Hormonal Regulation (Lecture 33)

Question 1

Metabolic regulation in complex organisms

Answer 1

Short term regulation (seconds, minutes) = achieved through allosteric control (ie. level
of metabolites) and post-translational modifications (ie. phosphorylation) of enzymes
under hormonal control (eg. catecholamines, insulin)

• Long term regulation (hours, circadian cycle, seasons, development, aging, etc.) =
  requires transcriptional regulation of metabolic genes

Question 2

Transcriptional Control of Metabolism

Answer 2

Requires specific signals to be transduced to nucleus where individual genes or entire gene
networks are targeted for regulation

Question 3

Important Aspects of transcriptional control of specific metabolic responses:

Answer 3

• Events upstream of transcriptional activity => signals involved (eg. glucagon, glucocorticoids) and their route to the nucleus (eg. signalling pathways, protein cleavage, direct activation)
• Molecular mechanisms by which transcription factors regulate gene expression
  Eg. recruitment of polymerase, enzymatic activity of coregulators
• Events downstream of transcription => depend on genes being targeted and which further signals are generated
  Eg. expression of metabolic enzymes or cascade of regulators

Question 4

Metabolic Transcription Factors

Answer 4

Bind DNA and receive signals to tell TFs to activate/repress transcription

Question 5

Nuclear Receptors

Answer 5

family of 48 ligand-responsive “zinc fingers” transcription factors

• Many members work as a metabolic sensor involved in all aspects of metabolism
  (carbohydrate, lipid, amino acid, etc.)
• Most NRs are directly activated by their ligands => hormones will enter nucleus from outside of cell and directly bind receptor
• Can also be regulated by post-translational modifications

Question 6

CREB (cAMP Response Element Binding)

Answer 6

CREM and ATFI

“leucine zipper” TF activated by phosphorylation by PKA in response to increased cAMP levels following glucagon action

• Considered first responders in activation of gluconeogenesis during fasting

Question 7

CEBP (CCAAT-enhancer binding protein)

Answer 7

basic “leucine zipper” TF
- Activity is constitutive
- Signal-independent high expression in liver plays major role in response to
fasting

Question 8

Forkhead Box Proteins

Answer 8

FoxO1, FoxO3, FoxO4, FoxO6, FoxA2)

involved in hepatic (liver) glucose production after nutrient deprivation

• Factors are activated by post-translational modifications such as phosphorylation and acetylation

Question 9

SREBP-1c (sterol response element binding protein)

Answer 9

bHLH-leucine zipper TF that targets genes involved in lipid metabolism

• Involved in cholesterol synthesis
• It is activated by proteolytic cleavage stimulated by sterols and unsaturated FAs

=> dormant in ER, low sterol levels activate protein which is trafficked to Golgi, recruties protease that cleaves twice and sends small part to nucleus for transcriptional activation of genes involved in sterol synthesis

Question 10

hREBP (carbohydrate response element binding protein)

Answer 10

bHLH-leucine zipper TF phosphorylated by PKA

• Responsive to glucose
• Phosphorylated ChREBP via PKA= inactive ChREBP
• Activated by removal of phosphate groups by PP2A phosphatase
• Regulates glucose and lipid metabolism

Question 11

Metabolic Coregulators

Answer 11

• Do not bind DNA, interact with TFs and or other coregulators
• TF binding proteins recognize and bind DNA sequence but coregulators are the moiety which activate or repress gene expression

Question 12

NCOA

Answer 12

nuclear receptor coactivator

Aka SRC, steroid receptor coactivator

Family of 3 members involved in all aspects of metabolism (lipid, carbohydrate, aa)

Question 13

NCOR 1 and 2

Answer 13

nuclear receptor corepressor

Two corepressors that often oppose action of NCOAs and other co-activator proteins

Question 14

RIP140 = aka NRIP1

Answer 14

• Action as repressor decreases mitochondrial biogenesis and oxidative metabolism in muscle
• Especially at rest

Question 15

PGC-1alpha and beta

Answer 15

master co-activators that interact with multiple transcription factors, most predominantly the nuclear receptors PPARs and ERRs

• Required for mitochondrial biogenesis, oxidative metabolism and heat production by brown fat

Question 16

KAT2A and B

Answer 16

lysine acetyltransferase Aka GCN5 and PCAF, which are histone acetyltransferases

• KAT2alpha acetylated PGC-1alpha to decrease its activity and blunting PGC-1alpha
  induced gluconeogenesis

Question 17

HDACs

Answer 17

large family of histone deacetylases with broad roles in metabolic control

• Recruitment of HDAC3 by nuclear receptor Rev-Erb-alpha (clock gene) inhibits expression of lipogenic genes

Question 18

CRTCs

Answer 18

family of 3 proteins that act as coactivators of CREB

Regulate CREB-dependent hepatic gluconeogenic program

Question 19

PRMTI

Answer 19

methylates nuclear receptors,

• in particular HNF-4alpha

Question 20

SRTI

Answer 20

a sirtuin, NAD+-dependent deacetylase that targets PGC-1alpha, ERRalpha and other coactivators

Affect mitochondrial function

Question 21

Nuclear Receptors

Answer 21

Are regulators of metabolism

Protypic TF to study gene regulation

Clinically relevant =>Targets of several drugs to control metabolism

Some are in cytoplasm, bind hormones and move to nucleus

Others are already bound to DNA in nucleus, awaiting hormonal signalling

Recruit coactivators which change expression of target genes

Transcribe genes that will affect metabolic activity

Nuclear receptors and other TFs do not have enzymatic activity; their function is to
recruit co regulatory proteins on DNA to activity transcription

Question 22

what are the ligands for nuclear receptors

Answer 22

hormonal ligans and metabolic ligands

Question 23

DHT

Answer 23

male steroid hormone, anabolic steroid for building muscle/strength

Question 24

Estrogen

Answer 24

female steroid hormone, involved in metabolic pathway, preparing for development of embryo, fat metabolism, change in metabolism associated with estrogen decrease at menopause often leads to weight gain

Question 25

cortisol

Answer 25

corticosteroid expressed in all tissues and regulate glucose | metabolism via glucocorticoid receptor

Question 26

Thyroid hormone

Answer 26

important in regulating metabolism; hyperthyroidism => high | metabolism, lose weight, opposite can also happen

Question 27

3 main elements of nuclear receptors

Answer 27

DNA. binding domain modulator ligand binding domain

Question 28

modulator function

Answer 28

the modulator is located at the N terminus and functions to modulate receptor activity. this is the target for phosphorylation and it receives signals through phosphorylation and acetylation. a trans activation through coregualttion interaction (AF-1) also occurs at this site

Question 29

DNA binding domain

Answer 29

DNA binding domain recognizes a specific sequence of DNA it is composed of zinc fingers and requires dimerization

Question 30

ligand binding domain

Answer 30

receives signals from ligands when bound to the nuclear receptor can form homo and hetero dimers transactivation (AF-2) where the coavtivators bind to the receptors for coregualtion interaction

Question 31

Nuclear receptors recognize specific DNA sequences near metabolic genes

Answer 31

Nuclear receptors recognizes hormone response element (HRE)

Question 32

methods of recognizing the HRE

Answer 32

Monomer can recognize extended half site Dimers can recognize two sites Homodimers act on inverted repeats Heterodimers act on direct repeats

Question 33

core motifs

Answer 33

AGGTCA (TGCCT) AGAACA (TGTTCT)

Question 34

Genome-wide identification of target genes

Answer 34

Must identify target genes of receptors Stimuli activate certain receptors => can observe mitochondrial biogenesis, fatty acid oxidation, oxphos, etc.

Question 35

when suing ChIP- sequencing (chromatic immunoprecipitation) to identify genome wide targets, the following stops are carried out:

Answer 35

(1) Extraction of DNA with factor bound (2) Fix factor to DNA with formaldehyde (3) Cut into small pieces (4) Use antibody against factor of interest, linked to beads (5) Immunoprecipitate beads, beads often magnetic (6) Add linkers to DNA, amplify by PCR, sequence & analyze

Question 36

what can be determined with ERR-alpha

Answer 36

the identification of metabolic pathways allows for the generation of a map of activators and inhibitors

Question 37

cistrome

Answer 37

entire set of binding events for a given transcription factor

Question 38

ChIP sequencing identifies

Answer 38

target genes of transcription factors and their cistrome also shows the binding spots, regulatory regions near metabolic genes that contains sites for TFs this indicated that transcriptional regulation of metabolic genes requires the coordination between several factors

Question 39

how do Nuclear Receptors require coregulators to modulate gene expression?

Answer 39

Nuclear receptors act as activators and repressors of metabolic genes Switch between corepressors and coactivator complexes allows TFs to act both negatively and positively on their targets in response to specific extracellular and intracellular signals ``` Intracellular = metabolite levels Extracellular = hormones ``` Factors involved in exchange of corepressor/activator complexes as well as TF are targets of multiple extracellular and intracellular pathways