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单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,2020/11/3,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,2020/11/3,*,General Mechanisms of Transcriptional Regulation,in Eukaryotes,Eukaryotic,Transcriptional,Activators:,激活蛋白的结构、作用方式,Eukaryotic,Transcriptional Repressors,Signal Integration and Combinatorial Control,Signal Transduction and the Control of Transcriptional Regulators,Gene Silencing by Modification of Histones and DNA,Epigenetic Gene Regulation,2020/11/3,0,UASs,(,upstream activation sequences,) =,enhancers,Each UAS is 17bp long, bound by a Gal4 dimer,The GAL System,in,Yeast,:,When lacking galactose, GAL genes are silent.,In,presence of galactose,(,and absence of glucose,), GAL genes are induced.,2020/11/3,1,(a) The DNA-binding domain of Gal4, without that protein,s activation domain, can still bind DNA but cannot activate transcription.,(b) Attaching,the activation domain of Gal4,to the,DNA-binding domain of the bacterial protein LexA,creates a,hybrid protein,that activates transcription of a gene in yeast,if,that gene bears a binding site for LexA.,Expression is measured using a reporter plasmid in which the,GAL1 promoter is fused to the E. coli lacZ gene,whose product (b-galactosidase) is readily assayed in yeast cells.,Transcription,activators,have,separate,DNA binding,and,activating,domains,:,the ,Domain s,GAL1 promoter,2020/11/3,2,Bacterial regulatory proteins,m,ostly use the,helix-turn-helix motif,to bind DNA target,Regulatory proteins : DNA binding regions,Homeodomain,One helix (for recognition, #3),fits in major groove and recognizes specific base pairs.,The other helix (#2),makes contacts with the DNA backbone, positioning the recognition helix properly and increasing the strength of binding,Eukaryotic regulators use a range of DNA binding domains,Homeodomain proteins (HTH):,helix-turn-helix,Zinc containing DNA-binding domain: zinc finger / zinc cluster,Leucine zipper motif,Helix-Loop-Helix proteins,HMG: interact with minor groove / alter DNA conformation,2020/11/3,3,zinc nger proteins,The zinc atom,interacts with,cysteine,and,histidine,residues and,serves a structural role,essential for,integrity of the DNA-binding domain,The,helix for recognition is presented to DNA by,sheet on the right.,The zinc is coordinated by the,two histidine,in the,helix and,two cysteine,in the,sheet,The zinc is coordinated by,four cysteine residues,2020/11/3,4,Leucine zipper protein,major groove,Leucine zipper motif,2020/11/3,5,The,dimerization surface,is formed from two helical regions:,the first is part of the same helix involved in DNA recognition;,the other is a shorter helix.,These two helices are separated by,a flexible loop,that allows them to pack together,Helix-Loop-Helix Proteins,*,2020/11/3,6,7,Activating Regions Are Not Well-Defined Structures,They are,adhesive surfaces,capable of interacting with,other protein surfaces,;,Not in a“lock and key”manner,;,a,re grouped on the basis of,amino acids content,.,Acidic region,: contain,both critical,acidic AAs,and,hydrophobic AAs,.,Glutamine-rich,region,Proline-rich,region,2020/11/3,7,DNA binding domain comprises three Cys2His2-like zinc fingers,The activation domains are composed of,serine/threonine,rich regions, flanked by regions rich in,glutamine,*,2020/11/3,8,*,2020/11/3,9,Eukaryotic activators,recruit polymerase,indirectly,1. Interacting with,parts,of the,transcription machinery,(GTFs, other proteins for RNAPII innitiation and elogation, etc.),2. Recruiting,nucleosome modifiers,/,remodelers,that alter chromatin structure, to help initiation,2020/11/3,10,Gal4 interacts with,mediator complex, which directly recruit RNAP II to gene promoters.,Gal4 binds to,TBP,and recruits the,TFIID,complex and, RNAP II to promoter.,The,mediator,act as a co-activator (facilitat,e,gene activation by TF,but,itself is,neither,part of the transcriptional machinery,nor,a DNA-binding protein),Activators interact with,parts of the transcription machinery,2020/11/3,11,Promoters bearing acetylated nucleosomes have,higher affinity for transcriptional machinery,Activators recruit nucleosome,modifiers,1. Acetylation can,alter the DNA-histone interaction,the,octamer slide,along the DNA to a new position.,2.,.,can,alter the interaction between adjacent nucleosomes,a more open chromatin.,3.,.,creates speci,fic,binding sites,on nucleosomes for proteins with,bromodomains,( eg., TFIID),2020/11/3,12,Activators recruit nucleosome,remodel,ers,to,“,Loosen” the chromatin structure,Sliding,2020/11/3,13,Requirement,of components of the transcriptional machinery,varies under different circumstances,Many proteins,can interact specifically with the,Gal4-activation domain,:,TBP, TFIIB, Gal11,(a component of Mediator),Cdk8, SWI/SNF,(nucleosome remodeler),SAGA,(Spt-Ada-Gcn5-acetyltransferase),Srb4,(another component of Mediator) and,proteasome components Sug1 and Sug2,-by 2006,EMBO reports VOL 7 | NO 5 | 2006,Gal4 functions in most eukaryotes, in many ways,*,2020/11/3,14,HSP70 gene from Drosophila, activated by heat shock, is controlled by,two activators,working together.,The,GAGA-binding factor,can recruit enough of the transcription machinery to the promoter for transcription initiation.,But, in the absence of,a second activator, HSF, most of the initiated polymerases,stall some 2550 bp down-stream from the promoter.,In response to heat shock, HSF binds to specic sites at the promoter and recruits,a,kinase,P-TEFb,(,positive transcription elongation factor,part of a larger complex, the SEC,super elongation complex), to the stalled polymerases.,A strong acidic activator like Gal4 is able to recruit PTEFb/SEC along with the rest of the machinery.,The kinase phosphorylates the Ser2 of the CTD heptad repeat of RNAPII,freeing the enzyme from the stall and allowing transcription to proceed through the gene.,*,2020/11/3,15,Transcriptional Repressor,In eukaryotes, most repressors,usually,do not,act,by,binding to sites that overlap with the promoter,and block binding of polymerase. (Bacteria often do so),1.,Most common:,Compacting the nucleosome,by,directly,removing/,adding some groups,or by,recruiting nucleosome modifiers,2.,Competing with the activator,for an overlapped binding site,3.,Binding to a site different from that of the activator, but,physically interacts with an activator,molecule,and,blocks its activating region,4.,Binding to a site upstream of the promoter,physically interacts with the transcription machinery,at the promoter to inhibit transcription initiation.,2020/11/3,16,In the presence of glucose,Repression of the,GAL1,gene in yeast,Mig1,binds to a site,between,the Gal4-binding site and the,GAL1,promoter,Recruits the,Tup1,protein,complex,Tup1,recruits,histone deacetylases,Directly interacts with,transcription machinery,Repress transcription,.,*,2020/11/3,17,2020/11/3,18,*,2020/11/3,19,*,2020/11/3,20,*,2020/11/3,21,22,Study protein/nucleic acid interaction,Gel retardation assay,/,electrophoretic mobility-shift assay,can be used qualitatively to identify,sequence-specific DNA-binding proteins,(such as transcription factors),Protein:DNA complexes migrate more slowly than free linear DNA fragments,in non-denaturing polyacrylamide or agarose gel electrophoresis,However, if circular DNA is used, the protein:DNA complex may migrate faster than free DNA,The order of component addition for the binding reaction is often critical.,Completed binding reactions are best electrophoresed immediately to preserve potentially labile complexes for detection.,2020/11/3,22,(A) Labelled oligonucleotide incubated with,increasing amounts of V47R POU mutant protein.,0, control DNA probe without protein.,Gel retardation assay of DNA-protein complexes.,(B) Quantification of gel retardation data.,Amount of free DNA as a function of log protein concentration,.,Relative affinity of protein to octamer probe (ATGCAAATGA) can be determined,FEBS Letters 412 (1997) 5-8,protein-DNA complex,free DNA,2020/11/3,23,24,1）,The protein protects DNA from attack by DNase. 2）Treat the DNA-protein complex with DNase under,mild conditions, so that,only one cut,occur per DNA molecule,on average.,Electrophoresis,autoradiograph,DNase I footprinting:,Identify the actual region of sequence with which the protein interacts.,The three lanes represent DNA that was bound to 0, 1, and 5 units of protein. The lane with no protein shows a regular ladder of fragments. The lane with one unit shows some protection, and the lane with 5 units shows complete protection in the middle.With sequence ladders, we can tell exactly where the protein bound.,only one cut,occur per DNA molecule,2020/11/3,24,Yeast two hybrid system:,to discover proteinprotein interactions,and,proteinDNA interactions,by using,yeast fusion proteins,transcription activation system,and,reporter genes,Transcription factor,When these two domanis are expressed as seperate proteins, the BD will still bind to DNA, but the AD is,NOT,in the right place to activate transcription,A split transcription factor,2020/11/3,25,BD,X,Y,AD,promoter,gene,Hybrid proteins as molecular,glue,To test if two proteins (X and Y) interact, they are expressed in,fusion,with the BD and the AD,BD,promoter,gene,X,Y,AD,If proteins X and Y bind to each other, the transcription factor is,reconstituted, and gene expression is activated,2020/11/3,26,AD,BD,GAL4-,AD,GAL4,and,Reporter genes,In many cases, the reconstituted transcription factor is the yeast GAL4 transcriptional activator.,reporter,gene,GAL4-,BD,GAL4-UAS,To monitor transcriptional activation, reporter proteins such as,b,-galactosidase,are expressed under the control of the GAL4-upstream activating sequence.,2020/11/3,27,Application of,the yeast two-hybrid system,Identifies,novel,protein-protein interactions,:,Which proteins can interact with an known protein?,Need to construct bait and prey plasmids to express fusion proteins,Identifies,mutations,that affect protein-protein binding,Can,identify interfering proteins,in known interactions,2020/11/3,28,Exploring protein-protein interactions: Pull-Down Assays,2020/11/3,29,2020/11/3,30,CHIP : chromatin immuno precipitation,CHIP-on-chip,2020/11/3,31,General Mechanisms of Transcriptional Regulation,in Eukaryotes,Recruitment of Protein Complexes to Genes by Eukaryotic Activators.,Transcriptional Repressors,Signal Integration and Combinatorial Control,Signal Transduction and the Control of Transcriptional Regulators,Gene Silencing by Modification of Histones and DNA,Epigenetic Gene Regulation,2020/11/3,32,Activators work together synergistically,(,When an effect is greater than additive,),to integrate signals,A,B,C,F,E,D,Signal Integration,and,Combinatorial Control,Numerous signals,may be required to switch a gene on;,Each signal is,transmitted,to the gene,by a separate regulator,.,2020/11/3,33,S1:,Multiple activators recruit a single component,of the transcriptional machinery,by touching the different part of the mediator complex.,S2:,Multiple activators each recruit a different component,of the transcriptional machinery.,These components binds to the promoter DNA,inefficiently,without help.,S3:,Multiple activators help each other,bind to their sites upstream of the gene they control.,Strategies of the synergy,a.“Classical” cooperative binding.,b. Both proteins interacting with a third protein.,c.,The first protein recruit a nucleosome remodeller whose action reveal a binding site for the second protein,.,d.,Binding a protein unwinds the DNA from nucleosome a little, revealing the binding site for another protein,.,2020/11/3,34,Example: the HO gene,of yeast,S. cerevisiae,only expressed in mother cells and at certain point in cell cycle, and,is controlled by two regulators: recruiting nucleosome modifiers and mediators.,SWI5: acts,only in the,mother cell,and binds,unaided,to multiple sites distant from the gene, which,recruit enzymes to open the SBF binding sites,SBF: only active at the,correct stages,of the cell cycle, and cannot bind the sites unaided,*,2020/11/3,35,Infection,Example:Cooperative binding of activators at human,b,-inter,f,eron gene.,Triggers,three activators (communicator),:,NFkB,IRF, and,Jun/ATF,Activators bind cooperatively to an enhancer located about 1 kb upstream of the promoter, forming,enhanceosome,The,human b-interferon gene,is activated in cells upon,viral infection,Transcription is activated to high level only when all the proteins are present and touching one another in just the right way,Role of HMGB as architectural factors,*,2020/11/3,36,Binding sites of activators at the human,b,-interferon gene,The conservation of the interferon- b enhancer DNA sequences,across species separated by 100 million years,.,The crystal structure of the enhanceosome,*,2020/11/3,37,When all of the regulatory proteins are bound and interacting correctly,they form a “landing pad,” a high-affinity binding site for the protein CBP,a co-activator protein that also recruits the transcriptional machinery.,The large CBP protein also contains an,intrinsic histone acetylase activity,that modifies nucleosomes and facilitates high levels of transcription,用肘轻推,nuc 2 is at the TATA box and transcription start site,37 bp off the TATA box,The B-interferon enhanceosome acts to move nucleosomes by recruiting the SWISNF complex,*,2020/11/3,38,Combinatory control,-,activators and repressors work together,-,complexity and diversity of eukaryotes,2020/11/3,39,E,xample: combinatory control of the mating-type genes from,S. cerevisiae,The cell types (the,a,and,haploid, and the,a,/,diploid) are defined by the sets of genes they express-,determined by mating-type regulators,: One ubiquitous regulator (Mcm1) and three cell-type-specific regulators (,a,1,1, and,2) , encoded by the MAT locus.,*,2020/11/3,40,2.,Signal Transduction,and,the Control,of Transcriptional Regulators,Signals,Transcriptional,regulators,signal transduction pathway,Mechanisms,I,:,Un,masking Activating Region by,:,a conformational change in the DNA-bound activator, revealing,a previously buried activating region,or by release of a masking protein,that previously interacted with, and eclipsed, an activating region.,through,binding,ligand directly,or,through a ligand-dependent phosphorylation.,Gal3 binds galactose,and ATP,2020/11/3,41,Lessons from Yeast-The GAL System:,When lacking galactose, GAL genes are silent.,In presence of galactose (and absence of glucose), GAL genes are induced.,*,2020/11/3,42,EMBO reports VOL 7 | NO 5 | 2006,Gal80 does not exhibit nucleocytoplasmic exchange,Galactose does not cause Gal3 entry into nucleus to account for the rapid Gal4-mediated gene activation,Nuclear-localized Gal3 is required for rapid induction,Stable Gal3Gal80Gal4 complex is not detectable in galactose-induced cells,Gal80 complexed with Gal4 on DNA does not exchange rapidly with free Gal80,Transient,association of Gal3 with Gal4-associated,Gal80: a plausible mechanism of the GAL gene switch,*,2020/11/3,43,*,2020/11/3,44,Phosphorylation of,Rb causes release of,it,from E2F and activation of the genes.,Mechanism II:,T,he masking protein,may,not only blocks,activating region,but also is itself (or recruits) a,deacetylase,actively represses the,gene.,Mutant Rb:,-inactivating mutation,-Rb gene deletion,T,he repressor Rb (,retinoblastoma protein),controls activity of E2F by,binding to it,-,both blocking activation,and,recruiting,a deacetylase,T,he mammalian activator E2F,binds sites,upstream of its target genes,.,uncontrolled cell cycle,2020/11/3,45,2. Signal Transduction and the Control of Transcriptional Regulators,Mechanisms: Transport into and out of the nucleus,:,When not active, many activators and repressors,are held in the cytoplasm,through,interaction with an inhibitory protein, or,with the cell membrane,. The signaling ligand causes them to,move into the nucleus,to activate transcription,;,A cascade of kinases cause,the phosphorylation of regulator,in nucleus;,The activated receptor is cleaved by cellular proteases, and,the c-terminal portion of the receptor,enters the nucleus and activates the regulator,2020/11/3,46,The STAT pathway,The MAP kinase pathway,The signal is then relayed (,分程传递,) to the relevant,transcriptional regulator,The transcriptional regulator control the,target gene expression,The signal is communicated to the,intracellular domain,of receptor,(via an,allosteric change or dimerization,),The ligand (“signal”) binds to an,extracellular domain,of a specific cell surface receptor,*,2020/11/3,47,Signaling mechanisms linking neuronal activity to gene expression and plasticity of the nervous system-,Annu Rev Neurosci. ;31:563-590,2. These pathways converge on preexisting transcription factors in the nucleus and lead to their activation through direct posttranslational protein modifications.,1. Calcium influx through neurotransmitter receptors or voltage-gated calcium channels leads to activation of calcium-regulated signaling enzymes, setting in motion several signal transduction cascades.,3. Several of the activity-regulated genes encode transcriptional regulators, which promote transcription of additional activity-regulated genes.,4.Genetic mutations in genes that encode the signaling molecules give rise to neurological disorders in humans ( yellow boxes).,3. Many other activity-regulated genes encode proteins working in dendrites or at synapses and thereby coordinate activity-dependent dendritic and synaptic remodeling within the neuron.,*,2020/11/3,48,General Mechanisms of Transcriptional Regulation,in Eukaryotes,Recruitment of Protein Complexes to Genes by Eukaryotic Activators.,Transcriptional Repressors,Signal Integration and Combinatorial Control,Signal Transduction and the Control of Transcriptional Regulators,Gene Silencing by Modification of Histones and DNA,Epigenetic Gene Regulation,2020/11/3,49,Gene “Silencing” by Modification of Histones and DNA,Transcriptional Silencing:,a specialized form of repression that can,spread along chromatin,switching off multiple genes,even those quite distant from the initiating event,without the need for each to bear binding sites for specific repressor,.,Transcriptional silencing,is a,position effect,: A gene is silenced because of where it is located, not in response to a specific environmental signal.,2020/11/3,50,Transcriptional silencing is associated with,m,odification of nucleosomes that alters the accessibility of a gene,to the transcriptional machinery and other regulatory proteins.,SIR2, 3, and 4 have been found as regulators of silencing (SIR stands for,S,ilent,I,nformation,R,egulator).,Rap1,recruits Sir complex to the temomere.,Sir2,deacetylates nearby nucleosome,The spreading of silencing is restricted/controlled,by,insulators and other kind of histone modifications,that block binding of the Sir2 proteins.,2020/11/3,51,Transcription can,also,be,silenced through chromatin methylation,by,histone methyltransferase,Methylation of H3K9 is a modification associated with silenced heterochromatin,in higher eukaryotes and in the yeast S. pombe. In contrast,methylation of H3K4 are associated with increased transcription.,In S. cerevisiae,methylation of specific lysine residues,in the tails of histones H3 and H4,help repression,of some genes and,block spreading of Sir2-mediated silencing,in others.,2020/11/3,52,When mammalian cell DNA is stained with,fluorescent dye, heterochromatin, which is,a densely condensed assembly of chromatin, becomes visible under bright light (left). The basic structure of heterochromatin is also found in fission yeast (right).,2020/11/3,53,The most common form of silencing,: a dense form of chromatin - “heterochromatin”.,Usually at particular regions of the chromosome, eg.,the telomeres,
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