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BioSci 145A Lecture 14 - 2/21/2002
Transcription factors III
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Finish up with identifying regulatory regions
Major families of transcription factors and their functions
– zinc finger genes
• nuclear hormone receptors
– helix-turn-helix
• homeobox genes
– helix-loop-helix
• myogenic genes
– bZIP proteins
•
Additional reading
– Evans (1988) Science 240, 889-895
– Blumberg and Evans (1998) Genes and Development
12, 3149-3155
•
Last year’s final exam is now posted.
– I will post answers in a couple of weeks after you
have had time to work through the questions
BioSci 145A lecture 14
page 1
©copyright
Bruce Blumberg 2000. All rights reserved
Identification of regulatory elements
•
Given a gene of interest, how does one go about studying
its regulation?
– First step is to isolate cDNA and genomic clones.
– Map cDNA to genomic sequence
• identify introns, exons
• locate approximate transcriptional start
– recognizing elements, e.g. TATA box
– 5’ primer extension or nuclease mapping
• get as much 5’ and 3’ flanking sequence as is
possible
– fuse largest chunk of putative promoter you can get
to a suitable reporter gene.
– Test whether this sequence is necessary and
sufficient for correct regulation
• how much sequence is required for correct
regulation?
– what is correct regulation?
» In cultured cells
» in animals?
– typical result is the more you look, the more
you find.
• questions are usually asked specifically. That is,
what part of the putative promoter is required for
activity in cultured liver cells?
– doesn’t always hold in vivo.
BioSci 145A lecture 14
page 2
©copyright
Bruce Blumberg 2000. All rights reserved
Identification of regulatory elements (contd)
•
Promoter mapping
– nuclease footprinting of promoter to identify regions
that bind proteins
– make various deletion constructs
• Previously made by ExoIII deletions or insertion
of linkers (linker scanning)
• typical method today is to PCR parts of the
promoter and clone into a promoterless reporter
– map activity of promoter related to deletions
• incremental changes in activity indicate regions
important for activity
– test elements for activity
BioSci 145A lecture 14
page 3
©copyright
Bruce Blumberg 2000. All rights reserved
Identification of binding proteins
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•
How to identify what factors bind to putative elements?
– examine the sequence
• does it contain known binding sites?
• if yes, do such proteins bind to the isolated
element in gel-shift experiments?
– do the elements bind proteins from nuclear extracts?
• gel shift (EMSA) experiments
– clone the elements into reporters with minimal
promoters.
• do these constructs recapitulate activity?
Biochemical purification of binding proteins
– tedious, considerable biochemical skill required
– two basic approaches
• fractionate nuclear extracts chromatographically
and test fractions for ability to bind the element in
EMSA
• DNA-affinity chromatography
– multimerize the element and bind to a resin
– pass nuclear extracts across column and
purify specific binding proteins
– protein microsequencing
– predict DNA sequence from amino acid sequence
• look in GENBANK database
• prepare oligonucleotides and screen library
BioSci 145A lecture 14
page 4
©copyright
Bruce Blumberg 2000. All rights reserved
Identification of binding proteins (contd)
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•
Biochemical purification of binding proteins (contd)
– advantages
• gold standard
• if you can purify proteins, this will always work
– disadvantages
• slow, tedious
• need good protein sequencing facility
• biochemical expertise required
• expense of preparing preparative quantities of
nuclear extracts
Molecular biological approaches
– oligonucleotide screening of expression libraries
(Singh screening)
• multimerize oligonucleotide and label with 32P
• screen expression library to identify binding
proteins
• advantages
– straightforward
– much less biochemical expertise required
– relatively fast
• disadvantages
– can’t detect binding if multiple partners are
required
– fair amount of “touch” required
BioSci 145A lecture 14
page 5
©copyright
Bruce Blumberg 2000. All rights reserved
Identification of binding proteins (contd)
•
Molecular biological approaches (contd)
– yeast one-hybrid assay
• clone element of interest into a reporter construct
(e.g. -gal) and make stable yeast strain
• transfect in aliquots of cDNA expression
libraries that have fragments of DNA fused to
yeast activator
• if the fusion protein binds to your element then
the reporter gene will be activated
• advantages
– somewhat more of a functional approach
– eukaryotic milieu allows some protein
modification
• disadvantages
– slow, tedious purification of positives
– can’t detect dimeric proteins
– sensitivity is not so great
AD
His
Bait elements
BioSci 145A lecture 14
page 6
©copyright
lacZ
Reporter(s)
Bruce Blumberg 2000. All rights reserved
Identification of binding proteins (contd)
•
Molecular biological approaches (contd)
– expression cloning (sib screening)
• clone element of interest (or promoter) into a
suitable reporter construct (e.g. luciferase)
• transfect (or inject, or infect, etc) pools (~10,000
cDNAs each) of cDNA expression libraries and
assay for reporter gene
• retest positive pools in smaller aliquots (~1000)
• repeat until a pure cDNA is found
– advantages
– functional approach
– presumably using the appropriate cell type
so modifications occur
– possibility to detect dimers with
endogenous proteins
– disadvantages
• VERY TEDIOUS
• very slow, much duplication in pools, extensive
rescreening is required
• could be expensive
BioSci 145A lecture 14
page 7
©copyright
Bruce Blumberg 2000. All rights reserved
Identification of binding proteins (contd)
– in vitro expression cloning (IVEC)
• Make small pools of cDNAs (~100)
• transcribe and translate cDNA libraries in vitro
into protein pools
• EMSA to test protein pools for element binding
• unpool cDNAs and retest
• advantages
– functional approach
– smaller pools increase sensitivity
• disadvantages
– can’t detect dimers
– very expensive (TNT lysate)
– considerable rescreening still required
– tedious, countless DNA minipreps required
BioSci 145A lecture 14
page 8
©copyright
Bruce Blumberg 2000. All rights reserved
Identification of binding proteins (contd)
– hybrid screening system 1
• begin with cDNA libraries in 384-well plates, 1
cDNA per well
• pool cDNAs using robotic workstation
• prepare DNA with robotic workstation
• transcribe and translate protein in vitro
• test for ability to bind DNA element using
sensitive, high-throughput assay
– fluorescence
– radioactive assay
• retest components of positive pools
• advantages
– very fast, only two steps required, ~ 2
weeks
– little work required
• disadvantages
– expense of robotics
– won’t detect dimers (unless 1 partner
known)
– expense of reagents (TNT, radionuclides,
fluorescent labels
BioSci 145A lecture 14
page 9
©copyright
Bruce Blumberg 2000. All rights reserved
Identification of binding proteins (contd)
•
– hybrid screening system 2
• prepare reporter cell line with element or
promoter driving reporter gene (e.g. luciferase)
• prepare cDNA pools as in system 1
• use robotic workstation to transfect cDNA
libraries into reporter cells
• assay for reporter gene
• advantages
– very fast
– truly functional approach
– use of cells allows modifications
– can detect dimers if one partner is already
present in cell
• disadvantages
– expense of equipment
OK, you have your element and binding protein, now
what?
– functional analysis depends on type of protein you
are dealing with
– goal will be to prove that this protein is necessary
and sufficient to confer regulation onto the promoter,
in vivo
• many just stop at works on the element
BioSci 145A lecture 14
page 10
©copyright
Bruce Blumberg 2000. All rights reserved
Transcription factors bind to regulatory elements
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•
The response element binding proteins you have carefully
identified are transcription factors.
– There are many types. The primary mode of
classification is via the type of DNA-binding
domains and intermolecular interactions
Features of transcription factors
– typically these proteins have multiple functional
domains
• can frequently be rearranged or transferred
– DNA-binding domains
• these domains take many forms that will be
discussed next time
• see also the list in TRANSFAC
http://transfac.gbf.de/TRANSFAC/
– Activation domains
• these are polypeptide sequences that activate
transcription when fused to a DNA-binding
domain
• these are diverse in sequence, 1% of random
sequences fused to GAL4 can activate
• many activation domains are rich in acidic
residues and assume an amphipathic -helix
conformation when associated with coactivator
proteins
• interact with histone acetylases that destabilize
nucleosomes and open chromatin
BioSci 145A lecture 14
page 11
©copyright
Bruce Blumberg 2000. All rights reserved
Transcription factors bind to regulatory elements (contd)
•
Features of transcription factors (contd)
– repression domains
• functional opposite of activation domains
• short and diverse in amino acid sequence
– some are rich in hydrophobic aa
– others are rich in basic aa
• some interact with proteins having histone
deacetylase activity, stabilizes nucleosomes and
condenses chromatin
• others compete with activators for the same
sequence and contacts with the transcription
machinery
– protein:protein interaction domains
• these are diverse in sequence but do contain
structural motifs
• leucine zipper
• helix-loop-helix
BioSci 145A lecture 14
page 12
©copyright
Bruce Blumberg 2000. All rights reserved
Regulating transcription factor activity (contd)
-catenin/
armadillo
BioSci 145A lecture 14
page 13
©copyright
Bruce Blumberg 2000. All rights reserved
Regulating transcription factor activity (contd)
•
How can the activity of a transcription factor be restricted
to a particular cell type or time?
– Factor is not generally present but synthesized only
where it is needed
• some developmental regulators
– The factor is present but must be modified to be
active
• heat shock factors - phosphorylated
• -catenin/armadillo - dephosphorylated
– A ligand is required for activity (or inactivity)
• nuclear hormone receptors
– The factor is localized to an inactive compartment
(e.g. cell membrane) and required cleavage for
activity
• sterol response factors (primarily cholesterol)
– The factor may be bound to an inhibitory factor in
the cytoplasm
• NF-B and I-B
– A dimeric factor can have multiple partners. Which
partner is present determines activity
• some dimers are active
• others are inactive
• eg bHLH and bZip proteins
BioSci 145A lecture 14
page 14
©copyright
Bruce Blumberg 2000. All rights reserved
Zinc finger genes
•
Zinc fingers are found in a variety of transcription factors
– two basic types
• Cys-His, consensus sequence is
cys-X2-4-cys-X3-phe-X5-leu-X2-his-X3-his
– typical gene has 3 or more fingers
– found in factors for Pol II and Pol III
• Cys-Cys, consensus sequence is
cys-X2-cys-X13-cys-X2-cys
– typical gene has only 2 fingers
– found in steroid hormone receptor
superfamily members
– may be involved in both DNA and RNA binding,
presence of finger does not indicate which
• eg TFIIIA binds DNA and RNA product
• eIF2 recognizes translational initiation sites
BioSci 145A lecture 14
page 15
©copyright
Bruce Blumberg 2000. All rights reserved
Zinc finger genes (contd)
•
purpose of fingers is to arrange residues such that zn ions
can be coordinated
– fingers may form -helical structures that fit into the
major groove of the DNA helix
– multiple fingers may act cooperatively to bind
nucleic acids
BioSci 145A lecture 14
page 16
©copyright
Bruce Blumberg 2000. All rights reserved
Zinc finger genes (contd)
•
•
BioSci 145A lecture 14
page 17
©copyright
cys-cys fingers in nuclear
receptors
– only 1st finger binds to
DNA
– second finger is
responsible for
protein:protein
interactions
– spacing between fingers
can vary quite a bit
finger 1 contains a regions
that determines target
specificity - P-box
– CGSCKA - AGAACA
– CEGCKG - AGTTCA
– these can be swapped
and change specificity of
the receptor
• used in ecdysoneinducible system
Bruce Blumberg 2000. All rights reserved
Hormonal signaling pathways
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•
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•
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Hormones are chemical messengers that coordinate
cellular activity
Can act in different ways
– endocrine - on distant cells
– paracrine - on neighboring cells
– autocrine - on cells which secrete them
Active at very low concentrations - typically less than 1
ppb (1 ppb ~= 3 nM)
Involved in numerous biological processes - many
hundreds of hormones
– reproduction - estrogen, testosterone, progesterone,
FSH, LH, activin
– metabolic rate - thyroid hormone, TSH, GH
– stress - glucocorticoids, ACTH, CRF
– blood pressure - aldosterone, renin, angiotensin,
vasopressin
– calcium homeostasis - vitamin D3, calcitonin, PH
Some vitamins or vitamin derivatives are hormones
– Vitamin A
• all-trans-retinoic acid
• 9-cis-retinoic acid
• 14-OH-retroretinol
– Vitamin D3
BioSci 145A lecture 14
page 18
©copyright
Bruce Blumberg 2000. All rights reserved
Nuclear hormone receptors
A/B
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•
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•
C
D
D
Domains are assortable and transferable
DNA-binding domain (DBD)
– responsible for direct binding to DNA
– discriminates half site sequence
– determines spacing between half sites
– contains an important dimerization motif
Ligand binding domain (LBD)
– responsible for ligand binding
– has a general dimerization motif
– contains an important transactivation domain
– may interact with amino terminus to modulate
activation
amino terminal region (A/B domain)
– contains an activation domain in many receptors
– may interact with other components of the
transcriptional machinery
– many receptors have alternative splicing or promoter
usage to yield different A/B domains
linker region (D) may influence activation, repression,
nuclear translocation or DNA-binding
BioSci 145A lecture 14
page 19
©copyright
Bruce Blumberg 2000. All rights reserved
F
Nuclear hormone receptors (contd)
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•
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•
bind to specific target DNA sequences
activate transcription of target genes upon ligand binding
-9
function at very low levels of ligand (~10 M or ~ ppb)
bind to small (~300d) lipophilic molecules
– steroids
– retinoids
– thyroid hormone
– vitamin D3
BioSci 145A lecture 14
page 20
©copyright
Bruce Blumberg 2000. All rights reserved
Nuclear hormone receptors (contd)
•
Many receptor ligands are related to cholesterol
– steroids
– bile acids
– oxysterols
– Vitamin D3
– ecdysone
can move freely through tissues
– penetrate to a target
– diffuse from a source
•
CH2OH
O
OH
HO
OH
1,25 dihydroxy D3
cortisol
O
HO
OH
I
NH2
HO
COOH
O
all-trans retinoic acid
COOH
I
I
tri-iodo thyronine
OH
OH
testosterone
O
BioSci 145A lecture 14
estradiol
HO
page 21
©copyright
Bruce Blumberg 2000. All rights reserved
Nuclear hormone receptors (contd)
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•
more orphan than known receptors
why study orphan receptors (not particularly easy)
– novel signaling pathways
– new developmental hormones
– target gene networks
– potential teratogens
– roles in adult physiology and endocrinology
– cancer treatment
BioSci 145A lecture 14
page 22
©copyright
Bruce Blumberg 2000. All rights reserved
Nuclear hormone receptors (contd)
•
Payoff from orphan receptor research so far (4 biotechs)
– LXR and FXR regulate cholesterol metabolism
• LXR diverts cholesterol into bile acid pathway
• FXR negatively regulates uptake of bile acids
– PPARs regulate fat metabolism
• PPAR is insulin sensitizer
– SXR and PXR regulate metabolism of steroids,
xenobiotics and environmental compounds
– CAR also mediates drug breakdown
BioSci 145A lecture 14
page 23
©copyright
Bruce Blumberg 2000. All rights reserved
Nuclear hormone receptors (contd)
•
Nuclear receptors interact with each other and DNA
– can form homodimers, heterodimers and monomers
– four possible modes of DNA binding
• IR, DR, ER, monomer
– steroid receptors very closely related, others not so much
• bind to HSPs and stay in cytoplasm until ligand
bound.
• GR, MR, PR and AR all bind each others response
elements. Significant crossover between pathways at
pharmacological levels of ligands (eg. anabolic
steroid use)
– RXR heterodimers is the largest and most diverse family
– monomeric orphan receptors may also dimerize and/or
interact with RXR depending on the response element.
BioSci 145A lecture 14
page 24
©copyright
Bruce Blumberg 2000. All rights reserved
Nuclear hormone receptors (contd)
Known ligands
RAR,,
TR ,
VDR
EcR
all-trans RA
thyroid hormone
1,25-(OH)2-VD3
ecdysone
Recent EX-orphans
PPAR ,,, 
FXR
BXR
LXR ,
fatty acids, eicosanoids
bile acids
benzoates
oxysterols
Activatable orphans
SXR/PXR
CAR ,
•
steroids, xenobiotics
androstans, xenobiotics
RXR is a common partner in >10 different pathways
– can be silent (non-permissive)
– can be active (permissive)
– offers another means to regulate dimeric receptors
• rexinoids (RXR selective compounds) are being
used clinically in treatment of diabetes, breast
cancer and other diseases.
– at least part of the reason that vitamin A levels are
tightly regulated in vivo
• too much or too little both very harmful,
particularly during development
BioSci 145A lecture 14
page 25
©copyright
Bruce Blumberg 2000. All rights reserved
Nuclear hormone receptors (contd)
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•
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P-box determines half-site specificity
– CEGCKGFF in many receptors
– therefore they all bind to the same or similar half
sites
– where does specificity come from?
spacing between half-sites encodes specificity
– 3-4-5 rule of Kaz Umesono
– DR-3
VDRE SXRE
– DR-4
TRE
SXRE, LXRE, FXRE, BXRE
– DR-5
RARE SXRE, CARRE
– DR-1
RXRE
PPRE
– DR-2
RARE
selectivity is not absolute but these provide a good model
for determining response elements
BioSci 145A lecture 14
page 26
©copyright
Bruce Blumberg 2000. All rights reserved
Nuclear hormone receptors (contd)
•
Transcriptional regulation by RXR heterodimers
– most, or all of these bind DNA in the absence of ligand
• unliganded receptor is a repressor
– effect of activators and repressors together ?
• ligand causes a conformational change that kicks
off corepressor
• liganded receptor can now recruit coactivators and
activate transcription
– coactivators and corepressors alter chromatin
conformation by modulating histone modification
– offers possible ways to specifically disrupt complex
BioSci 145A lecture 14
page 27
©copyright
Bruce Blumberg 2000. All rights reserved
Nuclear hormone receptors (contd)
•
How does one go about identifying orphan receptor
ligands?
– requirements
• receptor expression construct
• response element to make reporter
• cofactors (use tissue where receptor is active)
– good news!
• assay is very sensitive sub parts per billion
– analytical bad news
• chemistry requires parts per thousand-ppm
BioSci 145A lecture 14
page 28
©copyright
Bruce Blumberg 2000. All rights reserved
Nuclear hormone receptors (contd)
•
What are some effects of mutations in nuclear receptors?
– steroid receptors
• knockout of SF-1 removes adrenal/gonad axis
• human mutations in DAX-1 similar
• overproduction of adrenal steroids -Cushing’s
syndrome
• underproduction - Addison’s disease
• nonfunctional AR - testicular feminization
– genotypic males develop as females
externally but male internally (rumor about
well-known actress)
• nonfunctional ER
– male, osteoporosis, coronary artery
disease, continuous growth
– female lethal in utero, osteoporosis later
• nonfunctional MR- hypotension
• nonfunctional GR- hypertension and low renin
– other receptors
• thyroid hormone receptor - mutations can lead to
alterations in metabolism and ADHD
• vitamin D receptor - vitamin D resistant rickets
• retinoic acid receptor
– several types of leukemia result from fusion
of RAR to other transcription factors
– some are treatable with RA, others not
BioSci 145A lecture 14
page 29
©copyright
Bruce Blumberg 2000. All rights reserved