Gene-L14-Transcription VI: Signalling pathways & transcription

what is transcription regulated at RNA pol II promtor? how does it occur?

1. GTFs bind first and RNA pol II is recruited and positions DABFEH

2. activator proteins bind to regulatory DNA elements- enhancers promtors

3. cofactors and mediators- transmit signals from activators to the transcription machinery

regulation:

• activators DNA binding regulatory proteins

• cofactors- signal integration and bridging

• chromatin state

• PIC assembly and stability

how are transcription factors activated in cells? 3

• many are inducible- latent until activated1.

• signal transduction pathways- phosphorylation cascades

• direct activation at cell surface

• de novo synthesis

• translocate to the nucleus and bind enhancers/promtoros

how does a cell decide which genes to upregulate or down regulate in response to changing conditions?

• cells detect environmental or internal changes by signalling pathways

• these pathways can activate specific TFs

• activated TFs bind to gene specific enhancers and promotors- leads to selective gene expression

2 examples of immune/nutrient signalling and how this regulates gene uptake?

• detected by cellular sensors/receptors

• activates phosphorylation cascades

• IRF3 becomes phosphorylates- dimerises and translocates to the nucleus

• IRF3 activates transcription of IFN, pr inflammatory genes, antiviral response genes

• changes in nutrient levels- activate insulin receptor signalling pathways

• alters transcription

• regulates GLUT transporters for glucose uptake and glycogen storage

what are inducible TFs and how do they regulate gene expression? how do they exist, how’re they activated? what do they do?

• ITFs- allow cells to regulate transcription in response to environmental or signalling changes

• exist in latent forms in the cytosol or need to be synthesised- need additional signals to be activated

• they’re activated by signal transduction pathways or can be activated at receptor levels

• once activated they bind to enhancers and regulatory DNA elements and modify transcription of target genes

• IFs connect cell signalling and gene response

what are the main ways inducible transcription factors can be activated? 7

• Protein synthesis - the TF is made de novo in response to signs;

• Ligand binding - binding of a ligand activates the TF

• Covalent modification - e.g. phosphorylation activates the TF

• Addition of a second subunit - e.g. dimerisation

• Unmasking - removal of an inhibitory protein, e.g. NF-κB after loss of IκB

• Stimulation of nuclear entry

• Release from a membrane

what are common transcriptional regulatory loops- how do they control gene expression? 4

1. pos feedback loop

• gene a makes protein a

• protein a acts as TF and activates its own gene- more protein A made

2. neg feedback

• gene a makes protein A

• protein A represses its own gene

• reduces A

3. flip flop

• A repressed B when normally B represses A

• repressing B- strengthens A expression

4. feedforward

• TF a activates TF B

• A and or B then activate Z

how are glucocorticoid receptors activate to regulate transcription?

• steroid hormone glucocortioids and androgens are lipophilic- cross the plasma membrane

• bind intracellular receptors- nculear

• glucocorticoid receptor: ligand binding causes release from HSP90 chaperone complex

• causes dimerisation and translocation to the nucleus

• binds specific DNA sequences= hormone response eements

how does phosphorylation regulate transcription factor activity?

(1) Translocation to the nucleus- promotes or inhibits nuclear entry
– STAT1 and IRF3/7 - translocation and subunit interactions.
– NFAT: Regulates IL2 gene, also sites in IL4, TNFa, IL3 genes
• Induced by increasing intracellular Ca2+ flux.
• Ca2+ activates Ca2+/calmodulin-dependent phosphatase, dephosphorylates. NFAT is dephosphprylated and can nuclear translocation
• (2) DNA binding
– c-Myb: Phosphorylation by CK II decreases DNA binding - oncogenic activation if mutated
occurs when the phosphorylation site is mutated
– c-Jun: Dephosphorylation enhances DNA binding and subsequent txnl
activation.
• (3) Transactivation -

• increased by phosphorylation

• once bound to enhancer seuqnece- phosphorylation can enhance activation domain
  - NFkB, cJun, cMyc, CREB

how is IRF3 activated?

• viral dsDNA detected by cGAS- CPG systolic DNA sensor

• activates signalling cascade TBK1 kinase activation

• TBK1- phosphorylates IRF3- dimerises and translocated to the nucleus

• in the nucleus- binds ISRE- interferon stimulated response elements

• activate transcription of IFNs, pro inflammatory genes

how does IFNalpha/beta pathway activate STAT? how does IFN gamma do this?

• FNAalpha/beta bind to a shared receptor which activates JAK1 and Tyk2 kinases

• JAKs phoshphporylate STAT 1 and 2

• STAT1-2 heterodimer is made and IRF9 p48- makes the ISGF3 complex which is a TF

• ISGF3- translocates to the ncuelus

• binds ISRE- and antiviral genes ar transcribed by STAT1/2/IRF9=ISGF3

IFNgamma- activates JAK1/2- phosphorylate STAT1

• STAT1-jomodimer- translocates to the ncuelus

• binds GAS- distinct immune gene expression from ISRE pathway

how is NFKB activated and what regulates it nuclear translocation?

• NFKB is a stress/infection response TF

• inactive when NFKB(p65/50 dimers) is bound to IKBalpha in the cytosol

• stimuli by infections/cytokines/stress- activate the IKK complex

• IKK phosphorylates IKBalpha- IKBalpha is ubiqutinated by E3 ligase and degraded

• NFKB is released and binds to kb motif

how is NFKB activity measured?

1. loss of IKBalpha- it is phosphorylated if NFKB has been inactivated

2. p65 nuclear translocation- active nfkb has a p50/65 dimer- when activated moves from the cytosol to the nucleus so if p65 in the nculeus it is active

what is CBP/p300?

• a coactivator bridge- connects TF to RNA pol II machinery

• binds to NFKB, STATS, AP1 etc

• for NFKB: p65 subunit binds CBP/p300 and enhanced by phosphorylation of ser276 on p65

• CBP/p300 also binds TBP and TFIIB- helps bridge activators→basal transcription machinery and has histone acetylytransferase activity