Transcript Chapt 12 Transcription Activators in Eukaryotes Student learning outcomes: • Explain how gene-specific activators binding to DNA elements (enhancers) activate transcription. • Activators contain at least 2 domains:

Chapt 12
Transcription Activators in Eukaryotes
Student learning outcomes:
• Explain how gene-specific
activators binding to DNA
elements (enhancers)
activate transcription.
• Activators contain at least 2
domains: DNA-binding
domain (DBD) and
transcription-activation
domain (TAD).
Transcription factor p53
(tumor suppressor) binding DNA
12-1
• Describe some DBD and TAD motifs:
– Zinc modules,
– homeodomain, bZIP, bHLH (shared with prokaryotes)
– Examples of activators
• Explain how activators function by contacting GTFs,
recruiting pol II or contacting coactivators
• Describe how activators and coactivators can be
controlled by modifications including:
ubiquityation, sumoylation, phosphorylation, acetylation
• Important Figures: 1, 2*, 3, 4*, 6, 9, 10*, 12*,16, 18*, 20, 23,
24, 27, 33*, 34*, 35, 36, 37; Table 1
• Review questions: 1, 2, 3, 4, 5, 6, 9, 10, 13, 14, 17*, 18, 24,
12-2
28, 30, 31; Analy Q 1
12.1 Categories of Activators
• Activators can stimulate or inhibit transcription by
RNA polymerase II
• Composed of at least 2 functional domains:
– DNA-binding domain (DBD)
– Transcription-activation domain (TAD)
– Many also have dimerization domain
• Domain - independently folded region of protein
Ex. Fig. 13 Gal4
activator of yeast
12-3
DNA-Binding Domains (DBD)
DNA-binding domains - DNA-binding motifs:
– 3 Characteristic shapes for specific DNA binding
Zinc-containing modules: use Zinc ions to create
shape to fit a-helix of motif into DNA major groove
–
Zn fingers, Zn modules, Modules with 2 Zn and 6 cys
Homeodomains: about 60 amino acids - resemble HTH
(helix-turn-helix) proteins in structure and function;
ex. homeobox proteins regulate fruit fly development
bZIP and bHLH motifs: highly basic DNA-binding motif
linked to protein dimerization motifs
– Leucine zippers, Helix-loop-helix (ex. CCAAT enhancer-binding protein)
12-4
Transcription-Activating Domains (TAD)
Most activators have at least one of these domains:
– Acidic domains – ex. yeast GAL4 (11 acidic
amino acids out of 49 amino acids) in domain
– Glutamine-rich domains – ex. Sp1 has 2
regions that are 25% glutamine (Gln)
– Proline-rich domains – ex. CTF has domain
with 19 proline in 84 amino acids
12-5
12.2 Structures of DNA-Binding Motifs of
Activators
• DNA-binding domains well-defined structures (Ch. 9)
• X-ray crystallography shows how structures interact
with DNA targets
• Interaction domains often form dimers, or tetramers,
• Most DNA-binding proteins can’t bind DNA in
monomer form
• Compare common DNA binding motif in prokaryotes
12-6
Helix-Turn-Helix motif is common:
lac, trp, l, other phage repressors
l Family of Repressors
Repressors have
recognition helices (red)
fit major groove of
operator
• Helix-turn-helix motif
(HTH)
• Specificity of binding
depends on amino acids
in recognition helices
Fig. 9.2
9-7
High-Resolution Analysis of l
Repressor-Operator Interactions
Structural Features from co-crystal:
– Recognition helices of repressor
monomer (3, red) nestle into DNA
major grooves in 2 half-sites
– Helices approach, hold two monomers
together in repressor dimer
– DNA similar in shape to B-form DNA
– DNA bends at two ends of fragment as
it curves around repressor dimer
Figs. 9.6, 9.7:
Repressor dimer binding
to symmetric operator O1
9-8
Eukaryotic Homeodomains
have HTH motif
• Homeodomains contain
DNA-binding HTH (helixturn-helix) motif
• Recognition helix (3) fits
in DNA major groove,
makes specific contacts
• N-terminal arm nestles in
adjacent minor groove
• First identified Drosophila
developmental genes –
Fig. 8 Antennapedia mutant
has legs in place of antennae
Fig. 9 Drosophila homeotic
engrailed protein binding target
12-9
Eukaryotic bZIP and bHLH Domains
• bZIP proteins dimerize through leucine zipper motif
– Leu every 7 aa (one face); hydrophobic interactions
– adjacent basic regions embrace DNA like tongs
• bHLH proteins dimerize through helix-loop-helix motif
– basic parts of each long helix grasp DNA target site
• Ex. bZIP yeast GCN4 activator
Fig. 10 structure leucine zipper
Fig. 11 bZIP of yeast GCN4 activator
12-10
Eukaryotic bZIP and
bHLH Domains
• bHLH motif in activator myoD:
– HLH is dimerization
– Long helix 1 has basic region
that binds DNA
• Both bHLH and bZIP domains are found in Myc,
Max oncogenes; heterodimers bind to DNA like
HLH; extra dimerization potential from leu zippers
12-11
Zinc Fingers
Fig. 1 3D shape of 1
Xenopus Zn finger:
Zn blue; Cys yellow S;
His blue
• First described forTFIIIA
• 9 repeats of 30-aa element:
– 2 closely spaced Cys followed 12
aa by 2 closely spaced His
– Coordination of aa to metal helps
form finger-shape
– Protein rich in Zn: 1 Zn per repeat
– Specific recognition between Zn
Fig. 2; Zn finger has
finger and DNA in major groove
antiparallel b-strand (2 Cys)
12-12
and a-helix (2 His)
3 Zinc Fingers in Curved Shape bind DNA
Zif268 is mouse TFIIIAclass protein
Each Zn finger:
– antiparallel b-strand
contains 2 Cysteines
– 2 Histidines in a-helix
– Helix and strand
coordinated to Zn ion
aa of helix recognize bp
of major groove
Fig. 3
12-13
Zn module: GAL4 Protein of Yeast
• GAL4 regulates expression of genes for galactose
metabolism
• Binds target UAS (upstream activating sequence)
• Member of Zn-containing family of DNA-binding
proteins, Not zinc finger
• Each GAL4 monomer contains DNA-binding motif:
– 6 Cys coordinate 2 Zn ions in a bimetal thiolate cluster
– DNA recognition - short a-helix protrudes into major groove
– Dimerization motif on other end - a-helix forms parallel
coiled coil, interacts with a-helix on other GAL4 monomer
12-14
Zn module - GAL4 protein of yeast
Fig. 4 dimer of GAL4 binding DNA: Zn yellow
DNA recognition aa 8 to 40; linker 41-49; dimerization 50-64
12-15
Zn modules - Nuclear Receptors
• Nuclear hormone receptors interact with different
endocrine-signaling molecules
• Protein plus endocrine molecule forms complex
that functions as activator:
• Binds hormone response elements, stimulates
transcription of associated genes
• Ligands for these receptors are lipid-soluble,
cross membrane (steroids, retinoic acid, vitamin D)
12-16
Type I Nuclear Receptors
• Type 1 include Glucocorticoid (GR), Estrogen (ER)
• Reside in cytoplasm bound to protein Hsp90
• When receptors bind to hormone ligands:
–
–
–
–
Release cytoplasmic protein partners
Move to nucleus; dimerize
Bind to enhancers
Act as activators
Fig. 6
12-17
Glucocorticoid Receptor binding DNA
Fig. 6
structure
with half
sites
separated
by extra bp;
only 1
module
binds best
• DNA-binding domain with 2 Zn-containing modules
• One module has most of DNA-binding residues
• Other module has surface for protein-protein interaction to
12-18
form dimers
Types II and III Nuclear Receptors
• Type II nuclear receptors stay within nucleus
– Examples: thyroid hormone receptor (TR),
– vitamin D receptor (D3)
– Retinoic acid receptor (RAR) (more Ch. 13)
• Bound to target DNA sites
• Without ligands, receptors repress gene activity
• When receptors bind ligands, activate transcription
• Type III receptors are “orphan” whose ligands are
not yet identified
• All use a-helix for interaction with DNA sequences
12-19
12.3 Independence of Domains of Activators
• DNA-binding, transcription-activating domains of
activator proteins are independent modules
• Hybrid proteins with DNA-binding domain of one protein,
transcription-activating domain of another
• Hybrid protein still functions as activator
Fig. 18;
yeast
expressGal4
TAD on Gal4
or lexA DBD
12-20
12.4 Functions of Activators for pol II
• Eukaryotic activators recruit pol II to promoters
• Stimulate binding of general transcription factors
(GTFs) and pol II to promoter
• 2 hypotheses for recruitment:
– GTF cause stepwise build-up of preinitiation complex
– GTF and other proteins already bound to polymerase in
complex called RNAP holoenzyme
Kornberg lab quantitation of proteins tested models
12-21
Models for Recruitment
Fig. 14
12-22
Recruitment Model of GAL11P-containing
Holoenzyme
Fig. 16
• Ptashne found dimerization domain of GAL4 binds
to mutant form of Gal 11 (GAL11P) in holoenzyme
(GAL11P is potentiator) [GAL11 is part of mediator complex]
• Holoenzyme, plus TFIID, then binds to promoter,
activating gene
12-23
Recruitment Model of
GAL11P-containing
Holoenzyme
• Activation in some yeast
promoters appears to
function by recruitment of
holoenzyme
• chimeric activator proteins
with lexA DBD and other
domains recruit holoenzyme
Fig. 17
12-24
Recruitment of holoenzyme
may not be that common
• Kornberg evidence:
• TAP- epitope tags on components:
• Antibodies on immunoblot
quantified pol II, GTFs, mediator:
• pol II and some GTFs present in
much higher concentration
• Unlikely holoenzyme recruited as
unit
Fig. 18 Pol II about
3x more than
Mediator, TFIIH
12-25
12.5 Interaction Among Activators
• General transcription factors (GTFs) interact to form
preinitiation complex (PIC)
• Activators and GTFs also interact
• Activators interact with one another to activate gene
– Individual factors interact to form protein dimer facilitating
binding to single DNA target
– Specific factors bound to different DNA targets can
collaborate to activate gene
12-26
Importance of Dimerization to Activation
• Dimerization increases affinity between activator
and DNA target:
Some activators form homodimers (ex. GAL4)
Some form heterodimers
Ex. Products of jun and fos
Oncogenes form heterodimer,
bind AP-1 sites; heterodimer
Binds better than homodimers
Fig. 19; EMSA assay; Fos or Fos
Core mixed with Jun or Jun core
and AP-1 site; M = myc oncoprotein
12-27
Activators can Act at a Distance
• Bacterial and eukaryotic enhancers stimulate
transcription even though located some distance
from promoters
• Hypotheses explain ability of enhancers to act at a
distance
–
–
–
–
Change in topology
Sliding
Looping **
Facilitated tracking
Looping brings activators bound to different enhancers
closer to promoter
12-28
Hypotheses
of Enhancer
Action
Fig. 20:
a. Topology
b. Sliding
c. Looping
d. Tracking
12-29
Complex Enhancers
• Many genes have more than one activator-binding
site, -> respond to multiple stimuli
• Each activator that binds must interact with PIC
assembling at promoter, likely by looping out
intervening DNA
• Consider whole region upstream of promoter as an
enhancer (rather than each specific site for each
factor); different factors binding permit fine level of
control on expression
• Combinatorial code: final result of all activators,
inhibitors bound
12-30
Complex enhancer:
Control Region of Metallothionein Gene
Fig. 23
• Gene product helps eukaryotes cope with heavy
metal poisoning (small proteins rich in cys; bind metals)
• Turned on by several different agents
BLE = basal level enhancer; MRE = metal response element;
GRE = glucocorticoid response element; GC = Sp1 binding
12-31
Complex enhancers in development
Figs. 24, 25: sea urchin
Endo16 gene: different
activators and modules
at different times control
expression
Red = enhancers;
Blue = architectural factors
Ovals - activators
12-32
Architectural Transcription Factors
Architectural transcription factors: main purpose
to change shape of DNA control region so other
proteins bind successfully to stimulate transcription
HMG proteins bend DNA, help activators bind
(HMG, small nuclear proteins with high electrophoretic mobility)
ex. LEF-1 (T-cell receptor); HMGI (Y) for interferon B gene
12-33
Architectural Transcription Factor
Fig. 26 T cell
receptor a chain
• T cell receptor a-chain: enhancer elements upstream
• Elements bind to: Ets-1 LEF-1 CREB
– CREB (cyclic AMP response element binding protein)
– LEF-1 (lymphoid enhancer) not enhance transcription alone
– Activator LEF-1 binds minor groove of DNA through HMG
domain, induces strong bending of DNA
– DNA bending helps other activators bind, and interact with
activators and general transcription factors
12-34
Enhanceosome
• Complex of enhancer
DNA with activators
contacting DNA
• Ex. HMG helps bend
DNA so that it interacts
with other proteins
Fig. 27
HMG I(Y) plays similar role in human interferon-b control gene:
Activation seems to require cooperative binding of several
activators, including HMG I(Y) to form enhanceosome with a
12-35
specific shape
Insulators
prevent
activation
of unrelated
genes by
nearby
enhancers:
Fig. 28
• Enhancer-blocking activity: insulator between
promoter and enhancer prevents promoter activated
• Barrier activity: insulator between promoter and
condensed, repressive chromatin prevents promoter
repressed
12-36
Mechanism of Insulator Activity
• Sliding model
– Activator bound to
enhancer and
stimulator slides along
DNA to promoter
• Looping model
– Two insulators flank
an enhancer; when
bound, interact to
isolate enhancer
Fig. 29
12-37
12.6 Regulation of Transcription Factors
• Hormones binding nuclear receptors caused them to
move to nucleus, or to change from repressor to
activator (Ch. 13)
• Phosphorylation of activators allows to interact with
coactivators to stimulate transcription
• Ubiquitylation of transcription factors marks them for
– Destruction by proteolysis
– Stimulation of activity
• Sumoylation - attachment of polypeptide SUMO;
targets movement to compartments of nucleus
• Methylation and acetylation can modulate activity
12-38
Coactivators mediate signals from
activator to basal transcription apparatus
• CBP/p300 is common coactivator:
CREB binding protein/ 300kD protein
HAT activity, acetylates histones and others
• CREB (cyclic AMP responsive element binding
protein) binds CRE (Fig. 33)
• Nuclear receptors plus hormone bind DNA (Fig. 34)
• Both effects mediated through CBP
12-39
Coactivators do not stimulate basal
transcription, only activated
• Mediator: (Kornberg)
– Mediates activation; binds
CTD of pol II
– Found in yeast first
•
•
•
•
•
Many similar complexes:
SMCC/ TRAP
CBP/p300 (CREB-binding)
CRSP (for SP1)
SRC (steroid receptor co-)
Fig. 32
12-40
Coactivators Mediate Activation
Ex. CREB
stimulates
basal transcription
machinery via
CBP/p300
Protein Kinase A
functions in signal
transduction pathway
Phosphorylation of
CREB by PKA is
critical for activation;
Fig. 33
12-41
Activation of Nuclear Receptor-Activated
Gene uses CBP/ SRC coactivator
CARM1 methylates
proteins
Fig. 34
12-42
Ubiquitylation affects activation
• Ubiquitin - 76-aa polypeptide covalently
attached to Lys residues by Ubiquitin
ligase
• Ubiquitylation, especially
monoubiquitylation, of activators can
have activating effect
• Polyubiquitylation marks proteins for
destruction in proteasome, reduces or
abolishes activation
• Proteins from 19S regulatory particle of
proteasome can stimulate transcription
12-43
Sumoylation affects Activation
• Sumoylation - addition of one or more copies of
101aa polypeptide SUMO (Small Ubiquitin-Related
Modifier) to Lysine residues; similar to ubiquitylation
• Sumoylated activators targeted to a nuclear
compartment that keeps them stable,
but not activating targets
(PML nuclear bodies)
• Ex. PML protein in
•
promyelocytic leukemia;
• B-catenin and LEF-1 SUMO
Some proteins in
nuclear bodies
12-44
Acetylation affects Activators
• HAT - enzyme histone acetyltransferase, modifies
histone tails with acetyl groups (Ch. 13)
– Activity of CBP/p300
• Nonhistone activators and repressors can be
acetylated by HATs
• Acetylation has either positive or negative effects
12-45
Signal Transduction Pathways
• Begins with signaling molecule interacting with
receptor on cell surface (hormones, growth factors)
• Interaction sends signal into cell (second
messenger pathway)
• Often leads to altered gene expression
• Often protein phosphorylation passes signal from
one protein to another (PKA, MAPK, G proteins)
• Signal amplification occurs at each step
12-46
Three Signal Transduction Pathways that
share coactivator CBP/p300
12-47
Review questions
• 1. List three different classes of DNA-binding domains
found in eukaryotic transcription factors.
• 2. List three different classes of transcriptionactivation domains in eukaryotic transcription factors.
• 7. Explain the difference between type I and II nuclear
receptors, and give an example of each.
• 28. Draw diagrams to illustrate action of CBP as
activator of (a) phosphorylated CREB; (b) nuclear
receptor
12-48