gene therapy: still problems with long term expression etc. "" Myoblast therapy (satellite cells): problems with delivery, proliferation, viability" Embryonic stem cell therapy: problems with the source of cells and getting enough stem cells to differentiate down the muscle pathway" 6 10/14/11 Steps for Stem Cell Therapy:" 1.  Choose best stem cell (adult versus embryonic)" 2.  Achieve efficient differentiation into desired cell type" 3.  Control proliferation versus differentiation" 4.  Efficient delivery to affected tissue" Stem Cell" Mesoderm" Myoblast" Myocyte" Overview of Lecture #1" 1. Why study muscle development?
 2. Overview of Early Human Development
 3. Basis of transcriptional control 
 4. Introduction to Genes Regulating Skeletal "Muscle Development
 5. Where are these genes expressed in the " "developing embryo?" 7 10/14/11 A few hours after fusion of the two nuclei, the fertilized ovum divides The cells divide every 12-15 hours 8 10/14/11 The morula stage The blastocyst, surrounded by the zona pellucida, enters the uterus 9 10/14/11 The blastocyst frees itself of the zona pelucida - day 4 Embryonic stem cells Are derived from this stage At 8 days, the blastocyst (200 cells) lands on the uterine lining, the endometrium 10 10/14/11 Day12- The blastocyst (~2,000 cells) becomes anchored to the endometrium 3 Weeks - The 2 mm embryo has undergone gastrulation and the heart starts beating Future Head First somites Heart Region Neural Tube 11 10/14/11 4 Weeks - The 6 mm embryo contains somites and branchial arches, and the heart pumps blood to the liver and aorta Head Somites Heart 5 Weeks - The 1 cm embryo has developed eyes and limb buds Heart Limb buds 12 10/14/11 6 Weeks - The design for a human being begins to show Heart 7 Weeks - The fingers are starting to form 13 10/14/11 4 Months - The 5 cm fetus will grow to 10 cm 5 Months 14 10/14/11 The newborn baby Hand development: from 3 to 5 to 11 weeks 15 10/14/11 Foot development: from 4 to 9 weeks to 4 months Overview of Lecture #1" 1. Why study muscle development?
 2. Overview of Early Human Development
 3. Basis of transcriptional control 
 4. Introduction to Genes Regulating Skeletal "Muscle Development
 5. Where are these genes expressed in the " "developing embryo?" 16 10/14/11 Transcriptional control" •  The problem" –  Why are some genes expressed only in some cells?" –  How do we achieve precise, fine-tuned transcriptional responses " Model of the Preinitiation complex 1.  Multiple Activation Domains (AD) of transcription factors interact with Mediator simultaneously to regulate the frequency of transcription initiation. " 2.  AD–Mediator interactions regulate assembly of a preinitiation complex (PIC) composed of Pol II and the general transcription factors bound to promoter DNA. " 17 10/14/11 Transcription TFIID=yellow-brown, mediator=purple, RNA polymerase = blue-green, TFIIA/B=blue, TFIIH=green Website for movies about transcription and chromosome structure:
 WEHI-TV; DNA Molecular Animation
 http://www.wehi.edu.au/ education/wehi-tv/dna/index.html 18 10/14/11 DNA: Higher order structures •  Mammals have 2-3 billion bases of DNA –  Approximately 30,000 genes •  DNA has to be condensed to actually fit into the nucleus of the cell –  Condensation still has to allow for transcription factor access –  Tightly regulated process Histones •  Higher order packing of DNA is achieved through the association of DNA with histones The histone fold –  Core histones H3, H4, H2A, H2B –  Linker histones H1 and H5 Histone tail Site of multiple covalent modifications = regulation Dimer of H2A and H2B “handshake” 19 10/14/11 The Formation of the Histone Octamer" H3 tail Nature 389:251 260, 1997 Tails are turned outwards interaction with DNA Histone tails and chromatin packing •  To further pack the nucleosomes, a linker histone (H1) interacts with the inter-histone octamer DNA •  Interaction between histone tails allows further condensation of DNA into chromatin 20 10/14/11 DNA Chromosome Wrapping Nucleosomes need to be remodeled to allow for transcription and replication" 21 10/14/11 What types of proteins help remodel chromatin?! 1.  Chromatin remodeling ATPases " "" " "(SWI/SNF, BRG1)- Paper #3 " 2. Histone acetyl transferases (HATs = P300/CBP1)" What types of proteins prevent remodeling of chromatin?! 1. Histone deactylases (HDACs) - Papers #1 & 2" Nucleosome remodelers and HATs open chromatin: More transcription factors bind" 22 10/14/11 Assembly of preinitiation complex on open chromatin
 Chromatin remodeling ATPases are large complexes of multiple proteins" •  Yeast SWI/SNF" –  10 proteins" –  Needed for expression of genes involved in mating-type switching and sucrose metabolism (sucrose nonfermenting)." –  SWI/SNF complex interacts with chromatin to activate a subset of yeast genes." –  Is an ATPase" •  Mammalian homologs: hSWI/SNF" –  ATPase is BRG1, related to Drosophila Brahma (discussed in Paper # 3)" •  Other remodeling ATPases have been discovered." 23 10/14/11 Chromatin remodeling ATPases catalyze stable alteration of the nucleosome II: form a stably remodeled dimer, altered DNAse digestion pattern" III: transfer a histone octamer to a different DNA fragment" Nucleosome sliding" 24 10/14/11 CBP/p300 = HATs" •  CBP was first discovered as a cAMP response element binding factor (CREB) binding protein and therefore was named CREB binding protein!" •  p300 was discovered based on homology" –  CBP and p300 are highly homologous but not interchangeable" •  Roughly 300kDa proteins, with HAT activity" •  Knockouts of one or the other are embryonic lethal" –  Essential for normal growth and development" –  Role in many differentiation processes including muscle differentiation and adipogenesis" An integrator of transcriptional responses: Interaction of CBP/p300 with Transcription Factors! • Powerful HATS, acetylate H3 and H4 as well as TFs Goodman, Smolik Genes Dev. 2000 25 10/14/11 Histone deacetylases are repressors of transcription" •  Work locally on chromatin " –  Co-repressors" •  are not DNA-binding" •  are recruited to TF-complexes" – Make chromatin less permissive" Families of HDACs" 18 distinct human HDACs have been identified & are grouped into 3 broad classes : " •  Class I HDACs (HDAC1, -2, -3, -8 and -11) are homologous to the yeast transcriptional repressor yRPD3" •  Class II HDACs are homologous to yHDA1 (Papers #1 & 2)" –  subdivided into two subclasses:" »  IIa (HDAC4, -5, -7 and -9), nucleocytoplasmic shuttling, restricted expression" »  IIb (HDAC6 and HDAC10), " •  Class III HDACs are homologous to ySIR2 " –  Called ”sirtuins” " –  no homology to class I and II proteins" 26 10/14/11 HDACs Repress Transcription" Class IIa = HDAC-4, -5, -6, and -7" •  Expression is enriched in heart, skeletal muscle, and brain" •  Molecular mass is twice that of Class I, due to N-terminal extension
 NLS = Nuclear localization signal
 NES = Nuclear Export signal" MEF2 binding" Chromatin and HDAC" •  Reverse effect of acetylation" •  Histone deacetylases (HDACs) catalyze the deacetylation of lysine residues in the histone Nterminal tails" •  HDACs are found in large multiprotein complexes with transcriptional corepressors" 27 10/14/11 The Transcription puzzle Control of TF expression levels Transcriptional control Translational control Protein stability Protein modification Papers #1-4 Epigenetic effects Repressive chromatin structure Combinatorial effects TF synergy Cofactor cooperation RATE OF TRANSCRIPTION" TF TF Promoter/Enhancer Gene of Interest Overview of Lecture #1" 1. Why study muscle development?
 2. Overview of Early Human Development
 3. Basis of transcriptional control 
 4. Introduction to Genes Regulating Skeletal "Muscle Development
 5. Where are these genes expressed in the " "developing embryo?" 28 10/14/11 Overview of Differentiation" Note: The basic DNA sequence remains unchanged! Fertilized oocyte" Brain-specific " transcription factors" Muscle-specific " transcription factors" Chromatin" remodelling" Brain" Brain-specific mRNA" Brain-specific protein" Muscle" Muscle-specific mRNA" Muscle-specific protein" Skeletal muscle development: The First Factor Was Cloned By Differential Expression" Late 1980ʼs:" "" " " 5-azacytidine" Fibroblast cell " " -Housekeeping genes " "" " " " " " " "Skeletal muscle" -Housekeeping genes" -Muscle-specific genes" Subtractive cloning is a method used to isolate any clone present in one cell and not another." 29 10/14/11 Using subtractive cloning, MyoD was identified in 1989." Sequence Analysis:" •  Find the open reading frame of the cDNA: need an initiator Met (AUG) and a stop codon" •  Submit both the nucleic acid and protein sequences to Genbank and look for homologues or conserved proteins with similar sequences" Blast search with MyoD protein:" ! +++ +++ + +++ ++ ++ + + ! MyoD KRKTTNADRRKAATMRERRRLSKVNEAFETLK-RCTSSNPNQRLPKVEILRNAIRYIEGLQA! :..:.:::RR:. .:ER:R :.:..:F TL: : .. N:: :KV IL::A. Y:::LQA! N-myc NSDSEDSERRRNHNILERQRRNDLRSSFLTLRDHVPELVKNEKAAKVVILKKATEYVHALQA! " " " " Basic Helix-loop-helix" MyoD has a basic helix-loop-helix domain similar to the transcription factor Myc." 30 10/14/11 X-ray structure of MyoD •  Red = HLH •  Yellow = basic •  Blue= DNA Where is MyoD expressed?
 Probe duplicate blots containing RNA from different mouse tissues. " - MyoD - actin Therefore, MyoD is expressed only in skeletal muscle" 31 10/14/11 What is the function of MyoD?
 Hypothesis: MyoD is the gene activated by 5-azacytidine, controlling skeletal muscle development" Experiment: Express MyoD in fibroblast cells and look for transformation into muscle" Transfection of fibroblast cells:" DNA is taken up by eukaryotic cells when incorporated in a Ca2PO4 precipitate:" Mix Ca2PO4 and DNA" Add to cells" " " " " " " " " " " " "Incubate 9 hours " " and then wash" (Note: there are currently many different methods to carry out cellular transfection)" 32 10/14/11 Transient versus stable transfection" Transient transfection:" •  No integration of the plasmid into the chromosome and no selection used" •  No replication of the plasmid" •  Plasmid and expression is lost after 4 days" •  Higher expression levels obtained than with stable transfection" Stable transfection:" •  Integration of the plasmid into the chromosome (occurs during selection eg. Puromycin or G418)" •  Replication of the plasmid with the " " "chromosome" •  Plasmid and expression should be " " "maintained" •  Lower levels of expression than with transient" Use immunofluorescence to visualize the cells: 
 Fix cells with methanol" Incubate with primary Ab" Rat Anti-MyoD Ab Mouse Anti-Myosin heavy chain Ab MyoD Myosin heavy chain Incubate with secondary Ab" FITC-labeled Anti-rat Ab Rhodamine-labeled Anti-mouse Ab Rat-anti MyoD Ab Mouse - anti Myosin heavy chain Ab MyoD Myosin heavy chain 33 10/14/11 Find: MyoD converts cells into muscle!" MyoD is a member of the 
 Myogenic Regulatory Factor (MRF) Family" 34 10/14/11 Differentiation of skeletal myoblasts to myocytes and myotubes Differentiation" Myoblast" MyoD and MEF2C" Fusion" Myotubes" Myocytes" Myosin and Actin" The Second Factor was Cloned by Promoter Analysis 
 What is promoter/enhancer analysis?" Muscle Creatine Kinase Enhancer ß-Galactosidase Minimal promoter Reporter Gene 35 10/14/11 Promoter/Enhancer analysis" Muscle cell! Promoter is active! Non-muscle cell! Promoter is not active! 1.  Compare the expression of the promoter in muscle versus non-muscle cells (the correct promoter region will have higher activity in the muscle cell)" 2.  Create a series of deletions in the promoter to identify which sequences are essential for muscle-specific activity" Promoter “Bashing” ß-Galactosidase ß-Galactosidase ß-Galactosidase ß-Galactosidase ß-Galactosidase ß-Galactosidase ß-Galactosidase ß-Galactosidase ß-Galactosidase ß-Galactosidase ß-Galactosidase Muscle Activity + + + + - - + + + - - 36 10/14/11 Three essential sites found in the Muscle Creatine Kinase Enhancer " Muscle Creatine Kinase Enhancer 1 2 3 ß-Galactosidase Minimal promoter •  MEF1 = E box sequence that binds MyoD" •  MEF2 = A/T rich sequence " •  MEF3 = Later shown to bind the Six family of transcription factors" (MEF = myocyte enhancer factor)" The A/T rich sequence was used to screen a library and the MEF2 family of transcription factors were identified:" 37 10/14/11 Structure of MEF2A: MADS box homodimers" MEF2 and MRFs Act Cooperatively to Activate Muscle-Specific Genes" Inducing signal MEF2 Amplification and Maintenance Myogenic bHLH factors Activation MEF2 MEF2 MyoD E E box 38 10/14/11 Overview of Lecture #1" 1. Why study muscle development?
 2. Overview of Early Human Development
 3. Basis of transcriptional control 
 4. Introduction to Genes Regulating Skeletal "Muscle Development
 5. Where are these genes expressed in the " "developing embryo?" 39 10/14/11 (Pax7 is discussed in! Paper #4)! 40 10/14/11 Meox1 and Pax3/7 are expressed in the somites Meox1 Pax7 Myf-5, myogenin and MyoD are expressed in the myotome Myf-5 Myogenin 41 10/14/11 Overview of Lecture #1" 1. Why study muscle development?
 2. Overview of Early Human Development
 3. Basis of transcriptional control 
 4. Introduction to Genes Regulating Skeletal "Muscle Development
 5. Where are these genes expressed in the " "developing embryo?" 42 ...
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This note was uploaded on 12/18/2011 for the course BCH 4122 taught by Professor Ilonaskerjanc during the Spring '11 term at University of Ottawa.

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