2 Eukaryotic gene regulation occurs at several levels
3 1- Control at DNA level by DNA methylation Small percentages of newly synthesized DNAs (~3% in mammals) are chemically modified by methylation.Methylation occurs most often in symmetrical CG sequences.Transcriptionally active genes possess significantly lower levels of methylated DNA than inactive genes.Methylation results in a human disease called fragile X syndrome; FMR-1 gene is silenced by methylation.
4 2- Control at DNA level by Histone modifications(Chromatin Remodeling) Acetylation (乙酰化)by histone acetyl transferases (HATs) and coactivators leads to euchromatin formation; p53 acetylationMethylation by HDACs （去乙酰化酶）and corepressors leads to heterochromatin formation . Rb-E2F
5 3-Control at DNA level by gene amplification Repeated rounds of DNA replication yield multiplecopies of a particular chromosomal region.
6 4- Control at transcription initiation gene control region for gene X By using different sequences (promoter, enhancer or silencer sequences) and factors, the rate of transcription of a gene is controlledgene control region for gene X
7 5- Control at mRNA splicing (alternate splicing) (four exons)Calcitonin gene-related peptide6132 amino acidsReduces bone resorption37 amino acidsVasodilator
8 6- Control at mRNA stability Messenger RNA longevity can be influenced by several factors.Poly(A) tails seem to stabilize mRNAs.The sequence of the 3’untranslated region (3’UTR) preceding a poly(A) tail also seems to affect mRNA stability.Several short-lived mRNAs have the sequence AUUUA repeated several times in their 3’untranslated regions.For the iron (Fe 2+) transport protein transferrin receptor.A stem loop structure in the mRNA acts as an iron response element and binds a 90 kDa protein in the absence of iron. The RNA and iron binding regions of the protein overlap so in the presence of iron the 90 kDa binding protein can no longer bind to the mRNA iron response element and the stem loop no longer occurs. Since the stem loop is at the 3’ end of the mRNA , the loop is stabilising of the mRNA , protecting it from degradation. In the presence of iron, the loop disappears and the mRNA is degraded by 3’ exonucleases.s
9 6- Control at mRNA stability When this sequence is artificially transferred to the 3’untranslated region of more stable mRNAs, they, too, become unstable.Chemical factors, such as hormones, may also affect mRNA stablility.In the toad Xenopus laevis(非洲爪蟾) , the vitellogenin gene(卵黄生成素) is transcriptionally activated by the steroid hormone estrogen(类固醇激素 ) . However, in addition to inducing transcription of this gene, estrogen also increases the longevity of its mRNA.
10 6- Control at mRNA stability Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small, noncoding RNA molecules called microRNA (miRNAs).mRNA stability. When milk protein synthesis is stimulated in the mammary epithelium at child birth, the rapid increase in casein level arising from the pituitary hormone prolactin results from increased transcription of the casein gene but also from stabilisation of its mRNA. In fact, the stabilisation of mRNA is an essential component of the rapid build up of casein protein and this sort of regulation is evident in many situations in which the production of a particular protein needs to be increased to a high level.The mechanism is not well understood. The poly A tail protects the mRA from 3’ degradation. Histone mRNA (histones are produced during the DNA synthetic phase of the cell cycle) do not have a poly(A) tail and are unstable. The sequence of bases in the 3’ untranslated region, especially runs of As and Us can affect the stability of individual mRNAs. However, the enzzymes that break down the RNA are not well characterised.
11 7- Control at initiation of translation 5’ UTR3’ UTRAUGUAASpecific sequences make specific secondary structuresSpecific protein factors bind to these secondary structures
12 Doomed protein molecule ubiquitin protein ligase 8-Regulation by protein stabilityUbiquitin-dependent proteolysis.Protein molecule is tagged for degradation by attachment of a 20 kDa protein, ubiquitinATPCONHNH2+COOHCONH26S proteasomeDoomed protein moleculeubiquitin protein ligaseProtein stability. Ubiquitin system.20 kda protein ubiquitin is activated by ATPIt is linked by its C terminus to amino group on a lysine side chain in target protein.Enzyme is ubiquitin protein ligase.Up to 50 molecules of ubiquitin / target protein molecule.Ubiquitinylated protein molecule then degraded by proteosome. A large multiprotien complex (2000 kDa)The level of cell cycle regulatory proteins called cyclins are produced at the G1 to S phase boundary of the cell cycle. The proteins stimulate kinases which trigger DNA synthesis. Once this triggering has occurred, there is no further need for the cyclins and they are degraded by the ubiquitin system. This system allows for rapid removal of proteins. It is selective. Another method for degrading proteins, the lysozome is non-selective.It was originally thought that breakdown of proteins occurred in the lysozomes. However, reticulocytes which do not have lysozomes still break down abnormal proteins. The system involves a 76 amino acid residue protein called ubiquitin, because it is widespread in eukaryotic spp. It is also one of the most conserved proteins known, differing in only 3 AA between human and fruit fly.Attachment is to the C terminus of ubiquitin and this is transferred to the amino group of a lysine side chain of the protein. Many ubiquitins per target protein molecule.Proteasome is a 20 s/u multi protein complexcontaining at least 5 different proteolytic activities in the shape of a bi-capped hollow barrel. UBIQUITIN IS NOT DEGRADED.蛋白酶体系统(ubiquitin-proteasome system(UPS))主要由泛素激活酶(E1)、泛素交联酶(E2)、泛素连接酶(E3)和26S蛋白酶体组成,是降解细胞内蛋白质的主要途径对于许多细胞进程，包括细胞周期、基因表达的调控、氧化应激反应等，都是必不可少的。2004年诺贝尔化学奖.
13 Similarity of regulation between eukaryotes and prokaryote 1.Principles are the same:signals (信号),activators and repressors (激活蛋白和阻遏蛋白)recruitment and allostery, cooperative binding (招募，异构和协同结合)2. The gene expression steps subjected to regulation are similar, and the initiation of transcription is the most pervasively regulated step.
14 Difference in regulation between eukaryotes and prokaryote***** Pre-mRNA splicing adds an important step for regulation. (mRNA前体的剪接)The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart. (真核转录机器更复杂)Nucleosomes and their modifiers influence access to genes. (核小体及其修饰体)Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes. (真核基因有更多结合位点)
15 A lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integrationBacteriaYeastHuman
16 Core promoter Promoter Cis-acting elementPromoter Core promoter in eukaryote: TATA-box, Initiator (Inr) in prokaryote: -10 region, Inr Proximal elements of promoter in prokaryote: region in eukaryote: CAAT-box, GC-boxUPE: upstream promoter elementUAS: upstream activating sequenceTerminator (终止子)：A DNA sequence just downstream of the coding segment of a gene, which is recognized by RNA polymerase as a signal to stop transcription.
17 Enhancer (激活元件) : a given site binds regulator responsible for activating the gene. Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals. Activation at a distance is much more common in eukaryotes.Silencer （沉默子）A DNA sequence that helps to reduce or shut off the expression of a nearby gene.Insulators (绝缘子) or boundary elements (临界元件) are regulatory sequences between enhancers and promoters. They block activation of a linked promoter by activator bound at the enhancer, and therefore ensure activators work discriminately.
20 What is trans-acting factor? Usually they are proteins, that bind to the cis-acting elements to control gene expression.
21 These trans-acting factors can control gene expression in several ways: may be expressed in a specific tissue may be expressed at specific time in development may be required for protein modification may be activated by ligand binding
23 (3) Domains of trans-acting factors DNA binding domain DBDDNA结合结构域 transcription activating domain转录活化结构域
24 Topic 1: Conserved Mechanisms of Transcriptional Regulation 一、真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activatorsTopic 1: Conserved Mechanisms of Transcriptional Regulationfrom Yeast (酵母) to Mammals (哺乳动物)
25 The basic features of gene regulation are the same in all eukaryotes, because of the similarity in their transcription and nucleosome structure.Yeast is the most amenable to both genetic and biochemical dissection, and produces much of knowledge of the action of the eukaryotic repressor and activator.The typical eukaryotic activators works in a manner similar to the simplest bacterial case.Repressors work in a variety of ways.
26 Gal4 bound to its site on DNA 1. Eukaryotic activators (真核激活蛋白) have separate DNA binding and activating functions, which are very often on separate domains of the protein.*****Gal4 bound to its site on DNA
27 Eukaryotic activators---Example 1: Gal4***** Gal4 is the most studied eukaryotic activatorGal4 activates transcription of the galactose genes in the yeast S. cerevisae.Gal4 binds to four sites (UASG) upstream of GAL1(5'-CGGRNNRCYNYNYNCNCCG-3' ), and activates transcription 1,000-fold in the presence of galactoseThe regulatory sequences of the Yeast GAL1 gene.
28 Experimental evidences showing that Gal4 contains separate DNA binding and activating domains. Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription.Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor, LexA activates the transcription of the reporter gene. Domain swap experiment
29 Domain swap experiment Domain swap experiment***** Moving domains among proteins, proving that domains can be dissected into separate parts of the proteins. Many similar experiments shows that DNA binding domains and activating regions are separable.
30 Box1 The two hybrid Assay (双杂交) to study protein-protein interaction and identify proteins interacting with a known protein in cells*****Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4, respectively.Produce fusion proteins
36 Transcription factor motifs 2. Eukaryotic regulators use a range of DNA binding domains, but DNA recognition involves the same principles as found in bacteria. *****Helix-turn-helix ( HTH) (螺旋-转角-螺旋)Zinc finger （锌指）and zinc clusterLeucine zipper motif（亮氨酸拉链）Helix-Loop-Helix proteins （螺旋-突环-螺旋）: basic zipper and HLH proteinsTranscription factor motifs
38 Bacterial regulatory proteins Most use the helix-turn-helix (HTH：旋转-转角-旋转） motif to bind DNA targetMost bind as dimers to DNA sequence: each monomer inserts an a helix into the major groove.Eukaryotic regulatory proteinsRecognize the DNA using the similar principles, with some variations in detail.In addition to form homodimers (同源二聚体), some form heterodimers (异源二聚体) to recognize DNA, extending the range of DNA-binding specificity.
43 3. Activating regions (激活区域) are not well-defined structures*** The activating regions are grouped on the basis of amino acids content.Acidic activation region (酸性激活区域): contain both critical acidic amino acids and hydrophobic aa. yeast Gal4Glutamine-rich region (谷氨酰胺富集区): mammalian activator SP1Proline-rich region (脯氨酸富集区): mammalian activator CTF1
44 Eukaryotic Activators 二、真核转录激活蛋白的招募调控方式和远距调控特征Activation of the eukaryotic transcription by recruitment & Activation at a distanceTopic 2: Recruitment ofProtein Complexesto Genes byEukaryotic Activators
45 Eukaryotic activators (真核激活蛋白) also work by recruiting (招募) as in bacteria, but recruit polymerase indirectly in two ways: 1. Interacting with parts of the transcription machinery. 2. Recruiting nucleosome modifiers that alter chromatin in the vicinity of a gene.
46 1. Activators recruit the transcription machinery to the gene. The eukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes, such as the Mediator and the TFⅡD complex. Activators interact with one or more of these complexes and recruit them to the gene.
47 Chromatin Immuno-precipitation (ChIP) (染色质免疫共沉淀) to visualize where a given protein (activator) is bound in the genome of a living cell*****.)
48 Two types of Nucleosome modifiers : 2. Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome modifiers :Those add chemical groups to the tails of histones (在组蛋白尾上加化学基团), such as histone acetyl transferases (HATs)Those remodel the nucleosomes (重塑核小体), such as the ATP-dependent activity of SWI/SNF.
49 How the nucleosome modification help activate a gene?***** “Loosen” the chromatin structure by chromosome remodeling and histone modification such as acetylation, which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome.
50 Local alterations in chromatin structure directed by activators capable of bindingto their sites onDNA within anucleosome areshown boundupstream of apromoter thatis inaccessiblewithin chromain.The activator is shown recruitinga histone acetylase. That enzymeadds acetyl groups to residues withinthe histone tails. This alters thepacking of the nucleosomessomewhat, and also creates bindingsites for proteins carrying theappropriate recognition domains.(b)The activator recruits anucleosome remodeller, whichalters the structure ofnucleosomes around thepromoter, rendering itaccessible and capable ofbinding the transcriptionmachinery.
51 3. Action at a distance: loops and insulators Specific cis-acting elements called insulators (绝缘子) control the actions of activators, preventing the activating the non-specific genes
52 Insulators block activation by enhancers. a) A promoter activatedby activators boundto an enhancer.b) An insulator isplaced between theenhancer and thepromoter. Whenbound by appropriateinsulator- bindingproteins, activationof the promoter by theenhancer is blocked,despite activatorsbinding to the enhancer.c) The activator canactivate anotherpromoter nearby.d) The original promotercan be activated byanother enhancerplaced downstream.Insulators block activation by enhancers.
53 Transcriptional Silencing (转录沉默) Transcriptional Silencing is a specialized form of repression that can spread along chromatin, switching off multiple genes without the need for each to bear binding sites for specific repressor.Insulator elements can block this spreading, so insulators protect genes from both indiscriminate activation and repression。
54 4 Appropriate regulation of some groups of genes requires locus control region (LCR). Human and mouse globin genes are clustered in genome and differently expressed at different stages of developmentA group of regulatory elements collectively called the locus control region (LCR), is found kb upstream of the cluster of globin genes. It binds regulatory proteins that cause the chromatin structure to “open up”, allowing access to the array of regulators that control expression of the individual genes in a defined order.
55 Regulation by LCRs The human globin genes and the LCR that ensures their orderedexpression.(b) The globin genesfrom mice, which arealso regulated by anLCR.(C) The HoxD genecluster from themouse controlled byan element calledthe GCR which likethe LCRs appears toimpose orderedexpression on thegene cluster.Regulation by LCRs
56 Topic 3: Transcriptional Repressor & its regulation 三、真核转录阻遏蛋白（或抑制蛋白）及其调控Topic 3: TranscriptionalRepressor & its regulationIn eukaryotes, most repressors do not repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase. (Bacteria often do so)
57 Commonly, eukaryotic repressors recruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers, histone deacetylases (组蛋白去乙酰化酶) removing the acetyl groups]. Some modifier adds methyl groups to the histone tails, which frequently repress the transcription. This modification causes transcriptional silencing.
58 Three other ways in which an eukaryotic repressor works include: Competes with the activator for an overlapped binding site.Binds to a site different from that of the activator, but physically interacts with an activator and thus block its activating region.Binds to a site upstream of the promoter, physically interacts with the transcription machinery at the promoter to inhibit transcription initiation.
59 Competes for the activator binding Inhibits the function of the activator.Ways in which eukaryotic repressor work
60 Binds to the transcription machinery Recruits nucleosome modifiers (most common***)
61 A specific example: Repression of the GAL1 gene in yeast In the presence of glucose, Mig1 binds to a site between the USAG and the GAL1 promoter, and recruits the Tup1 repressing complex. Tup1 recruits histone deacetylases, and also directly interacts with the transcription machinery to repress transcription.
62 四、基于真核转录调控的前沿学科：信号传导 Signal transduction---A life science frontier centered on the eukaryotic transcriptional regulation.Topic 4: Signal Transduction (信号传导) and the Control of Transcriptional Regulators
63 1. Signals are often communicated to transcriptional regulators through signal transduction pathway 信号经常通过信号传导途径被运输到转录调节蛋白
64 Environmental Signals/Information (信号) 1 Environmental Signals/Information (信号) 1. Small molecules such as sugar, histamine (组胺). 2. Proteins released by one cell and received by another. In eukaryotic cells, most signals are communicated to genes through signal transduction pathway (indirect), in which the initiating ligand is detected by a specific cell surface receptor.
65 Signal transduction pathway*** 1. The initial ligand (“signal”) binds to an extracellular domain of a specific cell surface receptor2. The signal is thus communicated to the intracellular domain of receptor (via an allosteric change or dimerization )3*. The signal is then relayed (分程传递) to the relevant transcriptional regulator.4. The transcriptional regulator control the target gene expression.
66 JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity, allowing trans-phosphorylation.The activated JAKs subsequently phosphorylate additional targets, including both the receptors and the major substrates, STATs.STATs are latent transcription factors that reside in the cytoplasm until activated.
67 MAP kinases are activated within the protein kinase cascades called “MAPK cascade”. Each one consists of three enzymes, MAP kinase, MAP kinase kinase (MKK, MEK, or MAP2K) and MAP kinase kinase kinase (MKKK, MEKK or MAP3K) that are activated in series.A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues, and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2).
69 2. Signals control the activities of eukaryotic transcriptional regulators in a variety of ways. 信号通过不同方式控制转录调节蛋白的活性
70 Mechanism 1: unmasking an activating region: A conformational change to reveal the previously buried activating region.Releasing of the previously bound masking protein. Example: the activator Gal4 is controlled by the masking Gal80).Some masking proteins not only block the activating region of an activator but also recruit a deacetylase enzyme to repress the target genes. Example: Rb represses the function of the mammalian transcription activator E2F in this way. Phosphorylation of Rb releases E2F to activate the target gene expression***.
71 Activator Gal4 is regulated by a masking protein Gal80***
72 Mechanism 2: Transport into and out of the nucleus: When not active, many activators and repressors are held in the cytoplasm. The signaling ligand causes them to move into the nucleus where they activate transcription
73 Other Mechanisms #1: A cascade of kinases that ultimately cause the phosphorylation of regulator in nucleus (new) (Fig.19-4a).
74 Other Mechanisms #2: The activated receptor is cleaved by cellular proteases (蛋白酶), and the c-terminal portion of the receptor enters the nuclease and activates the regulator (new):(Fig.19-4c).