Computational Biology I LSM5191

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1 Computational Biology I LSM5191 Aylwin Ng, D.Phil Lecture 6 Notes: Control Systems in Gene Expression

2 Pulling it all together: coordinated control of transcriptional regulatory molecules

3 Simple Control: Lactose (lac) Operon in bacteria E. coli can use glucose or lactose as source of carbon and energy. In lactose-containing medium induction of lactose-metabolizing enzymes syn. In glucose-containing medium repression 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.

4 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 receptors (also known as nuclear receptors, e.g. Glucocorticoid receptors) in the cytoplasm. DNA binding Hormone-binding Glucocorticoid receptor

5 Absence of cortisol: Glucocorticoid receptor (GR) is bound in a complex with Hsp90 (heat-shock chaperon ) in the cytoplasm. Upon interaction with 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. LBD: Ligand-Binding Domain DBD: DNA-Binding Domain AD: Activation Domain Glucocorticoid Response Element

6 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 intracellular proteins. This constitutes the 1 st step in the intracellular stage of Signal Transduction. Receptor OUT Cell membrane IN Signaling cpd. P- phosphorylates protein Signal Transduction

7 Direct Signal Transduction Stimulation of cell surface receptor direct activation of a protein that influences transcription activity in nucleus. 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

8 e.g. 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

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

10 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).

11 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

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

13 MAP kinase signaling cascades

14 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

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

16 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

17 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

18 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.

19 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

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

21 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

22 Modeling genetic networks / circuits Modeling synthetic gene circuits derived from the bacteriophage λ gene expression control mechanism. Hasty J. et. al., 2002, Nature 420: Savageau M.A., 1974, Nature 252: Diagram adapted from Hasty et. al.

23 Interactome Complexity is achieved beyond the DNA level, through complex networks of gene expression control and interactions between gene products.