The Regulation of Eukaryotic Gene Expression

1
The Regulation of Eukaryotic Gene Expression

• ..using the example of PEPCK

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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

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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)

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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.

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PEPCK overexpression in muscle

• The youtube video
• http//www.youtube.com/watch?v4PXC_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.

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The Supermouse.

• Eats 60 more food than wild type mice
• Weighs 40 less than wild type mice
• Can run for gt4 h until exhaustion whereas the
  control littermates stop after only 10 min
• Has 2 3 fold less adipose tissue

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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.

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PEPCK overexpression in adipose tissue

• A less famous cousin mouse has the PEPCK enzyme
  overexpressed in adipose tissue.
• The results couldnt be further from supermouse!

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PEPCK overexpression in fat cells
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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.

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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.

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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
  lethalmice die within days of birth.

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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.
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The reaction!
Phosphoenol pyruvate
Oxaloacetate
15
Where does it fit in?
Glucose
Gluconeogenesis
Glycolysis
PEP
Pyruvate
OAA
LDH
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Glyceroneogenesis
PEPcarboxykinase
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Glyceroneogenesis
PEP
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PEPCK gene
TATA
CRE
TRE
GRE
PPARRE
IRE
-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

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Activating PEPCK activity in liver during
starvation

• Lets 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

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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

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Activating PEPCK activity in liver during
starvation

• The formation of the cortisolreceptor 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)

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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).

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Activating PEPCK activity in liver during
starvation

• It is thought that both the cortisolreceptor
  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.

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PEPCK gene
TATA
CRE
TRE
GRE
PPARRE
IRE
-300
-100
-400
-1000
Promoter and regulatory region
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blood
cytoplasm
nucleus
Cortisol binds to its receptor, exposing the NLS
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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?

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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 cortisolreceptor dimer and some
  HNFs bound.
• With different adipocyte specific nuclear factors
  you can get the reverse result.

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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)

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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)

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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)

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PEPCK gene
TATA
CRE
TRE
GRE
PPARRE
IRE
-300
-100
-400
-1000
Promoter and regulatory region
33
glucagon
Blood
Liver cytoplasm
Adenylyl cyclase
G protein
Glucagon receptor
GDP
Nucleus
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Glucagon binds to receptor
Blood
Liver cytoplasm
Adenylyl cyclase
GTP
GDP
Nucleus
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Adenylyl cyclase
Glucagon binds to receptor
Blood
Liver cytoplasm
GTP
ATP
cAMP
Nucleus
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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

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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
  dont 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 CANT

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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

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Summary Transcriptional Regulation of PEPCK

• Glucagon, a peptide hormone up-regulates PEPCK
• Glucagon cant 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

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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.

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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

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(No Transcript)
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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 3UTR 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.

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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

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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 dont even know the natural ligand for PPARg
  although the favoured candidates are fatty acids
  and their derivatives, in particular
  polyunsaturated fatty acids.

48
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.

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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

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Fat mice who are metabolically healthy
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Pharmaceutical applications TZDs

• act to up-regulate PEPCK synthesis in adipocytes,
  thus increase glyceroneogenesis ? more
  repackaging of FFAs in the adipocyte ? less FFAs
  in serum
• Stimulate adipogenesis (differentiation of new
  fat cells from fibroblasts) thus increasing the
  storage for FFAs and again lowering FFAs in serum.

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Implications of TZD treatment

• The patient may actually put on weight as
  adipogenesis is stimulated
• BUT the fat cells will be able to contain the
  FFAs and stop the release into the bloodstream.
• The increase in PEPCK activity will improve the
  fat storage in the adipocyte.

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Obesity other areas

• As well as elevated FFAs obese adipose tissue is
  often characterised by macrophage infiltration.
• Obesity is now considered to be a low grade,
  chronic inflammatory condition.
• The inflammatory response may account for the
  cardiovascular and diabetic symptoms associated
  with most sufferers.

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Obesity

• There is a strong link between nutrient sensing
  and pathogen sensing in an organism
• There has been very strong selection for
• strong immune response
• The ability to process and store energy
• In times of chronic nutrient overload the immune
  response may become overly sensitive

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Obesity other areas

• Some recent treatments for type-2 diabetes
  associated with obesity involve treating patients
  with anti-inflammatory drugs to reduce the
  inflammatory effects and so lessen the type 2
  diabetic symptoms.

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Obesity other areas

• Anti-TNF alpha treatments such as infliximab
  (often prescribed for rheumatoid arthritis and
  other inflammatory diseases) and even salicylic
  acid derivatives are being trialled.
• Metformin, the most commonly prescribed insulin
  sensitising drug, suppresses gluconeogenesis by
  inhibiting the expression of PEPCK and G6Pase

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For the final exam.

• ELMA will NOT be examined
• Material from the labs after the ELMA will be
  examined
• Beta galactosidase induction (gene expression)
• Protein purification

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For the final exam.The BCHM contribution

• All material covered in my lectures and Gareths
  lectures will be examined.
• I will place some reading material on the web and
  send it to your usyd email address. This material
  will also appear in the exam.