Download Eukaryotic gene expression

Eukaryotic gene expression
• Bacterial genes have a ground state that permits
transcription
– Without CAP site or operator, the sigma subunit will
locate a gene
• Eukaryotic genes require complex systems to turn
them on
– Chromatin structure must be relaxed in order for RNA
polymerase to gain access to DNA sequence information
– Eukaryotic genes are positively regulated. They are not
transcribed in the absence of active mechanisms.
– The regulatory components and systems are more
complex than bacteria
– Transcription is removed from translation
• There are no systems equivalent to attenuation in eukaryotes
The evidence for
• DNase I cleaves chromatin at
alteration of
the linker junctions between
chromosomal structure nucleosomes
during transcription
– When run on an agarose gel, the
DNA resulting from cleavage
forms ladders reflecting discrete
units increasing in size by 200
nucleotides
• This means that nucleosomes cover
the bulk of DNA and protect from
Dnase digestion
– But this procedure reveals the
structure of DNA, and not any
specific genes
DNAse digestion of heat shock genes
• When genes are identified
within these ladders, they are
found in two forms
– Genes that are not transcribed are
also found to form ladders in
response to DNAse I
– Genes that are transcribed are
fragmented into smaller pieces
• The nucleosomes are gone in the
upstream regions of genes
undergoing transcription
– A heat shock gene was digested
over time following heat shock
and the upstream region identified
with a specific probe
• Following heat shock, the control
regions of the gene become
hypersensitive to digestion
Where there are no nucleosomes
• The nucleosome free sites
are not throughout the
entire gene, but in certain
places called
hypersensitive sites
– Hypersensitive sites
correspond to regions of
DNA that bind transcription
factors
– Thus hypersensitive sites
are found upstream of the
coding region of genes
– They also may be found
wherever transcription
factors bind
• For eukaryotic genes, that
is not always just 5’ to the
transcriptional start
Other alterations
to transcriptionally
active DNA
• Loss of histone H1
– This is the histone that
exists between the
DNA/histone octomer coils
• Loss of methyl group from
5 methyl cytosine in CpG
islands
– Transcriptionally silent
DNA tends to have more 5
methyl cytosine than active
DNA
A clinical example
- thalassemia
• The shutdown of g globin is
due to methylation of the
upstream region of the genes
before and after birth
• In the human disease
thalassemia, b and d globin
chains are lost due to
mutation of the globin genes.
• One therapy involves
administration of 5
azacytidine
– Incorporation of this nucleotide
results in a loss of methylation
– This results in the activation of
fetal globin genes which
assume some of the oxygen
carrying capacity of the mutant
globin
Histone acetylation
Acetylation sites
Of Histone H4
• Histones have two functional
domains
– One for binding other histones
and wrapping DNA around the
nucleosome core
– The other is a modification site
for control of histone assembly
• Multiple lysine residues are
presented to the exterior of the
histone
• Histones are acetylated prior to
import into the nucleus
following their synthesis on
ribosomes
– They are actively assembled on
DNA by an enzymatic
mechanism
Acetylation near
transcriptionally active
genes
• A nuclear histone
acetylase further acts on
histones H3 and H4
– Increasing acetylation
decreases the affinity of
the histone octomer for
DNA
– This makes the DNA
more available for
binding interactions with
other proteins
– Histones are moved out
of the way by an ATP
driven process involving
a multiprotein complex
• Repressors may
stimulate deacetylation
• Activators may
stimulate acetylation
Eukaryotic promoters
• Why are they subject to positive regulation?
– The genes within are sequestered because of chromatin structure
– The size of the genome favors non-specific binding of regulatory proteins at
random
• In a diploid genome of 6 billion nucleotide pairs, a short sequence capable of binding
regulatory proteins would occur many times by chance
• So regulatory systems demand that multi-protein complexes form before a gene is
transcribed
– It is more efficient to negatively regulate the entire genome with a single
mechanism (chromatin structure) and then specifically turn on the set of genes
needed by the cell than to specifically negatively regulate every gene of a
eukaryote
• That would mean tens of thousands of repressors for each cell type
Promoters and Enhancers
• Promoters include, for example, TATAA boxes, GC boxes and
CAAT boxes that are responsible for positioning RNA polymerase II
at the beginning of a gene
– Polymerase II has no affinity for the TATAA box on its own.
– Assembly of a transcriptional complex depends on the sequence around the 5’
end of the gene
• Enhancers are sequences that are distant from the promoter but
positively affect its function
– They may be pointed in either orientation
Three classes of •
transcription factors
Basal (general)
transcription factors
– These interact directly with
RNA polymerase II or with
each other in building a
complex around the
promoter
– They also recognize the
promoter sequences
• The TATA box is highly
conserved
• The TATA binding protein +
transcription factors for
polymerase II (TF II) assemble
and provide the minimal
assembly for transcription
• But transcription still requires
a positive signal
– This complex marks the spot
where RNA polymerase is to
bind and begin transcription
Enhancer
binding proteins
• Also known as DNA binding
transactivators
– These bind enhancers that are far
away from the promoter
• They recognize the specific
enhancer sequence
• Some enhancer binding proteins
work on a large number of genes,
permitting coordinate control of
transcription
• Others are specific to a single
gene
– They then loop inward toward the
promoter so that the enhancer
binding protein can interact with
the basal transcription factors at
the promoter site
• Protein-protein interactions are
mediated through motifs such as
the leucine zipper and the helix
loop helix
Coactivator proteins
• These bind RNA
polymerase II complexes
and enhancer binding
proteins and mediate the
signaling between them
• RNA polymerase II may
carry the coactivator
proteins with it as it
transcribes
• Coactivators are necessary
for transcription
The process of
transcriptional
activation
• Remodeling chromatin
– May involve
• Demethylation of 5 methyl C
• Acetylation of histones
• Binding of basal transcription
factors
• Transactivator binding enhances
the remodeling of chromatin and
facilitates opening up chromatin
structure
– This helps other enhancer binding
proteins to interact with exposed
DNA sequence
• Transactivators interact with
coactivators and help RNA
polymerase position itself on the
transcription complex at the
TATA box
Induction and
repression
• Inducibility and especially
repression is not as common a
phenomenon in eukaryotic cells
– Especially higher eukaryotic cells
• The larger the organism, the more
stable the environment a cell
experiences
– So it needn’t respond to radical
changes in the environment
• However some transcriptional
regulation is still necessary
– Transactivators can serve the function
of inducers or repressors
– A repressor generally inhibits the
function of an inducer by some
mechanism
• Competitive binding
– To DNA
– To basal transcription factors
• Directly binding the activator
Induction and
repression
• Binding to a small
molecule can result in an
increase or decrease in the
ability of a transactivator
to work
– Steroid hormone receptor
becomes a functional DNA
binding transactivator in
response to binding its
ligand
• Binding to a ligand displaces
HSP90 (a heat shock protein)
and permits translocation to
the nucleus and subsequent
DNA binding
AD: activator domain
DBD: DNA binding domain
LBD: ligand binding domain
– In the absence of ligand, it
interferes with transcription
and thus becomes a
repressor
• The GAL genes of yeast
A specific example
– These are a set of individual genes under coordinate control
• Eukaryotes don’t have operons
– Each gene has a promoter and set of enhancers (called UAS)
• Turning on one of the GAL genes means activating a set of enhancer
binding proteins and coactivators that turn on all of the other GAL genes
– The products of the genes are needed for importation of galactose and
its metabolism
The GAL genes can be repressed
• The logic is the same as
with bacteria
• When glucose is
present, there is no
necessity to make
galactose importation
and metabolizing
enzymes, so the genes
are shut down
– This repression
overrides induction
Induction
• Gal4p is a transactivator
that induces transcription at
a GAL locus by interacting
with the coactivator
assembly at promoter
– In the absence of galactose,
Gal4p is sequestered by
another regulatory protein
Gal80
– Gal4p is displaced from
Gal80 by Gal3p when Gal3p
binds galactose
– Thus in contrast to bacterial
inducers, the ligand binding
and the DNA binding
proteins are not the same
Minimum structure
of enhancer binding •
proteins
Each must
– Bind its target DNA
– Bind the promoter complex and/or
activating proteins
• The ability of a protein to
perform each function is due to
functional domains in the
protein
– The domains permit interaction
with other proteins and specific
recognition of DNA sequence
– In addition to DNA and protein
interaction domains, there are 3
common types of activator
domains
• Acidic – Gal 4p
• Glutamine rich – SP1
• Proline rich - CFT1
GAL 4p
• This has a domain
resembling a zinc finger
– Instead of two cys and two his
coordinating a Zn , it has 6
cys residues
• It is a homodimer, bound by
interactions of two coiled
coils
• The two zinc fingers interact
with a palindromic sequence
• The protein is controlled by
another domain that is rich
in aspartic and glutamic acid
residues
– This was identified by
constructing mutants of the
Gal4p gene that substituted
other amino acids in this
domain
– The mutants lost function
• SP1 binds the GC box
– GC boxes are located close to the TATA sequence
– SP1 is a very common enhancer binding protein
• Many genes lack a GC box
– There are 3 Zn fingers for DNA binding
– Two glutamine rich activator domains
• CTF1 binds the CAAT box
– The DNA binding domain is unique and is neither helix turn helix or a zinc
finger
– The activation domain is proline rich
SP1 and CTF1
Domain
swapping
• Since the domains for DNA
binding and activation are
distinct, their domains may be
separated on the level of DNA
– By taking a domain for DNA
binding and adding it to a domain
for activation, a new protein may
be engineered
– This binds the DNA sequence
specified by one gene, and
responds to the signals of another
– Such experiments permit the
manufacture of proteins with
unique control abilities
• Although not therapeutically
useful right now, they are
important experimental tools in
defining the way that genes
respond to external signals.
• Gene expression in
multicellular organisms is
often controlled by
intercellular signaling
– Some genes are directly
responsive to environmental
stimulus however
• UV induction of DNA repair
enzymes
• Stress response (heat shock)
genes
• Signaling takes two forms
– Hormones may be bound by
• Membrane bound receptors
• Diffusible receptors
Regulated gene
expression
Diffusible
receptors
• Diffusible receptors act by
directly binding a hormone
and then moving into the
nucleus
– Hormone binding induces a
conformational change that
permits the receptor to act as a
transcriptional activator
• Diffusible molecules can be
transactivators
– “trans” means something that
acts on a gene that originates
from another site. In this
fashion they resemble the
activators of bacteria
• However hormones are made by
one cell in order to command a
transcriptional response in
another cell
• Bacterial effectors are nutrients
or their metabolites or analogs
Steroid
hormones
• These are
– endocrine hormones
– hydrophobic molecules that are
synthesized using cholesterol as a
precursor
– made by certain cell types and
secreted in response to
biochemical, developmental or
neurological signals
– carried by the blood from their cell
of origin to target cells either
dissolved or by a protein carrier
• Many are too hydrophobic to
dissolve directly in blood
• They enter a cell by dissolving
in the plasma membrane and
diffusing to their receptor
The steroid
hormone receptor
• This is a DNA binding protein
with a hormone binding domain
at the carboxyterminal end of
the protein
• There are several related types
– Each receptor has a specific
complement of transcription
factors it must interact with which
vary from one receptor to another
and one cell type to another
• They are all related in structure
– The DNA binding domain
contains two Zn fingers and is in
the middle of the protein
– The domain that interacts with
transcription factors is amino
terminal and varies in structure
• The hormone binding region is
highly variable in structure
– Each must recognize and bind its
cognate ligand
• Loss of responsiveness to a
hormone can be caused by
changes in any of the three
domains
M
U
T
A
T
I
O
N
S
– Hormone-ligand complexes may
serve either positive or negative
regulatory functions
• Mutations prevent transcriptional
activation or repression in
response to hormone binding
– Mutation in the androgen
binding domain of the androgen
receptor creates androgen
unresponsiveness
– Mutation in the DNA binding or
transcription activation domains
would mean the protein could
bind androgen, but nothing
would happen
» This results in
developmental
abnormalities such as XY
females
» To the left are four XY
siblings suffering from
androgen insensitivity
The cis
elements
• Cis refers to sequence involved
in gene expression
– Trans elements interact with cis
elements but arise from other genes
– The glucocorticoid responsive
element (GRE) and estrogen
responsive element (ERE) share
sequence homology
• The cis elements that are
important in hormone
responsiveness are the binding
sites for the hormone-receptor
complexes
– Hormone responsive elements: HRE
– These are direct repeats that interact
with the Zn finger domains
• The consensus sequences for these
receptors are very similar
• This reflects the similarity in the Zn
finger domains among the various
receptors
Receptor –
DNA binding
• Following binding of a
hormone, the receptor
diffuses to the nucleus and
binds the HRE
• The receptor is a dimer
– Each subunit of the dimer
binds to one of the two repeat
elements
– The strength of binding is
determined by the variation of
the HRE away from the
consensus sequence
– The stronger the binding
between the receptor and
HRE, the longer the receptor
will remain bound and the
longer transcription will be
activated
• Binding is an all or none
event
• If bound, activation due to
the receptor is full
Phosphorylation of
transcription factors
• Transcription factors are subject to
phosphorylation on serine and
threonine residues
• This is the result of second
messenger activation of serinethreonine kinases or ras activation
• In abnormal, though common,
conditions, such as mutations or
viral infections, gene expression is
deregulated and genes are
inappropriately expressed because
of deregulated phosphorylation
mechanisms
– This is because second messengers
activate a complex cascade of
enzymatic steps that can be perturbed
at many different points
– Here the transcription factor Elk-1 is
activated through phosphorylation
Repression
• This occurs at the transcriptional and translational
level
– Genes are usually turned off as a default at the
transcriptional level
• But this does not mean the mRNA is gone
– It could have been stabilized through sequestration
– Translational regulation permits rapid responsiveness
• The primary transcript of a gene may take several minutes to
synthesize because of its size
• It also must be spliced and transported to the ribosomes
• A sequestered transcript that is released in response to a signal
is faster
Translational
repression affects
more than just
ribosomal
proteins
• The distribution of mRNA
within some cells creates a
distribution of protein
inside a cell
– This results in intracellular
protein gradients that are
important in development
Regulatory
mechanisms of
translational
initiation
1. Inhibition of initiation factors
through phosphorylation
– eIF phosphorylation inhibits its
function and can be reversed
through dephosphorylation
2. Inhibition of initiation factors
by binding to specific factors
– Interference with eIF4E and eIF4G activity by 4E-BP’s.
3. Inhibition of specific mRNA
by binding of inhibitory
proteins to sequences in the
3’untranslated region
Phosphorylation of
eIF-2 inhibits its
activity
• Maturation of red blood
cells involves a stage in
which reticulocytes translate
mRNA left behind after the
loss of the nucleus
• Reticulocytes regulate the
amount of globin
synthesized by
phosphorylating eIF-2
– When there is heme
deficiency globin synthesis is
wasteful since hemoglobin
cannot be synthesized
• Low heme activates HCI
which phosphorylates eIF-2
– Phosphorylated eIF2 binds
eIF2 binding protein and is
unavailable for translational
initiation
eIF4E inhibition
• eIF4E is necessary to bind
the 5’ CAP in order to from
an initiation complex for
translation
– Normally it binds eIF4G
• Maskin binds eIF4E
(preventing it from binding
eIF4G) when it is bound to
an mRNA through
interaction with CEPB
Developmental control •
of gene expression
The study of fruit fly
development resulted in
the discovery of a number
of genes involved in
human disease
– Although fruit fly
development is greatly
different in the processes
leading to the final form,
the activation of genes and
the structure of gene
products and their
participation in the
formation of patterns and
structures have parallels in
human gene regulation and
the structure of human
regulatory gene products
Fly development is controlled by
gene expression
• The conceptually difficult part of this is to
understand how a single cell can create multiple,
morphologically different structures starting from
a seemingly symmetrical, undifferentiated state
merely by dividing.
• It is easier to think of the process in parts and then
add up the whole than to see a cell turn into a fly
and attempt to understand the entire process at
once
• Maternal genes
Three gene families
are responsible for
early development
– Made by the female and
exist within the egg at
the time of fertilization
• Responsible for
establishing the polarity of
the early embryo
• Zygotically acting genes
– Segmentation genes
• These establish a
repeating pattern of body
segments
– Homeotic genes
• These establish the
identity of the segments
Polarity
• This is a distinction in
structure established
between two poles.
– The distinction needn’t be
great, only a morphological
difference is enough to
create polarity
– Without polarity, further
structures would have no
way of organizing
themselves
• Segments would be
repeating structures that
are all the same
– Establishing polarity is thus
the earliest developmental
event
• Polarity is actually
established by the
assymetry of the egg
• This yields pole cells on
one end of the zygote
• This is obvious in the formation
of the fly abdomen
Segmentation
– Repeating abdominal segments are
very similar in appearance
– However this patterning extends
from end to end of the fly
• The patterns are given different
identities by homeotic genes
• Thus the head and abdomen begin
as segments similar to abdominal
segments
– But polarity makes them
different, and therefore the genes
that are expressed within each
segment differs
– Segments are created and further
divided into smaller segments
• Gap genes create the largest
segments
• Pair rule and segment polarity
genes subdivide the largest
segments
• These give rise to the dramatic
mutants of Drosophila
• Once segments are established with
the proper polarity, homeotic genes
create unique structures
• Mutation of a particular homeotic
gene results in the formation of a
structure that is due to the action of
another homeotic gene
– The homeotic genes are controlled by
their position within a gradient of polarity
– So if one segment was destined to give
rise to antennae, but lacked the homeotic
gene due to mutation, it would create the
next most available structure
Homeotic genes
Antennaepedia
• Antennaepedia represents the
mutation of a gene that
would create an antennae
– Antennapedia is a transcription
factor that coordinately
controls expression of genes,
that when expressed result in
an antennae
• In its absence, the next most
similar structure is a leg
– The normal leg is also formed
in the next segment
– The gene encoding the protein
controlling leg development is
expressed at lower
concentrations in the head
segment than antennapedia
gene, but in the absence of
antennapedia, it is the most
highly expressed protein
capable of activating genes
that result in a structure
Key genes are
expressed early
• Maternal genes establish
polarity due to formation of
gradients
– Front to back and top to bottom
gradients establish anterior
posterior and dorsal ventral
gradients
• When cells are formed in the
blastoderm, they form within an
environment in which the
concentration of transcription
factors will vary along one axis
– This varies the type and numbers
of genes that are expressed within
any cell
• To the left is bicoid RNA
(upper) and bicoid protein
(lower) in the early embryo
– The RNA gradient is present in the
egg and establishes the protein
gradient
• Bicoid is a maternal gene that
controls expression of
segmentation genes
A few examples of
developmental
genes
– It is a transcription factor that
activates segmentation genes
– And a translational repressor
• It appears in the anterior of an egg,
and its concentration falls of
towards the posterior
• The gradient is maintained during
formation of the larvae
• Experiments with bcd mutants
– (a) A failure of bicoid to be expressed
means a fly develops with two
posteriors rather than an anterior and a
posterior
– (b)injecting cytoplasm from a normal
embryo rescues the embryo (makes an
anterior)
– ( c) injecting bicoid mRNA also
rescues
• It represses translation of caudal
in the anterior of the fly larvae
What bicoid does
– Caudal is a transcription factor
found uniformly throughout the
larvae, and it creates the posterior
end
• And activates expression of
hunchback
– Hunchback is a transcription factor
that creates the anterior end
• The bicoid gradient means it has
these effects only in the anterior
end
• Without bicoid, caudal is not
repressed in the anterior end and
hunchback is not transcribed
Nanos
• This is a translational
repressor that is found at
highest concentrations in the
posteror of a fly larvae
• It acts in concert with the
uniformly distributed
pumilio gene product to
translationally repress
hunchback
• This results in establishment
of high caudal gene product
and inactivated hunchback
mRNA, meaning the
posteriorizing effect of
caudal dominates
Some Gap genes
• Gap genes create a gross
form of segmentation in
the early embryo
• They are overlayed onto
the pair-rule gene
expression to create
complex transcriptional
signals
• These are the expression
patterns of the hb-z, Kr
and kni genes
fushi tarazu
and eve
• These are two segmentation
genes known as pair rule genes
that split a segment in two
– Ftz establishes the “pair rule”
• Two segments form out of one
• Without it the fly forms 7 rather
than 14 segments
• Ftz (blue) is expressed in each
segment, in the anterior half of
the segment
– This expression pattern is again the
result of the action of an anterior
posterior gradient, but now within
each segment
• Eve (brown) for even-skipped
are expressed in the posterior
half of each segment
• Both ftz and eve are
homeodomain transcription
factors that control expression of
genes expressed in the segments