Computational Biology I LSM5191

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1 Computational Biology I LSM5191 Aylwin Ng, D.Phil Lecture 4 Notes: Gene Regulation & Control

2 Do all cells in an individual have the same DNA content?

3 PART I: CONTROL OF GENE EXPRESSION

4 REGULATION OF GENE EXPRESSION Any of these stages could be used to regulate expression of specific genes in particular tissues. But in general, the primary control of gene expression is at the level of transcription. DNA Start Exon1 Intron Exon2 Termination *Transcription m 7 Gppp Addition of 5 cap Cleavage & addn of polya tail at 3 end m 7 Gppp A (A) 200 RNA splicing m 7 Gppp A (A) 200 Transport to cytoplasm Translation Protein

5 TRANSCRIPTIONAL CONTROL Regulatory Sequence Elements Short regulatory elements Enhancers or Enhancer Elements Locus control regions Transcriptional activators

6 Short regulatory elements Elements commonly found in many genes e.g. TATA, CCAAT and Sp1 boxes. Elements found only in specific genes, e.g. heat-shock element found only in genes whose transcription is increased in response to elevated temperature. Heat inducible Hsp 70 Non-heat inducible tk Heat-shock element Chimeric gene tk Pelham, 1982, Cell 30: Heat-inducible transcription

7 Sequences present in the upstream region of hsp70 gene also found in other genes Element Name Consensus sequence Other genes containing sequences TATA box TATA A/T A A/T Very many genes. CCAAT box TGTGGCTNNNAGC CAA α- and β-globin, albumin, HSV tk, cellular oncogenes: c-ras, c-myc, etc. Sp1 box GGGCGG Metallothionein IIA, type II procollagen, dihydrofolate reductase, etc. CRE T/G T/A CGTCA Somatostatin, fibronectin, α-gonadotrophin, c-fos, etc. AP2 box CCCCAGGC Collagenase, MHC class 1 antigen H-2K b, metallothionein IIA. Heat-shock consensus CTNGAATNTTCTAG A Heat-inducible genes hsp83, hsp27, etc. Adapted from Latchman, D., 1998, Gene regulation, Stanley Thornes Publ.

8 Enhancer or Enhancer Elements These elements can activate a promoter when placed: up to several kb from promoter, in either orientation relative to promoter, upstream or downstream of the transcribed region, or within introns. Genes exhibiting tissue-specific expression found to contain enhancers. A tissue-specific enhancer can activate the promoter of its own or another gene only in one particular tissue and not others: Enhancer is transferred to unrelated gene Gene active in cell type A, not in cell type B Promoter X transcription unit X enhancer Cell type A Promoter Y Promoter Y transcription unit Y Cell type B Promoter Y enhancer + transcription unit Y enhancer transcription unit Y Enhancer is active; Activates promoter High level transcription Enhancer inactive Low level transcription

9 Enhancer or Enhancer Elements Case example: Tissue-specific expression of Insulin gene in vivo. Insulin gene enhancer element linked to gene encoding large T antigen (Ag) of SV40 virus. This construct introduced into a fertilized mouse egg. Egg returned to oviduct of mouse. Expression of large T Ag analyzed in all tissues of the transgenic mouse (using specific antibody). Expression of large T was detectable only in the pancreas (specifically in the ß cells of the pancreatic islets which produce insulin). Enhancer is therefore capable of conferring the specific pattern of insulin gene expression on an unrelated gene in vivo. Hanahan, D., 1985, Nature 315:

10 Enhancer or Enhancer Elements Enhancer elements often contain sequences (motifs) similar to sequences found adjacent to promoters. Octamer motif (ATGCAAAT) of heavy chain Immunoglobulin (Ig) enhancers is also found in Ig promoters. Possible mechanisms of action: By changing chromatin structure leading to nucleosome displacement, By direct interaction with the proteins of the transcriptional apparatus: (a) (b) (c) Enhancer Enhancer Protein factor binds to Enhancer element & slides along DNA Protein factor binds & contacts apparatus by looping out of intervening DNA Bound protein Enhancer Transcription start site Protein factor binds to Enhancer element & contacts transcriptional apparatus via other proteins

11 Models (a) and (b) cannot explain the following finding: Immunoglobulin enhancer activates equally well 2 promoters located 1.7kb and 7.7kb away on the same DNA molecule Models (a) & (b) would postulate that the sliding of factors or the assembly of connecting molecules would stop at the 1 st promoter. E 1.7kb P1 6kb P Atchison & Perry, 1986, Cell 46: Model (c) readily explains the observation showing the critical importance of DNA structure on the action of enhancers. Region between SV40 enhancer & promoter: Removal of multiples of 10 bases (1 helical turn) Removal of bases corresponding to half a helical turn Activity. Activity disrupted. Takahashi et al., 1986, Nature 319:

12 Enhancer-binding proteins bend DNA Enhancer-binding proteins actually bend the DNA so that interactions can occur between regulatory proteins bound at distant sites on DNA. e.g. T-cell receptor α chain gene enhancer. LEF-1 factor binds to a site at the centre of this enhancer, bends DNA, brings other regulatory factors into close proximity: X X Y Y X LEF-1 X Y Y LEF-1

13 LEF-1 LEF-1 Werner and Burley, 1997, Cell 88:

14 LOCUS CONTROL REGIONS (LCRs) LCRs are sequences (additional to promoters & enhancers) that are necessary for high-level gene expression. Influence expression of adjacent genes in a position-independent manner, i.e. regardless of the position of the genes in the genome. Act in a tissue-specific manner. LCR elements have been identified in α- and β- globin gene clusters, the major histocompatibility (MHC) locus, CD2 and lysozyme genes.

15 LOCUS CONTROL REGIONS (LCRs) In the β-globin gene cluster, LCR is located 10-20kb upstream of the β-globin genes. Its deletion leads to a lack of expression of any genes in the cluster. In Humans, this leads to a lethal disease (Hispanic thalassaemia), in which no functional haemoglobin is produced DNase I sensitivity ε Gγ Aγ ψβ δ β LCR

16 LOCUS CONTROL REGIONS (LCRs) LCRs are rich in sequence motifs also found in promoters & enhancers. LCRs function by affecting chromatin structure: When gene is introduced transiently into cells (I.e. exogenous DNA is not packaged into chromatin) LCR has no effect on gene activity. When gene is an integral part of the chromosome LCR affects gene activity. LCR induces DNase I hypersensitivity in adjacent regions (e.g. β-globin cluster). DNase I hypersensitivity is characteristic of active or potentially active genes. Gene lacking LCR will be subject to the influence of adjacent regulatory elements which might repress its expression by directing its organization into a closed chromatin conformation.

17 TRANSCRIPTIONAL CONTROL Transcription Factors (Transcriptional Activators) Proteins that bind to DNA in a sequence-specific manner and regulate the level of transcription. transcriptional activators have characteristic structural features: Helix-turn-helix motif Zinc finger motif The Leucine zipper References: Travers A., 1993, DNA-protein interactions, Chapman & Hall Ptashne, M. & Gann, A., 2002, Genes & Signals, CSH Lab Press. Ptashne, M , Genes & Regulation, CSH Lab Press. Branden, C & Tooze, J., 2000, Introduction to protein structure. 2 nd Ed, Garland Pub.

18 Transcriptional Activators Genome-wide comparison of transcriptional activator families across Eukaryotes No. of members Transcriptional activator f Adapted from Tupler et al., 2001, Nature 409:832 amilies

19 Helix-turn-Helix motif Many transcriptional activators with this type of DNA-binding domain are called homeodomain proteins. Name derived from a group of Drosophila genes (homeotic genes) in which the conserved sequence encoding this structural motif was 1 st observed. Mutations in these homeotic genes transformation of one body part into another during fly s development. These genes encode regulatory proteins activate or repress activity of other genes encoding proteins req.d for development of certain structures. e.g. The Engrailed (Eng) protein binds the identical sequence recognized by Ftz (another homeodomain protein) and blocks gene induction by Ftz. Eng α-helix α-helix Turn DNA binding Adapted from Harrison, 1991, Nature 353:715

20 Zinc finger motif This motif is common in eukaryotic proteins. Est.d 1% of all mammalian genes code for zinc finger proteins. At least 6 different versions of this motif. The first identified was the Cys 2 His 2 finger. Consensus sequence: Tyr/Phe-X-Cys-X 2-4 -Cys-X 3 -Phe/Tyr-X 5 -Leu-X 2 -His-X 3-4 -His This structure binds one Zn 2+ ion through the 2 Cys and 2 His side chains. The transcriptional activators, TFIIIA (for the gene encoding 5S RNA of the ribosome) was the 1 st to be identified bearing this motif. Cys 2 His 2 Cys Cys Zn His His

21 Cys 4 Zinc finger motif The second type is the Cys 4 zinc finger. Found in more than 100 transcriptional activators. Steroid receptor superfamily or now known as nuclear receptors. 2 groups of 4 critical Cys bind a Zn 2+ ion. Cys 2 His 2 proteins generally contain 3 or 4 repeating finger units and bind to DNA as monomers. Cys 4 proteins generally contain only 2-finger units and bind to DNA as homodimers or heterodimers. The yeast Gal4 protein exhibits the Cys 6 zinc finger motif.

22 Leucine zipper Motif present in many transcriptional activators. Contains the hydrophobic leucine at every 7 th position in the C-terminal portion of their DNA-binding domains. These proteins bind to DNA as dimers. Dimers form via hydrophobic interactions between the C-terminal regions of the α-helices, forming a coiled-coil structure. Hydrophobic side chains form a stripe down one side of the α-helix. Hydrophobic stripes make up the interacting surfaces between the helices in the coiled-coil dimer. L L L L L L L L Basic DNA-binding domain

23 Fos and Jun proteins: Examples of transcriptional activators bearing the leucine zipper motif: Jun can bind as a homodimer to the AP1 recognition sequence, TGAGTCAG, transcriptional induction of phorbol esters. Fos cannot bind to DNA alone, but can form a heterodimer with Jun. Jun-Fos heterodimer binds AP1 with 30-fold greater affinity than Jun homodimer.

24 Modular nature of Transcriptional activators transcriptional activators have modular structures (e.g. with a DNA-binding and activation domains). Classic domain-type structure seen in yeast transcriptional activators GCN4. GCN4 induces genes encoding enzymes of amino-acid (a.a.) biosynthesis in response to a.a. starvation. Expt: 60a.a. region (containing DNA-binding site) introduced into cells binds GCN4-responsive genes but fails to activate transcription. This only confirms the DNA-binding domain. A functional test is needed to identify the activation domain.

25 Identify activation domain: Perform Domain-swap experiment to locate activation domain of FactorA: Link various regions of FactorA to DNA-binding domain of FactorB FactorA FactorB Where is activation domain? DNA-binding domain DNA-binding domain Reporter gene activation Response element & binding site for FactorB Reporter gene

26 An extreme example of ( parasitic ) modularity: Herpes simplex virus (HSV) VP16 protein activates the transcription of viral immediate-early genes during lytic infection. VP16 contains a potent activation region. VP16 contains no DNA-binding domain & therefore cannot bind DNA itself. But following infection, VP16 complexes with cellular Oct-1 protein, which binds the sequence, TAATGARAT, in the viral promoters, and Activation is achieved. VP16 ACTIVATION Oct-1 DNA-binding domain Binding site

27 General features of Activating Domains: Do not show strong a.a. sequence similarity amongst transcriptional activators. But in many cases, activating domains contain a very high proportion of acidic a.a., a region of strong negative charge. E.g. 17 acidic a.a. residues were found in the 82-a.a. N-terminal activating domain of the glucocorticoid receptor. E.g. 17 acidic a.a. residues were found in the 60-a.a. activating domain of GCN4. Hence activating domains also known as Acidic Blobs or Negative Noodles. It has been suggested that activating domains adopt an α-helical or an antiparallel β-sheet conformation.

28 TRANSLATIONAL CONTROL Regulation at the level of translation. Significance of Translational Control: Translational control tends to occur in situations where very rapid responses are required. Translational control viewed as supplementing the regulation of transcription, to meet the requirements of particular specialized cases. E.g. following heat shock, it is necessary to: Shut down rapidly enzyme and structural protein synthesis, Rapidly synthesize heat-shock proteins.

29 TRANSLATIONAL CONTROL Some interesting examples of how control is mediated by untranslated region of mrna: Ferritin expression: Control is mediated by sequences in the 5 untranslated region of ferritin mrna. Sequences in this region can fold into a stem-loop structure. Stem-loop structure is stabilized by the Iron-response-element binding protein (IRE-BP) interacting with this structure. Presence of Iron: IRE-BP binds iron and dissociates from the stem-loop in the process, stem-loop structure unfolds, enhanced translation of gene encoding ferritin. IRE-BP Stem loop + Fe Nascent polypeptide IRE-BP Fe ribosome Start of translation Start of translation

30 TRANSLATIONAL CONTROL Transferin receptor expression: Control is mediated by sequences in the 3 untranslated region (important for the stability) of the transferin receptor mrna. Sequences in this region can also fold into a stem-loop structure. Stem-loop structure is stabilized by the Iron-response-element binding protein (IRE-BP) interacting with this structure. Presence of Iron: IRE-BP binds iron and dissociates from the stem-loop in the process, stem-loop structure unfolds, RNA becomes susceptible to nuclease degradation at a rapid rate. Nascent polypeptide IRE-BP Stem loop 5 3 ribosome + Fe IRE-BP Fe Stem loop unfolds 5 3 RNA is rapidly degraded

31 PART II: CONTROL SYSTEMS in Gene Expression Putting it all together: coordinated control of transcriptional regulatory molecules

32 Simple Control: Lactose (lac) Operon in bacteria E. coli can use glucose or lactose as source of carbon and energy. In glucose-containing medium repression of lactose-metabolizing enzymes syn. In lactose-containing medium induction of lactose-metabolizing enzymes syn. Enzymes induced in the presence of lactose are encoded by the lac operon. lacy gene encodes lactose permease (pumps lactose into cell), lacz gene encodes β-galactosidase (splits lactose glucose + galactose), laca gene encodes thiogalactoside transacetylase (function not well understood). These genes (in the operon) are coordinately regulated. I P O Z Y A Another gene (adjacent to lac control region), laci, encodes the lactose repressor. In the absence of lactose: the repressor binds to the (O)perator, blocking the binding of RNA polymerase to (P)romoter. In the presence of lactose/allolactose: The repressor binds to allolactose, structural change to HLH motif can t bind operator, RNA pol can now gain access to the Promoter syn of lacz,y,a-encoded products.

33 Simple Control: Control by Lipid-soluble Steroid Hormones Steroid hormones are signaling compounds that coordinate a range of physiological activities in eukaryotic cells. Lipid-soluble steroids (e.g. cortisol) can directly penetrate the cell membrane. Once inside cell, steroid hormone interacts with specific steroid receptor (also known as nuclear receptor) protein (e.g. Glucocorticoid receptor) in the cytoplasm. DNA binding Hormone-binding Glucocorticoid receptor

34 In the absence of cortisol, Glucocorticoid receptor (GR) is bound in a complex with Hsp90 (heat-shock chaperon ) in the cytoplasm. Upon interaction with its ligand (cortisol), GR undergoes a conformational change: Hsp90 is released. Receptor with bound cortisol translocates into the nucleus. Receptor functions as a transcription activator. DNA-binding domain (Zn finger motif) binds to the glucocorticoid response element (GRE). Activation Domain stimulates transcription of genes. Glucocorticoid Response Element LBD: Ligand-Binding Domain DBD: DNA-Binding Domain AD: Activation Domain

35 Signaling mediated by cell surface receptors Many other extracellular signaling compounds: Cannot penetrate cell membrane, or Lack specific transport mechanism for their uptake. Signaling is transmitted by binding to specific receptor proteins that span across the cell membrane: Binding conformation change in the receptor, Inducing a series of biochem. events within the cell, e.g. phosphorylation of an intracellular protein. This constitutes the 1 st step in the intracellular stage of Signal Transduction. Receptor OUT Cell membrane IN Signaling cpd. P- phosphorylated protein Signal Transduction

36 Direct Signal Transduction Stimulation of cell surface receptor direct activation of a protein that influences transcription activity. Direct system used by many cytokines (e.g. interleukins and interferons). Binding of cytokines to their cognate receptors: activation of transcriptional activator called STAT (signal transducer & activator of transcription). If receptor is a member of the tyrosine kinase family activate STAT directly i.e. phosphorylation of single tyrosine residue (near C-term) of STAT. If receptor is a tyrosine-kinase-associated receptor activation via JAKs (Janus kinases), which auto-phosphorylate activate STAT. Receptor (Tyr kinase family) Receptor (Tyr kinase-associated) STAT dimerizes activation of JAK involves Dimerization

37 Signal Transduction Pathways activated by Interferon (IFN-γ) IFN-γ is secreted by antigen-activated T- helper lymphocytes. Binding of IFN-γ to its receptor induces oligomerization of the IFN-γ -receptor subunits IFNGR1 and IFNGR2, Phosphorylation and activation of Jak1, Jak2, IFNGR1 and Stat1. Stat1 homodimers translocate to the nucleus, bind to γ-activated sequence (GAS) elements and, regulate gene expression with other transcriptional activators (e.g. BRCA1 and MCM5). Several other signal-transduction pathways are activated also in parallel with the Jak Stat1 pathway in response to IFN-γ (shown in the small box). Concensus for DNA-binding 5 -TTN 5-6 AA-3 Adapted from Ramana et. al., 2002, Trends Immunol, 23:96

38 Numerous genes regulated by IFN-γ in macrophages Adapted from Ramana et. al., 2002, Trends Immunol, 23:96

39 Complex Signal Transduction Cascades Activation of receptor represents just the 1 st in a series of steps that eventually lead to one or more transcriptional activators or repressors being switched on or off. MAP (mitogen activated protein) kinase system A minimal signaling module consists of: a MAP kinase (MAPK), a MAP kinase kinase (MKK or MEK), & a MAP kinase kinase kinase (MKKK or MEKK). Signals are transmitted through the module by sequential phosphorylation and activation of these components arranged in a signaling cascade of ser/threonine kinases. Different groups of MAP kinases are activated by different signaling modules that are composed of distinct protein kinases. Three major groups of MAP kinases have been identified by molecular cloning: the extracellular signal regulated kinases (ERKs), the p38 MAP kinases, & the c-jun amino-terminal kinases (JNKs).

40 Mitogen receptor Signal transduction from a mitogen receptor Activated receptor recruits Raf (a protein kinase), Initiates a cascade of phosphorylations: MEK MAPK Rsk Activated MAPK translocates into nucleus and switches on (by phosphorylating) ELK-1 and c-myc MAPK Rsk activates SRF (serum response factor) by phosphorylation. ELK-1, c-myc & SRF are transcriptional activators. MAPK

41 Mammalian MAP kinase signaling pathways Adapted from Dong et al., 2002, Annu. Rev. Immunol 20:55

42 MAP kinase signaling cascades

43 Complex Signal Transduction Cascades The Ras System Ras family of proteins are important intermediates in signal transduction pathways initiating from activated receptor tyrosine kinases (RTKs). Ligands for RTKs include NGF (nerve growth factor), PDGF (platelet-derived growth factor), FGF (fibroblast growth factor), EGF (epidermal growth factor) and Insulin. Ras is a GTP-binding switch that alternates between active state (with bound GTP) and an inactive state (with bound GDP). [1 & 2] Ras activation is facilitated by guanine nucleotide exchange factor (GEF). GEF facilitates dissoc. of GDP from Ras. [3 & 4] GAP (GTPase-activating protein) accelerates the hydrolysis of bound GTP regenerate inactive Ras.GDP

44 Adapter protein & GEF establish link between RTKs and Ras GEF activity of Sos Second messenger pathways Raf MAP kinase pathway

45 Complex Signal Transduction via second messengers Some signal transduction pathways transfer an external signal to the nucleus using an indirect mechanism via second messengers. Second messengers transduce signals from cell surface receptors in several directions, so that a variety of cellular activities respond to one signal. Second messengers: camp (cyclic AMP), cgmp, IP 3, DAG, calcium ions. Levels of camp or cgmp (regulated by cylase and decylase activities), control the activities of various target enzymes. e.g. camp activates protein kinase A phosphorylates CREB (camp ) interacts with p300/cbp modify histones / nucleosome positioning / affect chromatin structure. e.g. Activation of phospholipases cleave phosphatidylinositol-4,5-bisphosphate (PIP 2 ) to give inositol-1,4,5-trisphosphate (IP 3 ) and 1,2-diacylglycerol (DAG). IP 3 increases intracellular [Ca 2+ ] activates p300/cbp IP 3 increases intracellular [Ca 2+ ] activates calmodulin

46 Complex Signal Transduction via second messengers Phospholipase C cleaves PIP 2 to give IP 3 and DAG. IP 3 increases intracellular [Ca 2+ ], which recruits Protein kinase C (PKC) from cytosol to membrane. At membrane, PKC is activated by DAG. Activated PKC then phosphorylates several cellular enzymes. Animation clip: 2 nd messengers Activation via calmodulin

47 Control of immune cell repression & activation Interplay of the various signal transduction pathways to bring about control Stimulating CD4 + Th1 cells with peptide antigen on MHC class II molecules, but in the absence of co-stimulatory signal via CD28, does not activate Th1 cells, but instead, drive them towards an unresponsive state (anergy). Interleukin-2 (IL-2) production is severely impaired (20-fold decrease). The cause: a block in p21 ras activation (due to either inhibited msos activity or altered Ras-GAP function). decrease in signaling through ERK and JNK pathways, decrease in c-fos and JunB induction, transactivation by AP-1 is diminished, transcription activation of the IL-2 gene impaired.

48 Cross-talk between classical & alternative MAPK pathways thought to occur between GTP-p21ras and GTP-Rac Adapted from Schwartz, 1997, Curr Opin Immunol 9:351

49 Adapted from Gerondakis et al., 1998, Curr Opin Immunol 10:353

50 A model gene expression regulatory network Colored circles represent distinct transcriptional activators. Rectangular ovals represent potential target genes in the genome. The color of the rectangular oval indicates which transcriptional activator is regulating its expression in response to the environmental stimulus; in addition, arrows point from each transcriptional activator to its regulated genes. Note that this model can be thought of as an individual regulatory network or as a collection of regulatory networks. Wyrick JJ & Young RA, Curr Opin Genet Dev 2002, 12(2):130-6