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BioSci 145A Lecture 13 - 2/19/2002
Transcription factors II
Topics we will cover today
– Hybrid tetracycline regulated system
– implications of transgenic technology
– Principles of gene regulation
– Identification of regulatory elements
– Identification of regulatory element binding proteins
– Functional analysis
– Transcription factors - introduction
– Modulation of transcription factor activity
transcription factor resources
• detailed transcription factor database
• collected information about homeobox genes
• nuclear receptor resource
– nuclear transport Nakielny and Dreyfuss (1999) Cell
99, 677-690.
– Nuclear pore structure Daneholt (1997) Cell 88, 585588.
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Regulated expression - hybrid tetracycline systems
– references
• Kringstein et al (1998) PNAS 95, 13670-75
• Baron et al (1999) PNAS 96, 1013-1018
• Blau and Rossi (1999) PNAS 96, 797-799
– utilizes highly engineered tet and reverse tet proteins
to get specific effects
properties of the system
– what happens when one puts proteins into the cell that
respond differently to the same effector compound?
• If they can dimerize with each other
• can not dimerize with each other
• or if they do not dimerize and bind to different
and non-overlapping operator sequences
– different function, can heterodimerize
• in this case, a fair number will make unproductive
heterodimers and interfere with desired effect
• this will also be problematic even if the two
dimers have been engineered to bind different
• so for maximum effect, we must prevent
heterodimerization between effectors that
– can bind to different sequences
– have different functions (activator vs
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Regulated expression - hybrid tetracycline (contd)
Heterodimers possible
same DNA targets
different DNA targets
Heterodimers not possible
functional discrimination
– Opposite function - same DNA target
• use pure tetR and VP16-rtetR
• at low dox, the repressor will dominate
• as dox increases, the repressor will dissociate and
VP16-rtetR will activate
• increases dynamic range of activation ~105 fold
– increases sensitivity as well
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Regulated expression - hybrid tetracycline (contd)
– Opposite function - different DNA target
• use std tet with one type of tetO to regulate gene
• and rev tet with another type of tetO to regulate
gene B
• in the absence of dox, gene A will be activated
while B will be silent
• in the presence of high dox, gene A will be
inactivated and gene B will be activated
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Regulated expression - hybrid tetracycline (contd)
– activator and repressor
• very sensitive regulation of responsive gene
• tightly regulated expression over 5 logs
• can readily measure effects of subtle changes in
gene expression
– how much change in expression is required
to get effects?
– Do effects differ at different levels of
– Activator/repressor two targets
• can create “conditional mutants” that have one
activity at low levels of effector substance and
another at high levels
• can regulate two different genes or two alleles of
a single gene
– mutually exclusive expression
– or expression of neither
• can repeatedly switch between two states and
observe effects at high resolution
• possible to perturb intracellular equilibria in
small increments and follow the effects on
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Regulated expression - hybrid tetracycline (contd)
Caveats and pitfalls
– best done with stable cell lines
– much more versatile than ecdysone
– possible to fine tune expression of a single gene, or
two genes with unprecendented resolution
– can make conditional mutants without genetics
• eg in model systems not amenable to genetics
such as Xenopus or chicken
– can make conditional replacements in vivo
• knock the repressible tetO into an endogenous
• introduce a transgene under the control of the
activatable tetO
• breed these mice with a line expressing the dual
tet repressors
• increasing dox will inactivate the endogenous
gene and activate the transgene
– technically demanding
– multiple steps required
– may not work as well as predicted due to complexity
– virus-based systems may not work in ES cells
– Clontech’s TRE-effector plasmids have high
background - need to be remade
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Regulated gene expression - summary
what system do you need to use?
– Transgenic animals?
– Gene therapy?
– Cell culture?
How much tolerance is there for modulation of non target
– Eg glucocorticoids, estrogens, progestins and
thyroid hormones are very important physiologically
and cannot be modulated without collateral effects in
• however, these are very good in the early
embryo of model organisms
How stringent must the regulation be?
– The more stringent the requirements for control the
greater the likelihood that complex techniques will
be required
• eg hybrid tetracycline
Is there a need for regulating multiple genes?
– Hybrid tet is the only way to go
What are the commercial implications?
– Licenses may be required for various technologies
• tet is controlled by BASF
• ecdysone by the Salk Institute
– depending on the license agreement, one technique
might be preferred (eg ecdysone)
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Gene transfer technology - implications
Genetics and reverse genetics
– gene transfer and selection technology speeds up
genetic analysis by orders of magnitude
– virtually all conceivable experiments are now
• all questions are askable
– much more straightforward to understand gene
function using knockouts and transgenics
• gene sequences are coming at an unprecedented
rate from the genome projects
• Knockouts and transgenics remain very
expensive to practice
– other yet undiscovered technologies will be
required to understand gene function.
Clinical genetics
– Molecular diagnostics are becoming very widespread
as genes are matched with diseases
• huge growth area for the future
• big pharma is dumping billions into diagnostics
– room for great benefit and widespread abuse
• diagnostics will enable early identification and
treatment of diseases
• but insurance companies will want access to
these data to maximize profits
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Gene transfer technology - implications (contd)
gene therapy
– new viral vector technology is making this a reality
• efficient transfer and reasonable regulation possible
– long lag time from laboratory to clinic, still working with
old technology in many cases
– The Biotech Death of Jesse Gelsinger. Sheryl Gay
Stolberg, NY Times, Sunday Magazine, 28 Nov 99
protein engineering
– not as widely appreciated as more glamorous techniques
such as gene therapy and transgenic crops
– better drugs, eg more stable insulin, TPA for heart attacks
and strokes, etc.
– more efficient enzymes (e.g. subtilisin in detergents)
– safe and effective vaccines
• just produce antigenic proteins rather than using
inactivated or attenuated organisms to reduce
undesirable side effects
metabolite engineering
– enhanced microbial synthesis of valuable products
• eg indigo (jeans)
• vitamin C
– generation of entirely new small molecules
• transfer of antibiotic producing genes to related
species yields new antibiotics (badly needed)
– reduction of undesirable side reactions
• faster more efficient production of beer
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Gene transfer technology - implications (contd)
transgenic food
– gene transfer techniques have allowed the creation of
desirable mutations into animals and crops of
commercial value
• disease resistance (various viruses)
• pest resistance (Bt cotton)
• pesticide resistance
• herbicide and fungicide resistance
• growth hormone and milk production
– effective but necessary?
– negative implications
• pesticide and herbicide resistance lead to much
higher use of toxic compounds
• results are not predictable due to small datasets
• at least one herbicide (bromoxynil) for which
resistance was engineered has since been banned
plants as producers of specialty chemicals
– still very underutilized since plant technology yet
lags behind techniques in animals
– great interest in using plants as factories to produce
materials more cheaply and efficiently
• especially replacements for petrochemicals
– plants and herbs are the original source of many
pharmaceutical products hence it remains possible to
engineer them to overproduce desirable substances
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Principles of gene regulation
Why does gene expression need to be controlled anyway?
– Primary purpose in multicellular organisms is to
execute precise developmental decisions so that
• proper genes are expressed at
– appropriate time
– correct place
– at the required levels
• so that development, growth and differentiation
proceed correctly
– maintenance of homeostasis
• produce required substances in appropriate amounts
– nutrients, cofactors, etc.
• degrade undesired substances from
– diet
– metabolism
– injury
• inter and intracellular signaling processes
Where are the control points?
– Activation of gene structure
– initiation of transcription
– processing of the transcript to mRNA
– transport of mRNA to cytoplasm
– translation of mRNA
– processing and stability of protein
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Principles of gene regulation (contd)
Activation of gene structure
– genes are active only in cells where they are
– structure of gene determines whether it is can be
transcribed or not
– activation of an active structure may be one of the
first steps in gene regulation
• modification of DNA
– methylation of DNA inactivates genes
– active genes are hypomethylated
• modification of histones
– methylation and acetylation of histones
activates gene expression
» acetylase activates
• active genes are in an open, hypomethylated
coformation. associated histones are
– one of the primary responsibilities of cell-type
specific transcription factors is to facilitate the
formation of an active chromatin conformation
• majority of alleged co-activator and co-repressor
proteins are relatively non-specific modifiers of
chromatin conformation that interact with
specific factors targeting chromatin remodeling
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Principles of gene regulation (contd)
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Principles of gene regulation (contd)
Initiation of transcription
– Once the DNA template is accessible, the next
requirement is to form the initiation complex
• although other forms of regulation are important,
the majority of regulatory events occur at the
initiation of transcription
– genes under common control share response
elements (aka cis-cting elements, enhancers)
• these sequences are presumed to be recognized
by specific protein(s)
• the protein(s) functions as a transcription factor
needed for RNA polymerase to initiate
• the active protein is only available when the
gene is to be expressed
– response elements are often cell-type or tissuespecific
• because binding proteins are cell-type specific
• but this is a tautology
– each gene has multiple response elements
• each regulatory event depends on the binding of
a protein to a particular response element
• any one of these can independently activate the
• combinatorial regulation by multiple elements
and proteins is a central mechanism by which
levels of gene expression are modulated
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Principles of gene regulation (contd)
– cis-acting control elements can be located many
kilobases away from the transcriptional start site
• in intergenic regions
• in introns
• some elements may be quite close to TATA box
or other intitiator elements
– cis-acting elements are responsible for allowing the
recruitment of TBP and assembly of the initiation
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Transcription factors and the preinitiation complex
Model for cooperative assembly of an activated transcription initiation
complex at the TTR promoter in hepatocytes.
– Four activators enriched in hepatocytes plus ubiquitous AP-1 factors
bind to sites in the hepatocyte-specific enhancer and promoterproximal region of the TTR gene
– Activation domains of the bound activators interact extensively with
co-activators, TAF subunits of TFIID,Srb/mediator proteins and
general transcription factors. This causes looping of DNA and
formation of stable initiation complex
– Highly cooperative nature of complex assembly prevents initiation
complex from forming in other cells that lack all four of the
hepatocyte-enriched transcription factors.
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Principles of gene regulation (contd)
processing of the transcript to mRNA
– RNA is synthesized as an exact copy of DNA
• heterogeneous nuclear RNA (hnRNA)
– hnRNA gets capped and polyadenylated
– introns are spliced out by the spliceosome, a large
complex of RNA and proteins.
• exons can also be spliced out as well. Alternative
splicing may produce proteins with new functions.
– Molecular mechanisms underlying alternative
splicing are still only poorly understood
– regulation of alternative splicing is important in
the CNS and for sex determination
– splice junctions are read in pairs
• spliceosome binds to a 5’ splice donor and scans for
a lariat sequence followed by a 3’ splice acceptor
• mutations in either site can lead to exon skipping
– principle underlying gene trapping
– mRNA is now ready for transport to cytoplasm
– some organisms perform trans splicing between mRNAs
• another way to generate mRNA diversity
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Principles of gene regulation (contd)
transport of mRNA to cytoplasm
– capping, polyadenylation and
splicing of mRNA are
prerequisites to transport
– macromolecules are specifically
transported bidirectionally though
nuclear pores
• direction controlled by
nuclear import and export
signals in macromolecules
– fully processed mRNAs are
packaged into ribonucleoprotein
particles, mRNPs
• hnRNP proteins contain
nuclear export sequences
– These are transported through the
pore complex, unwinding as they
do so
– On the cytoplasmic side of the
pore, the mRNA is stripped from
the RNP by binding to ribosomes
– those with signal sequences are
paused and subsequently
associate with ER
– those without are translated
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Principles of gene regulation (contd)
translation of mRNA
– by default, mRNAs are all translated
– efficiency of translation is important for protein
• regulatory genes tend to be poorly translated
– two primary mediators of efficiency
• consensus around the ATG
– optimum is ACCACCATGG
– most important factor is a G following
ATG (A gives about 40% of protein
– underlined sequence will give very high
levels of translation - NcoI site
• stability of mRNA in the cytoplasm varies
– many short lived mRNAs have multiple
copies of the sequence AUUUA in 3’ UTR
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Principles of gene regulation (contd)
• stability of mRNA (contd)
– others mRNAs are specifically degraded,
e.g. transferrin
– in the absence of iron, a specific protein
(IRE-BP) binds to a region of the
transferring mRNA containing AUUUA
– this protects the mRNA from degradation,
transferrin is synthesized and iron
– iron binds to IRE-BP and dissociates it
from mRNA
» AUUUA mediates degradation
BioSci 145A lecture 13
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
– 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
– 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 13
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 13
Bruce Blumberg 2000. All rights reserved
Identification of binding proteins
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
• 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
• 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 the database
• prepare oligonucleotides and screen library
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Identification of binding proteins (contd)
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
• advantages
– straightforward
– much less biochemical expertise required
– relatively fast
• disadvantages
– can’t detect binding if multiple partners are
– fair amount of “touch” required
BioSci 145A lecture 13
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
• disadvantages
– slow, tedious purification of positives
– can’t detect dimeric proteins
– sensitivity is not so great
Bait elements
BioSci 145A lecture 13
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 slow, much duplication in pools, extensive
rescreening is required
• could be expensive
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Identification of binding proteins (contd)
– in vitro expression cloning (IVEC)
• transcribe and translate cDNA libraries in vitro
into small pools of proteins (~100)
• 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 13
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
– little work required
• disadvantages
– expense of robotics
– won’t detect dimers (unless 1 partner
– expense of reagents (TNT, radionuclides,
fluorescent labels
BioSci 145A lecture 13
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
– 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 13
Bruce Blumberg 2000. All rights reserved
Transcription factors bind to regulatory elements
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 (next time)
Features of transcription factors
– typically these proteins have multiple functional
• 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
– Activation domains
• these are polypeptide sequences that activate
transcription when fused to a DNA-binding
• 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
• interact with histone acetylases that destabilize
nucleosomes and open chromatin
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved
Transcription factors bind to regulatory elements (contd)
Features of transcription factors (contd)
– repression domains
• functional converse 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
– protein:protein interaction domains
• these are diverse in sequence but do contain
structural motifs
• leucine zipper
• helix-loop-helix
BioSci 145A lecture 13
Bruce Blumberg 2000. All rights reserved