Molecular Evolution and
the Origins of New Genes
Carol Eunmi Lee
Evolution 410
University of Wisconsin
Outline: Where do new genes come from?
1. Gene Duplications and Differentiation
– Example: Evolution of steroid hormone
receptors
– The ancient estrogen receptor
2. Specific Example: Evolution of the
aldosterone - Mineralocorticoid Receptor (MR)
complex
– Evolutionary pathway of permissible mutations
Evolution of Steroid Receptor
Gene Families
• Evolutionary History of Function
• Gene Duplication leading to
evolutionary origins of novel functions
• How mutations interact to modify
function
The origin of new genes
Gene Duplication followed by differentiation
(neofunctionalization, subfunctionalization,
etc.) lead to new gene families:
Examples:
Receptors, Enzymes, Developmental genes, etc.:
–
–
–
–
–
–
Hox clusters
Osmoregulatory ion uptake enzymes (ATPases)
Cytp450s (detoxification enzymes)
Olfactory genes
Opsin genes
Hemoglobin
Gene Duplications
• Main source of novel genes
Sources of Genetic Variation
(type of mutation)
• Gene duplications,
followed by differentiation
• End up with “gene family”:
different opsin genes,
hemoglobin, ATPases,
etc.
Fate of Duplicated Genes
Loss of function of
extra gene copy
New function of
extra gene copy
Partition of function
between the gene copies
What are Steroid Receptors?
Steroid Hormone Receptors
• Intracellular receptors (typically cytoplasmic)
that bind to ligands (e.g. steroid hormones)
• The ligand (hormone)-receptor complex are
transcription factors
• These transcription factors initiate signal
transduction, which lead to changes in gene
expression
(hormone)
(hormone-receptor
complex)
transcription factor
Steroid Hormones are lipid soluble, bind to cytoplasmic
receptors and then enter the nucleus
Steroid Hormones are
lipid soluble, bind to
cytoplasmic receptors
and then enter the
nucleus
Hormone (ligand) + Hormone Receptor (ER) form a complex
that acts as a transcription factor, binding to DNA and
regulating gene expression
Evolutionary History of Steroid Receptors
Glucocorticoid
receptor
Baker, ME. 2001. Adrenal and sex steroid
receptor evolution: environmental implications.
Journal of Molecular Endocrinology 26:119–
125
Mineralocorticoid
receptor
Sex steroid
response
probably arose
in the early
Cambrian
Progesterone
receptor
Androgen
receptor
Estrogen
response
evolved in
jawless fish or
tunicates (early
chordates)
Eel ERb
Human ERb
Trout ERa
Xenopus ERa
Human ERa
Estrogen
Receptors
the most ancient of
the adrenal and
sex
steroid receptors
• More Ancestral receptors
tend to be less specific…
Glucocorticoid
receptor
• Specificity for particular
ligands often evolves over
time
Mineralocorticoid
receptor
• A gene encoding one
receptor might
duplicate and evolve
specificity for different
ligands
Progesteron
e
receptor
Androge
n
receptor
Eel ERb
Human ERb
Trout ERa
Xenopus ERa
Human ERa
Estrogen
Receptors
the most ancient of
the adrenal and sex
steroid receptors
Pollutants, pharmaceuticals,
pesticides, plastics, etc. often have
estrogenic effects because:
Estrogen receptors are
fairly nonspecific
• They are ancestral receptors
• Bind to multiple ligands,
~12 estrogens (estradiol, estriol,
estrone…etc.)
Estrogen Receptor-alpha;
Ligand-binding domain complexed
to estradiol
So... many compounds will bind to estrogen receptors,
more so than to testosterone receptors or other steroid
hormone receptors
Estrogens in Humans
• Estrone
• Estradiol
• Estriol
• Several others
Estrogenic
Compounds
Many chemicals in
the environment
(pesticides,
plastics) will bind to
estrogen receptors,
which are ancient
and non specific
hormone receptors
The Estrogen Receptor Relative Binding Affinities of 188 Natural and
Xenochemicals: Structural Diversity of Ligands
Toxicoogical Sciences (2000) 54: 138-153
Robert M. Blair et al.
Abstract
We have utilized a validated (standardized) estrogen receptor (ER) competitive-binding
assay to determine the ER affinity for a large, structurally diverse group of chemicals. Uteri
from ovariectomized Sprague-Dawley rats were the ER source for the competitive-binding
assay. Initially, test chemicals were screened at high concentrations to determine whether a
chemical competed with [3H]-estradiol for the ER. Test chemicals that exhibited affinity for
the ER in the first tier were subsequently assayed using a wide range of concentrations to
characterize the binding curve and to determine each chemical's IC50 and relative binding
affinity (RBA) values. Overall, we assayed 188 chemicals, covering a 1 × 106-fold range of
RBAs from several different chemical or use categories, including steroidal estrogens,
synthetic estrogens, antiestrogens, other miscellaneous steroids, alkylphenols, diphenyl
derivatives, organochlorines, pesticides, alkylhydroxybenzoate preservatives (parabens),
phthalates, benzophenone compounds, and a number of other miscellaneous chemicals. Of
the 188 chemicals tested, 100 bound to the ER while 88 were non-binders. Included in the
100 chemicals that bound to the ER were 4-benzyloxyphenol, 2,4-dihydroxybenzophenone,
and 2,2′-methylenebis(4-chlorophenol), compounds that have not been shown previously
to bind the ER. It was also evident that certain structural features, such as an overall ring
structure, were important for ER binding. The current study provides the most structurally
diverse ER RBA data set with the widest range of RBA values published to date.
Estrogen binding affinities for 188 chemicals
•
A ring structure is of primary importance
With the exception of 4 chemicals (kepone,
norethynodrel, 5a-androstane-3a, 17b-diol, and 5aandrostane-3b,17b-diol)
•
Phenol ring (Hydroxyl group on the phenyl
[=benzene]): Thought to act as a hydrogen bond donor
and acceptor in the ER-binding site
•
Longer side chains
•
In general, chemicals with 2 ring structures separated
by 2 carbon atoms (steroidal and synthetic estrogens
and diphenyl ethanes) have higher affinities than
chemicals with a single ring structure or 2 rings
separated by one carbon atom (but the latter
compounds might still bind, perhaps not as strongly).
Estrogenic
Compounds
Many chemicals in
the environment
(pesticides,
plastics) will bind to
estrogen receptors,
which are more
ancient and non
specific hormone
receptors
Evolution of Function of
Hormone Receptors
How does specificity for particular ligands
evolve?
Fate of Duplicated Genes
I will now give an example of gene
duplication followed by evolution of
increased specificity of function
Loss of function of
extra gene copy
New function of
extra gene copy
Partition of function
between the gene copies
Question:
• How would specificity for a new ligand
(hormone) arise after gene duplication?
•
How would an integrated molecular system
evolve, such as the functional interaction
between a hormone and receptor?
•
That is, how could a new hormone evolve if a
receptor is not present, and visa versa?
Example
• Evolution of function of the aldosterone Mineralocorticoid Receptor (MR) complex
• How did this ligand-receptor relationship
evolve?
Bridgham et al. 2006. Science. 312:97
• Aldosterone is thought to be a recently
derived hormone, and a tetrapod
(vertebrates with four feet) specific
hormone, absent in more anciently
derived species
• Mineralocorticoid receptor (MR) and the
Glucocorticoid receptor (GR) descended from a
gene duplication deep in the vertebrate lineage
(~450+ mya) and now have distinct signaling
functions
• In most vertebrates, GR is activated by the stress
hormone cortisol to regulate metabolism,
inflammation, and immunity
• MR is activated by aldosterone to regulate
reabsorption of ions and water and secretion of
potassium in the kidneys. MR can also be
activated by cortisol
The gene duplication
event leading to MR
and GR occurred
>450 million yrs ago
How did these two hormone
receptors evolve distinct functions
(binding to different ligands)?
Background
• Functional assays indicate that the ancestral (basal)
receptors are activated by very low doses of aldosterone,
cortisol, and 11-deoxycorticosterone (DOC); they are
similar in this respect to MRs of tetrapods and teleosts
(Fig. 2 -next slide)
• The only receptors insensitive to aldosterone are the GRs
of tetrapods and teleosts
• Given these results, the most parsimonious scenario is
that AncCR (Ancestral receptor) was capable of being
activated by aldosterone (even though it would not yet
have existed) and that aldosterone sensitivity was lost in
the GRs of bony vertebrates (see Fig. 1)
GRs are not the aldosterone receptor
The GR and CRs of
more ancestral fishes do
respond to aldosterone
The GRs of bony
fishes and
tetrapods (which
evolved later) do
not respond to
aldosterone
• How might have the aldosterone-MR
partnership have evolved?
• If the hormone is not yet present, how could
selection drive the receptor’s affinity for it?
• Conversely, without the receptor, what selection
pressure could guide the evolution of the
ligand?
Test Hypothesis:
• Joe Thornton’s lab performed gene resurrection
to experimentally examine the function of the
ancestral corticoid receptor (AncCR)
• Inferred the maximum likelihood (ML) amino acid
sequence of AncCR’s ligand-binding domain (see
Fig. 1)
• Synthesized the AncCR-LBD sequence and
expressed it in cultured cells; using a reporter
assay
Results
• AncCR is a sensitive and
effective aldosterone
receptor (Fig. 3A)
• This result is surprising, because aldosterone
has long been considered a tetrapod-specific
hormone (evolved later)
• Aldosterone is absent from the plasma of
lamprey and hagfish (more ancient vertebrates)
(Fig. 3B)
Ancestral receptor is activated by aldosterone
But, aldosterone is absent
in the blood of basal fish
• WHY would the ancient corticoid
receptor respond to a not yet existing
hormone (aldosterone)?
• And how would the specificity between
MR and aldosterone evolve?
Fig. 4. Evolution of specific aldosterone-MR signaling by molecular
exploitation. (A) Synthesis pathway for corticosteroid hormones. Ligands
for the ancestral CR and extant MRs are underlined; cortisol, the ligand for
the tetrapod GR, is overlined. The terminal addition of aldosterone is in
green. Asterisks, steps catalyzed by the cytochrome P-450 11bhydroxylase enzyme; only the tetrapod enzyme can catalyze the step
marked with a green asterisk. (B) MR’s aldosterone sensitivity preceded
the emergence of the hormone. The vertebrate ancestor did not synthesize
aldosterone (dotted circle), but it did produce other corticosteroids (filled
circle); it had a single receptor with affinity for both classes of ligand. A
gene duplication (blue) produced separate GR and MR. Two changes in
GR’s sequence (red) abolished aldosterone activation but maintained
cortisol sensitivity [see (C)]. In tetrapods, synthesis of aldosterone
emerged due to modification of cytochrome P-450 11b-hydroxylase. mya,
million years ago. (C) Mechanistic basis for loss of aldosterone sensitivity
in the GRs. Phylogenetically diagnostic amino acid changes that occurred
during GR evolution were introduced into AncCR-LBD by mutagenesis.
Dose-response is shown for aldosterone (green), DOC (blue), and cortisol
(red). The double mutant (bottom right) has a GR-like phenotype. Arrows
shows evolutionary paths via a nonfunctional (red) or functional (green)
intermediate.
Extant MRs retain the ancestral phenotype, so the
specificity of the MR-aldosterone relationship is
actually due to the secondary loss of aldosterone
sensitivity in the GR (Fig. 4B), rather than evolution
of specificity for MR.
• Ancestral receptor was less specific:
activated by aldosterone, cortisol, and11deoxycorticosterone (DOC)
After gene duplication:
• MR retain binding capacity for aldosterone
• GR loses binding capacity for aldosterone
Which Mutations?
• The Thornton Lab explored which sequence
changes are on the branch where
aldosterone sensitivity was lost
• Introduced all four single GR-diagnostic
states and all six two-fold combinations into
AncCR-LBD using mutagenesis and
determined their effect on receptor function
Which Mutations?
• Replacement of Serine106 with Proline (S106P) and
Leucine111 with Glutamine (L111Q) conferred a GRlike phenotype
Non permissible
mutation, because
having mutation L111Q
first rendered the
protein non functional
Permissible
mutations; S106P
had to happen first
When each mutation
was introduced in
isolation, it was
discovered that both
are required to yield
the GR phenotype
• L111Q alone radically reduces activation by all ligands tested
• S106P reduces aldosterone (green) and cortisol (red)
sensitivity, but this receptor remains highly DOC-sensitive (blue)
• In the S106P background, L111Q further reduces aldosterone
sensitivity but now restores cortisol response to levels
characteristic of extant GRs
What are these mutations
doing at the structural level?
• Instead of using the ancestral AncCR, the
structures of AncGR1 and AncGR2 were
compared to determine the mechanism by which
these two substitutions shift function
• Ancient GR1 and GR2 were reconstructed using
homology modeling and energy minimization
based on the AncCR and human GR crystal
structures
2007. Science 317:1544
The major structural difference
between AncGR1 and AncGR2
involves Helix 7 and the loop preceding
it, which contain S106P and L111Q and
form part of the ligand pocket (Fig. 2B).
In AncGR1 and AncCR, the loop’s position
is stabilized by a hydrogen bond between
Ser106 and the backbone carbonyl of
Met103.
The movement of helix 7 dramatically repositions
site 111, bringing it close to the ligand
In this conformational background,
L111Q (leucine to glutamine) generates
a hydrogen bond with cortisol’s C17hydroxyl, stabilizing the receptorhormone complex. Aldosterone and
DOC lack this hydroxyl, so the new bond
is cortisol specific
Replacing Ser106 with proline in the derived GRs
breaks this H bond and introduces a sharp kink into
the backbone, which pulls the loop downward,
repositioning and partially unwinding helix 7
The two substitutions
destabilize the receptor complex
with aldosterone or DOC
Achieves stability with cortisol,
switching preference to cortisol,
and not aldosterone
This mode of structural evolution is termed “conformational
epistasis” because one substitution remodels the protein
backbone and repositions a second site, changing the
functional effect of substitution at the second site
Permissible Evolutionary Pathways:
Permissive substitutions stabilized specific
structural elements, allowing them to tolerate later
destabilizing mutations that conferred a new
function
Some evolutionary pathways are permissible,
whereas others are not. Sometimes, some
mutations must occur first.
Evolution of specificity of function
• Structural studies of human GR have shown that these
two residues change the architecture of the ligand-binding
pocket and alter contacts with steroid in ways that exclude
aldosterone and facilitate cortisol activation
• Results indicate that aldosterone specificity of MR arose
from two crucial Amino Acid replacements in the GRs that
wiped out ancestral sensitivity to aldosterone
• These changes result in evolution of a more
specific endocrine response, allowing electrolyte
homeostasis to be controlled without also
triggering the GR stress response
Molecular Exploitation
• Functional interaction between aldosterone and
mineralocorticoid receptor evolved by a stepwise selective
process
• Ancestral gene resurrection demonstrates that long before the
hormone evolved, the receptor’s affinity for aldosterone was
present due to its similarity to more ancient ligands (probably
DOC) – also, ancestral receptor was less specific…
• Two amino acid changes in the ancestral sequence resulted in
the evolution of present-day receptor specificity
• Results indicate that tight interactions could evolve by
molecular exploitation—recruitment of an older molecule,
previously constrained for a different role, into a new
functional complex
1. Evolution of mineralocorticoid and glucocorticoid receptors from an
ancestral receptor is an example of:
a) Evolution occurring through gene duplication followed by
increase in specificity of function of GRs
b) Mutations occurring independently, leading to the same amino
acid substitutions in both receptors (Parallel Evolution)
c) Evolution occurring through gene duplication followed by
subfunctionalization between MRs and GRs for aldosterone
specificity
d) Evolution by genetic linkage
e) Evolution occurring through gene duplication following by
mutational accumulation
2. P, Q, and R represent different mutations leading to evolution of
an enzyme from LDH-B x to LDH-B z. The Q mutation leads
to loss of function of the enzyme, but the R mutation stabilizes
the loss of function of Q. Which of the following is NOT likely
to be a possible evolutionary pathway?
a) P, PR, PQR
b) R, RP, RPQ
c) P, PQ, PQR
d) R, RQ, PQR
3. A hypothetical ancestral hormone receptor is broadly sensitive to
many different ligands (hormones). Particular mutations change
the sensitivity of the receptor to three ligands, shown in the
graphs below. Which receptor possesses mutation(s) that
would NOT be considered permissible?
The graphs below show the effects of mutations on sensitivity of corticoid
hormone receptors to aldosterone (solid green line), DOC (dashed blue
line) and cortisol (dashed red line).
(1)
(2)
(3)
(4)
4. Which of the following is the most permissible evolutionary pathway
toward lowered sensitivity to aldosterone, while maintaining relatively
high sensitivity toward DOC and cortisol?
(a) 1 --> 2 --> 4
(b) 1 --> 3 --> 4
(c) 4 --> 3 --> 1
(d) 1 --> 3 --> 2
(e) 4 --> 2 --> 3
5. Which of the following is FALSE regarding the figures on the
previous slide?
(a) AncCR is sensitive to aldosterone
(b) AncCR is also activated by cortisol
(c) The L111Q mutation rendered the receptor less functional
overall
(d) The S106P mutation resulted in reduced sensitivity to
aldosterone relative to other hormones
1: a
2: c
3: b
4: b
5: d