The Regulation of Eukaryotic Gene Expression

1. The Regulation of Eukaryotic Gene Expression ..using the example of PEPCK

2. PEPCK • This is an acronym for an enzyme • PhosphoEnol Pyruvate CarboxyKinase • This enzyme is ONLY regulated by gene expression! • No allosteric activators, covalent modification etc • No activation by cAMP, inhibition by insulin etc

3. PEPCK • The enzyme is expressed in liver, kidney, adipose tissue and to a lesser extent in muscle • It is a key enzyme in gluconeogenesis (the synthesis of new glucose, usually from lactate, pyruvate or alanine) and glyceroneogenesis (the synthesis of glycerol, usually from lactate, pyruvate or alanine)

4. Why choose PEPCK? • It is an enzyme. Why would this be good? • It is not post-translationally regulated. Why would this be good? • A number of hormones influence gene expression in different tissues.

5. PEPCK overexpression in muscle • The youtube video • http://www.youtube.com/watch?v=4PXC_mctsgY • is of a mouse with PEPCK overexpressed in muscle only. • This mouse hit the popular press in 2007 and put Case Western Reserve University in Cleveland Ohio on the map! • Earl Sutherland, the discoverer of cAMP also hailed from Case Western.

6. The Supermouse…. • Eats 60% more food than wild type mice • Weighs 40% less than wild type mice • Can run for >4 h until exhaustion whereas the control littermates stop after only 10 min • Has 2 – 3 fold less adipose tissue

7. PEPCK overexpression in muscle • This mouse was leaner than wild type mice, ran for longer and lived longer! • They were also more aggressive. • The overexpression had switched the muscle fuel usage to fatty acids with little lactate production.

8. PEPCK overexpression in adipose tissue • A less famous cousin mouse has the PEPCK enzyme overexpressed in adipose tissue. • The results couldn’t be further from supermouse!

9. PEPCK overexpression in fat cells

10. PEPCK overexpression in adipose tissue • These mice are obese although metabolically healthy (as measured by glucose tolerance and insulin sensitivity) until you put them on a high fat diet. • Then you see insulin resistance and diabetes emerging.

11. PEPCK overexpression in liver • Leads to altered glucose tolerance • Insulin resistance, NIDDM • Increased gluconeogenesis causes increased hepatic glucose production which is released into the blood stream • This caused increased insulin secretion but ultimately insulin resistance.

12. PEPCK Knock out in liver • Surprisingly these mice can maintain blood glucose under starvation conditions • They develop liver steatosis (fatty livers) probably because of impaired oxidation of fatty acids • A total PEPCK knock out in all tissues is lethal…mice die within days of birth.

13. Why the dramatically different outcome for the mouse when PEPCK is overexpressed in different tissues?It is after all the same enzyme catalysing the same reaction.

14. GTP GDP The reaction! Phosphoenol pyruvate Oxaloacetate

15. Where does it fit in? Glucose Gluconeogenesis Glycolysis PEP Pyruvate OAA LDH

16. Glyceroneogenesis PEPcarboxykinase

17. Glyceroneogenesis PEP

18. cAMP response element Glucocorticoid response element PPAR response element Insulin response element Thyroid response element PEPCK gene TATA PPARRE IRE GRE TRE CRE -300 -100 -400 -1000 Promoter and regulatory region

19. PEPCK regulation in liver • PEPCK activity is highest in liver during starvation • Glucocorticoids such as cortisol and glucagon both activate the expression of the PEPCK gene in liver • The glucocorticoids are steroid hormones whereas glucagon is a peptide hormone

20. Activating PEPCK activity in liver during starvation • Let’s consider the glucocorticoid response first. • Cortisol is the active glucocorticoid hormone. • Pharmaceutical analogues are cortisone (converted to cortisol by a dehydrogenase) and the synthetic analogues prednisone and dexamethasone • Often administered for their immunosuppressive properties

21. Activating PEPCK activity in liver during starvation • Cortisol is produced and released by the adrenal gland….it travels through the circulation and can pass through the cell plasma membrane (unlike peptide hormones) • Once inside the cell it binds to a cytosolic receptor in specific cells

22. Activating PEPCK activity in liver during starvation • The formation of the cortisol:receptor complex exposes a nuclear localisation signal • The complex moves to the nucleus • It binds as a dimer to the glucocorticoid response element (a sequence of DNA upstream of a number of genes including PEPCK)

23. Activating PEPCK activity in liver during starvation • The binding of this complex greatly enhances the frequency of initiation of the basal transcription apparatus (RNA pol II with all the bits). • Other protein factors (coactivators) also bind. These factors reside in the nucleus of liver cells and are known as hepatic nuclear factors (HNFs).

24. Activating PEPCK activity in liver during starvation • It is thought that both the cortisol:receptor complex and one or more of the HNFs need to be bound for effective enhancement. • This is important for the tissue specific nature of the PEPCK up-regulation.

25. cAMP response element Glucocorticoid response element PPAR response element Insulin response element Thyroid response element PEPCK gene TATA PPARRE IRE GRE TRE CRE -300 -100 -400 -1000 Promoter and regulatory region

26. cortisol Cortisol receptor HNFs NLS NLS RNA pol II NLS TATA blood cytoplasm nucleus Cortisol binds to its receptor, exposing the NLS

27. Differing response to glucocorticoids in different tissues • While cortisol up regulates PEPCK transcription in the liver. • It down regulates PEPCK in adipose tissue. • The same gene (single copy in the genome) with the same promoter and regulatory regions! How is this possible?

28. PEPCK down regulation by cortisol in adipose tissue • We are not sure! The accepted logic at present is that for effective up regulation in the liver you need both the cortisol:receptor dimer and some HNFs bound. • With different adipocyte specific nuclear factors you can get the reverse result.

29. Activating PEPCK activity in liver during starvation • During starvation glucagon is secreted by the alpha cells of the pancreas (it is synthesised there) • Glucagon is a peptide hormone which cannot cross the plasma membrane • It binds to a cell surface receptor (a G-coupled protein receptor)

30. Activating PEPCK activity in liver during starvation • The binding of glucagon to this receptor causes a conformational change, associations of subunits and ultimately the activation adenylyl cyclase. • This causes an increase in cAMP activates Protein Kinase A  moves to the nucleus  phosphorylates transcription factors (CREBs)

31. Activating PEPCK activity in liver during starvation • The phosphorylated CREBs then bind to the CRE (cAMP response element) site on the DNA •  effective enhancement of PEPCK transcription (amongst other genes you need up regulated in starvation)

32. cAMP response element Glucocorticoid response element PPAR response element Insulin response element Thyroid response element PEPCK gene TATA PPARRE IRE GRE TRE CRE -300 -100 -400 -1000 Promoter and regulatory region

33. R R C C Protein kinase A glucagon Blood Liver cytoplasm Adenylyl cyclase G protein Glucagon receptor GDP Nucleus

34. R R C C Protein kinase A Glucagon binds to receptor Blood Liver cytoplasm Adenylyl cyclase GTP GDP Nucleus

35. P R R C C CREB P R R CREB CREB P CREB C C Adenylyl cyclase Glucagon binds to receptor Blood Liver cytoplasm GTP ATP cAMP Nucleus

36. PEPCK down regulation by Insulin What we know….. • Insulin inhibits the basal PEPCK transcription apparatus • Insulin antagonizes the induction of PEPCK expression by glucagon or glucocorticoids

37. PEPCK down regulation by Insulin • It is thought that intermediates in the insulin signalling pathway are involved. • In spite of all we know about insulin we still don’t know exactly how insulin inhibits the transcription of PEPCK. • It would be nice to say that an intermediate produced by insulin signalling phosphorylated a transcription factor which binds to the IRE…. BUT I CAN’T

38. Summary: Transcriptional Regulation of PEPCK • Use the liver in starvation as the context • PEPCK needs to be up-regulated to make glucose (GLNG) to maintain blood glucose and thus to supply the brain with fuel • In adipose tissue it has the role of making glycerol for the packaging of fatty acids to triglycerides

39. Summary: Transcriptional Regulation of PEPCK Cortisol, a steroid hormone, up-regulates PEPCK Cortisol can enter the cell (because it is hydrophobic enough) where it binds to a cytosolic receptor NLS unmasked  enters nucleus  dimerises  binds to GRE

40. Summary: Transcriptional Regulation of PEPCK • Glucagon, a peptide hormone up-regulates PEPCK • Glucagon can’t enter the cell  binds to G-coupled protein receptor  activates adenylyl cyclase  cAMP↑  binds to Protein kinase A  R subunits dissociate from C subunits  C subunits enter nucleus  phosphorylate CREB  dimerise and bind to CRE

41. Post transcriptional regulation of PEPCK • Glucocorticoids and cAMP also stabilise the PEPCK mRNA in the liver cytoplasm. • Insulin destabilises it. • mRNA stability contributes significantly to the overall up or down regulation of gene expression. • PEPCK is normally very unstable. • mRNA stability is measured by its half life.

42. Why would it be advantageous for an mRNA sequence like PEPCK to be unstable? • If PEPCK is only regulated by gene expression it is difficult to down regulate the sequence at the level of synthesis if the mRNA persists in the cytoplasm. • This also applies to the Trp operon enzymes

43. cytoplasm Translation Processed mature mRNA AAAAAAAAA 3’ AAAAAAAAA 3’ Nucleus 5’ MeG 5’ MeG Processing Primary transcript Transcription DNA

44. PEPCK mRNA stability • A sequence at the 3’ UTR of PEPCK mRNA has been identified which “destabilises” the mRNA. • If that sequence is inserted into the 3’UTR of other more stable mRNAs, such as globin, the half life reduces significantly. • We are yet to determine how cAMP or cortisol stabilises this mRNA.

45. PEPCK gene expression in adipose tissue • Another response element becomes significant, the PPARRE • Peroxisomal Proliferator Activator Receptor (PPAR) Response Element • There in fact 4 PPARs; one of the ones of interest to adipocytes is PPARg, the other is PPAR d • liver has PPARa and PPARg

46. RXR RXR PPARg PPARg PPARg activates the transcription of genes involved with adipogenesis and fat storage cytoplasm Nucleus

47. Pharmaceutical applications • A new group of insulin sensitizers, the thiazolidinediones (TZDs) act on PPARg. • The most commonly prescribed are Rosiglitozone and Piogliterzone • These are artificial ligands for PPARg. • We don’t even know the natural ligand for PPARg although the favoured candidates are fatty acids and their derivatives, in particular polyunsaturated fatty acids.

48. RXR RXR PPARg PPARg TZDs TZDs are artificial ligands for PPARg. These are used as insulin sensitising agents. cytoplasm Nucleus

49. Pharmaceutical applications • They work to sensitize the body to insulin in an interesting way. • Insulin resistance is thought, in part to be brought on by elevated free fatty acids (FFA) in the serum interfering with insulin signalling. • Elevated FFAs are commonly associated with obesity which gives one of the putative links between obesity and insulin resistance.

50. Pharmaceutical applications • Obesity is characterised by lots of large adipocytes which become leaky, hence losing weight is one of the most effective ways of enhancing insulin sensitivity. • There are some mice that, although fat are metabolically healthy (remember the PEPCK mouse) • They have adipocytes that can contain the FFAs