Control o f of Gene Expression Expression Partha Roy 1

Transcription

1 Control of Gene Expression Partha Roy 1

2 Why do these two cell types look so different? Neuron Red blood cell Different kinds of differentiated cells must express at least some different proteins to carry out specialized jobs specific to the cell-type (is it the DNA that t is different between these two types of cells?) 2

3 Genetic composition must be the same in different differentiated cells Adult cow Harvest somatic cells Adult cow Fuse somatic cell with egg Cell division embryo Unfertilized egg remove chromosome Reconstituted zygote Transfer to foster cow Genetic info from a differentiated somatic cell is sufficient for forming a complete organism calf (adapted from Alberts et al.) 3

4 Regulation of gene expression is important because: 1) To selectively express proteins in different kinds of cells that are responsible for carrying out specific and distinct functions. 2) To modulate cell function in response to environmental cues. 4

5 Different levels of control of gene expression cytoplasm nucleus Stability control Degradation DNA Transcriptional control primary RNA transcript mrna mrna protein RNA processing control RNA transport And localization control Translational control 5

6 Final mrna available for translation = mrna transcribed - mrna degraded (transcriptional control) (mrna stability control) Q: Is it the difference in the rate of transcription or mrna stability that is responsible for cell-specific differences in some protein levels? Genes that require rapid induction and repression: SHORT half life Genes that do not require rapid induction and repression: LONG half life 6

7 Nuclear Run-ON assay: Measures the rate of transcription initiation RNA pol II Nascent mrna strands Isolate cell nuclei in cold condition (freezes RNA pol II and synthesized mrna strands on DNA) Add 1 radiolabeled, 3 unlabeled nucleotide; Incubate the nuclei at 37 degree for 5-10 min elongation of the existing strands (No de novo transcription initiation in isolated nuclei) * mrna degradation occurs in cytoplasm Hybridize labeled mrna to cdna probe; Autoradiography (signal proportional to transcript 7 level) Carey and Smale

8 Nuclear Run-on Assay : shows transcriptional control between cell types Blot cdnas corresponding to liver specific proteins onto nylon membranes Nuclear run-on with isolated nuclei from mouse liver, kidney and brain Only liver blot shows hybridization. Intensity of the spots correlates with the level of active transcription. A- actin, α and β T- tubulins 8 Lodish 5e

9 Outline Structural feature of transcriptional regulation unit Mechanisms of Transcriptional regulation 9

10 Structural Features of a typical transcription unit Prokaryote vs. Eukaryote Experimental identification ifi i of these structural features 10

11 Prokaryotic Transcription unit Half of bacterial genes are arranged in clusters of genes having functional relation (operon) Promoter (binding site for pol II, Initiation factors) Gene clusters Operator (binding site for control elements e.g repressor / activators) mrna (polycistronic) o c) Individual proteins control elements are fairly close to the start site 11

12 Eukaryotic Transcription unit Distant control sequences and elements repressor a muti-unit it protein complex transcription Proximal control sequence and elements Single gene single mrna 12 Lodish 5e

13 Analyses of transcription unit 1) Mapping the start site of transcription RNAse Protection 2) Identification of control elements 13

14 +1 -y +x Hind3 site +1 RNAse Protection assay Bacteriophage promoter Construct a plasmid including region of gene containing putative start site (can be isolated by PCR of genomic DNA) and a promoter downstream of it. -y +x First cut with Hind3 to linearize plasmid, And then perform in vitro transcription (by adding radiolabeled UTP, RNA polymerase, buffer) +1 +x 3 5 Generate antisense RNA probe 14 Carey and Smale

15 3 +1 +x 5 RNAse Protection assay (continued) AAAAA Hybridize with mrna (hybridizes only +1 to +X region) x 5 AAAAA Digest the unhybridized region (single stranded RNA) by RNAse A Creates a x-nucleotide RNA-RNA hybrid undigested Run on gel and autoradiography to determine the size (determine x determine start site) digested 15 Carey and Smale

16 How do we identify the control regions of DNA regulating gene transcription? You need to analyze (measure) gene transcription with or altered control regions 1) Need to measure the transcription of a gene that is not expressed endogenously. 2) Drive the expression of reporter gene (not expressed otherwise) with or without altered control regions: Measure gene activity (proportional to the gene expression). Readout (quantifiable) Reporter gene: LacZ (b-galactosidase activity) GFP (fluorescence) luciferase (light) 16

17 5 deletion analysis : shows proximal upstream elements are important Reporter gene: lacz, luciferase GFP 17 Lodish 5e Progressive deletion of upstream elements decreases gene expression

18 Linker scanner mutagenesis: maps important control regions Transcription level TATA box Random nucleotide cassette Conclusions: 1) distinct control regions 2) sequence specificity Lodish 18 5e

19 Modular characteristics of transcriptional activators (repressor) Activation (or repression) domain DNA-binding domain Mediator CTF Pol II Transcriptional Activator (or repressor) DNA CTF: core transcription factors DNA binding domain interacts with regulatory promoter Activation (repression) domain interacts with mediator and other proteins Repressors are functional inverse of activators 19

20 Experimental demonstration of modular nature of transcriptional activator Expression of lacz in yeast cells expressing either WT or mutant Gal4 showing distinct DNA binding and activation domains 20 Lodish 5e

21 Three techniques to identify (and map) DNA-protein interactions 1) DNAse-I foot-printing 2) Electrophoretic-mobility shift assay (EMSA) 3) DNA affinity chromatography 21

22 DNAse-footprinting assay 1) Label control regions of DNA with radioactive tag. 2) Label DNA with or without different fractions of nuclear extract. 3) Cut with low conc. of DNAse-I (one random cut/ DNA molecule). 4) Run rxn products on a gel 22 Principle: DNA, when bound to protein, becomes resistant to cleavage by DNAse I and produces foorprints (missing bands) on gel electrophoresis. Lodish 5e

23 Gel-filtration nuclear fractions 9,10,11 contain proteins that bind to DNA NE no nuclear extract FT flow through 23 Lodish 5e

24 Electrophoretic mobility shit assay Pi Principle: il (Protein+DNA) complex migrates slowly l compared to free DNA in gel electrophoresis Protein fractions that contain putative DNAbinding proteins 24 Lodish 5e

25 Purification of sequence-specific DNA-binding protein Step 1 (purification) Nuclear extract Affinity matrix containing ii a specific DNA sequence elute Step 2 (in vitro transcription) Template DNA (containing binding site for X) + pol II, general transcription factors + radiolabeled dilbldribonucleotide +/- X Analyze the transcript level on gel electrophoresis by autorad (presence of X should increase the transcript level) Sequence, clone (X) Confirm In vitro transcription activity ty Confirm In vivo transcription activity 25

26 Step 3: Confirm in-vivo transcription activity Cotransfect plasmids coding 1) suspected transcription Factor (X) and 2) a reporter gene at the downstream of binding element for X increased reporter transcription Lodish 26 5e

27 Prokaryotic transcription control 27

28 General scheme of prokaryotic transcriptional control Negative regulation bound repressor prevents transcription X Pol II can t bind gene OFF repressor Ligand binding to repressor displaces repressor - pol II Pol II binds gene ON - ligand 28

29 Positive regulation Bound Activator promotes transcription Pol II binds gene ON Ligand binding prevents activator from DNA binding X Pol II can t bind gene OFF activator - pol II - ligand 29

30 Gene regulatory proteins can act as both activator or repressor Positive ii regulation Binding of an Activator at the upstream of gene 1 facilitates pol II binding promotes transcription of gene 1 Pol II binds gene ON Shifting of binding site of the same factor at the upstream of gene 2 prevents pol II binding and represses transcription of gene 2 1 Activator/ repressor - pol lii X 2 Pol II can t bind gene OFF 30

31 Dual control of gene transcription (example: lacz) -lactose +glucose (low camp) CAP lacr X lacz No transcription +lactose +glucose (low camp) CAP polii lacr lacz lactose Low transcription +lactose -glucose (high camp) camp CAP polii lacr lacz lactose High transcription CAP- catabolite activating protein (+ve regulator) lacr lac repressor ( -ve regu;ator) 31

32 Eukaryotic transcription control 32

33 Two levels of control needed for transcription initiation: 1) Decondense chromatin structure 2) Assembly of protein complexes to initiate transcription (pol II, general transcription factors, mediator, activators, coactivators etc.) either proximal or distant factors relative to the start t site transcription ti lodish 33

34 Decondensation of actively transcribing chromatin as demonstrated by increased sensitivity to DNAse1 Southern blot with globin specific probe lodish 14 day erythroblast actively transcribes globin gene decondensed and suscesptible to DNAse I; corresponding regions of chromatin in undifferentiated non-erythroid cells not actively transcribing globin are less susceptible to DNAse I. 34

35 1) Chromatin remodeling occurs by the actions of : Chromatic remodeling complex Covalent modification i of histone proteins 35

36 Covalent modification of histone tails H2A tail H4 tail H2B tail H3 tail H2A tail H4 tail H2B tail H3 tail Nt termini itails of fthe four core histone subunits are conserved tails contact DNA on neighboring nucleosome Lysine -ve charge on DNA alberts 36

37 Types of histone modification Acetylation of lysines (neutralizes +ve charge; decondensed chromatin transcriptional activation) Deacetylation ti of flysines (condensed dchromatin transcriptional repression) Methylation of lysines (prevents acetylation - repression) Phosphorylation of serine (transcription) 37

38 Histone acetyl transferase (HAT) Histone Acetylated histone Histone deacetylase (HDAc) GCN5, Rpd3- Coactivators (do not directly bind to DNA) lodish 38

39 2) Assembly of transcription preinitiation complex Multiprotein complex forming on enhancer also create favorable DNA conformation for binding of other transcription factors Ordered binding and interactions between activators and coactivators (including mediator bridges gap between activators and transcription unit initiation unit) recruit and position pol II and core transcriptional factors to initiate transcription. Also, the binding is co-operative (i.e binding of one factor allows binding of a second factor more readily). lodish 39

40 Regulation of transcription factor activity Whether or not a specific gene is expressed is dependent on the concentrations (transcription control) and activities of transcription factors that t interact t with the regulatory sequences of the gene. 40

41 Hormone-dependent activation of transcription factors Small Lipid soluble molecule can freely diffuse though plasma and nuclear membranes and interact with nuclear receptors (act as transcription factors) Binds to hormone Activation domain DNA-binding Ligand binding Nuclear receptor type transcription factor (ex: estrogen receptor, steroid receptor) 1) Hormone binding induces conformational change of nuclear receptor. 2) In some cases, hormone binding domains acts as a repressor domain in the absence of ligand binding. 41

42 Model for hormone dependent gene activation by homodimeric Nuclear receptor lodish Nuclear factor is sequestered in cytoplasm by inhibitors; hormone binding displaces the inhibitor; nuclear factor can now translocate to nucleus and induce gene expression. In some cases, deletion of ligand binding domain activates it. 42

43 Demonstration of nuclear translocation of receptor in response to hormone stimulation β-galactosidase: used as a reporter gene Dex: dexamethasone (gluococorticoid hormone) Immunofluorescence of β-galactosidase (β-galactosidase is also transported) 43

44 How are different cell-types created? (cells need dto memorize patterns of changes in gene expression) 44

45 Two proteins that repress each other s synthesis and stimulate their own expression determine two inheritable states Lytic cycle phage Lysogenic cycle No lamba repressor High lamba repressor High cro No cro ci off cro ci mrna cro off cro Lamda repressor Lamda repressor positively regulates its own transcription and shuts off cro transcription; cro binds at a different site on the operator and shuts off lambda repressor transcription alberts 45

46 Expression of a critical regulatory gene (master gene) can trigger expression of a whole set of downstream genes Example: muscle development Treating C3H10T½ fibroblast cells with 5-azacytidine (a C-derivative that can not be methylated), causes these cells to spontaneously differentiate into muscle. 5 Azacytidine Skeletal muscle Pg 914, lodish 46

47 47

48 (myogenic determinant) One of the four proteins identified lodish 48

49 myod-transcription Myoblast precursor cells of muscle (differs from muscle in terms of expression of several structural genes) signal factor MEF 2 Expression of Myo D (master gene) (myocyte enhancement factor) Binding of MyoD And MEF enhance Transcription activity Muscle structural genes (contains myod binding Muscle development element) 49

50 Developmental decision can be reinforced by: chromosomal modification which can be stably inherited by daughter cells Example: methylation of C in CG inheritable and makes it transcriptionally silent. CH 3 ACATCGT TGTAGCA CH 3 CH 3 ACATCGT TGTAGCA DNA replication ACATCGT TGTAGCA CH 3 Recognized by Maintenance methylase Methylated cytosine base pairs with Guanine CH 3 ACATCGT CG TGTAGCA CH 3 CH 3 ACATCGT TGTAGCA CH 3 Methylated C is recognized by proteins that cause chromatin remodeling by recruiting i remodeling complex, HDACs locks into transcriptionally silent state. alberts 50