Download Functional variation of MC1R alleles from red

© 2001 Oxford University Press
Human Molecular Genetics, 2001, Vol. 10, No. 21 2397–2402
Functional variation of MC1R alleles from red-haired
individuals
Eugene Healy, Siobhán A. Jordan, Peter S. Budd, Ruth Suffolk, Jonathan L. Rees1 and
Ian J. Jackson*
MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK and 1Department of
Dermatology, University of Edinburgh, Lauriston Building, The Royal Infirmary, Edinburgh EH3 9YW, UK
Received June 8, 2001; Revised and Accepted July 30, 2001
Red hair in humans is associated with variant alleles
of the αMSH receptor gene, MC1R. Loss of MC1R
function in other mammals results in red or yellow
hair pigmentation. We show that a mouse bacterial
artificial chromosome (BAC) which contains Mc1r
will efficiently rescue loss of Mc1r in transgenic mice,
and that overexpression of the receptor suppresses
the effect of the endogenous antagonist, agouti
protein. We engineered the BAC to replace the
mouse coding region with the human MC1R
sequence and used this in the transgenic assay. The
human receptor also efficiently rescued Mc1r deficiency, and in addition, appeared to be completely
resistant to the effects of agouti, suggesting agouti
protein may not play a role in human pigmentary
variation. Three human variant alleles account for
60% of all cases of red hair. We engineered each of
these in turn into the BAC and find that they have
reduced, but not completely absent, function in
transgenic mice. Comparison of the phenotypes of
αMSH-deficient mice and humans in conjunction with
this data suggests that red hair may not be the null
phenotype of MC1R.
INTRODUCTION
The wide range of hair and skin colour in mammals, including
humans, is partly due to different contributions of two melanin
types, black/brown eumelanin and red/yellow phaeomelanin
(1–3). Eumelanin synthesis is stimulated in melanocytes by
activation of the α melanocyte stimulating hormone (αMSH)
receptor, MC1R (4). Mutations of MC1R produce altered
pigmentation in mice and other mammals (5–11). Loss of function of MC1R results in a red coat in guinea pigs, horses and
pigs, a yellow coat in mice and either red or yellow hair
(depending on the breed) in dogs. Point mutations within
MC1R that confer constitutive or ligand-independent activity
result in a dominant black phenotype in mice, cattle, sheep,
foxes and possibly in dogs. An endogenous antagonist of
MC1R, agouti protein, binds to the receptor and results in
phaeomelanin synthesis in the melanocyte (12–14). In mice,
agouti is expressed in a pulse during hair growth, resulting in a
yellow subapical band in the otherwise black hair. Overexpression of agouti in mutant mice produces a dominant
yellow phenotype, whereas loss of agouti results in hairs that
are uniformly black (11). Surprisingly, mice that lack proopiomelanocortin (POMC), the precursor of αMSH, are not
yellow but are quite dark (15), suggesting that there is substantial ligand-independent signalling from MC1R. Furthermore,
these mice show an agouti pattern on their hairs, indicating that
agouti protein modulates this signalling.
Human MC1R sequence variants are associated with red hair
and fair skin in the Caucasian population (16–19). These
variant alleles are extremely common; in northern European
populations <50% of the MC1R genes encode the ‘wild-type’
or consensus protein. Three alleles in particular, Arg151Cys,
Arg160Trp and Asp294His together make up 22% of the
MC1R genes and account for 60% of all cases of red hair. In
most families MC1R alleles behave as normal recessive mutations, although there are a few individuals with red hair in
whom no MC1R variant can be found. Rare individuals lack
POMC and therefore lack αMSH, and consequently have red
hair, adrenal insufficiency and obesity because of lack of
stimulation of the whole family of melanocortin receptors (20).
However, this does not seem to be the explanation for the vast
majority of red-haired individuals without MC1R variants, and
other, yet to be identified genes are probably playing a role.
When introduced into cells in culture, the common red-hair
associated variants produce a very severely impaired cAMP
response to αMSH (21,22). However, these experiments
provide only a crude measure of receptor function, relying on a
single assay (cAMP level) of activation in non-melanogenic
cells. Therefore, we have developed a sensitive in vivo assay
using transgenic rescue of Mc1r mutant mice.
We find that Mc1r-containing bacterial artificial chromosome (BAC) transgenes efficiently rescue or over-rescue the
yellow phenotype of Mc1r-deficient mice in a copy number
dependent manner. Human MC1R also rescues efficiently, and
the human receptor is much less sensitive to the antagonistic
action of mouse agouti protein, suggesting that agouti may not
*To whom correspondence should be addressed. Tel: +44 131 467 8409; Fax: +44 131 343 2620; Email: [email protected]
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
Present address:
Eugene Healy, Dermatopharmacology, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, UK
2398 Human Molecular Genetics, 2001, Vol. 10, No. 21
Table 1. Pigmentation and transgene copy number in transgenic recessive
yellow lines rescued by Mc1r
Line
Coat colour
Transgene copy number
A134
Agouti
1
A139
Agouti
1
A139.3
Agouti
1
A135.2
Agouti
2–3
A139.1
Dark agouti
3–4
A138.1
Very dark agouti
3–6
A139.2
Dark agouti
ND
Coat colour was determined on the first hair cycle. In general, the altered
pigmentation was maintained throughout subsequent hair cycles, with some
loss of darker (eumelanin) pigmentation after 9 months.
play a significant role in human hair colour. BAC transgenes
containing any of three common human MC1R variant alleles
result in partial rescue of coat colour. The common alleles that
result in red hair in humans alter melanin synthesis in vivo but
they are not complete loss-of-function mutations.
RESULTS
The BAC, CITB-484H6, contains the mouse Mc1r gene and
∼65 and 50 kb flanking the gene on the 5′ and 3′ sides, respectively. We injected the BAC into mouse embryos homozygous
for the recessive yellow (Mc1re) mutation. Nine positive transgenic founders gave rise to seven lines, all of which showed
rescue of the coat colour from yellow to agouti or darker
(Table 1). In addition to darkening of hair, the skin of the ear
and tail was also rescued from pale to darkly pigmented
(Fig. 1A) consistent with a role for MC1R in interfollicular as
well as follicular pigmentation. Comparison of BAC copy
number with the pigmentation in the rescued lines indicated a
dosage effect (Table 1). The agouti pattern of dorsal hair
pigmentation is characterized by a stripe of yellow phaeomelanin below the tip of a black, eumelanic, hair. This is
produced by a pulse of synthesis of agouti protein near the start
of the hair growth cycle. Transgenic Mc1re mice carrying one
or two copies of the BAC transgene were agouti in colour, indicating that the transgenic MC1R responds normally to agouti
protein. Animals carrying three or four copies had coats darker
than wild-type, in which the sub-apical yellow band was
reduced in length, whereas in mice with up to six BAC transgene copies the transgenic hairs are almost entirely black. It
appears that the antagonist effect of agouti at MC1R can be
overcome by increased copy number (and presumably overexpression) of the Mc1r gene. This could be due to summation
of a low level of constitutive signalling by MC1R even in the
presence of agouti, or to saturation of a limited pool of agouti
protein by increased receptor number.
We modified the BAC by recombination in Escherichia coli,
and replaced the mouse Mc1r coding region with that encoding
human consensus MC1R, upstream of an internal ribosome
entry site (IRES) and a lacZ reporter (23). Transgenic lines
were produced containing this modified BAC in recessive
Figure 1. Coat colour of recessive yellow (homozygous Mc1re) mice rescued
with Mc1r transgenic BAC. (A) Top, non-transgenic, agouti mouse; bottom,
transgenic recessive yellow mouse rescued with BAC encoding mouse Mc1r
(line A138.1, three to six copies). (B) Left, non-transgenic recessive yellow
mouse; right, transgenic mouse rescued with BAC carrying human wild-type
MC1R (line A167, five to seven copies). (C) Left, transgenic recessive yellow
mouse rescued with BAC encoding human wild-type MC1R (line A168, three to
five copies); right, non-agouti mouse. Note the non-agouti mouse has yellow fur
behind the ears, which is black in the transgeneic animal. (D) Top, non-transgenic recessive yellow mouse; bottom, transgenic mouse rescued with BAC
encoding Arg151Cys MC1R variant. (E) Bottom, non-transgenic recessive
yellow mouse; top, transgenic mouse rescued with BAC encoding Arg160Trp
MC1R variant. (F) Right, non-transgenic recessive yellow mouse; left,
transgenic mouse rescued with BAC encoding Asp294His MC1R variant.
yellow mice. Copy number analysis indicated that these
animals contained approximately four and six copies of the
transgene, respectively (Table 2). We assayed expression of
the human transgene by comparison with the endogenous
Mc1r (Table 2) and found the levels of the human MC1R
mRNA equivalent to an output per gene of ∼50% of the endogenous gene (i.e. four transgene copies express mRNA equivalent to two endogenous copies), possibly because the
IRES:lacZ destabilizes the mRNA. Expression of human
MC1R-encoding mRNA not only rescues the Mc1re mutant
phenotype but in both lines the hair colour is rescued from
yellow to entirely black with no sign of any phaeomelanin
synthesis (Fig. 1B and C, and Fig. 2). In addition, the ear and
tail skin shows over-rescue to a black colour. This degree of
over-rescue is much greater than that produced by up to four or
more copies of the BAC encoding mouse Mc1r. Human MC1R
is reported to be ‘super-sensitive’ to melanocortin peptides;
assayed as a cAMP response in transfected cells, it is eight
times more sensitive to αMSH and about 200 times more
Human Molecular Genetics, 2001, Vol. 10, No. 21 2399
Table 2. Pigmentation of human wild-type and variant MC1R transgenic recessive yellow lines
Line
MC1R construct
Coat coloura
Transgene copy numberb
Total transgene expressionc (N)
A167
Wild-type
Black
5–7
3.40 ± 0.37 (5)
A168
Wild-type
Black
3–5
1.60 ± 0.80 (8)
A184
Arg160Trp
Grey
2
0.48 ± 0.22 (4)
A185.2
Arg151Cys
Grey
2
0.58 ± 0.25 (6)
A186
Asp294His
Dark yellow
1
0.35 ± 0.14 (6)
A207
Asp294His
Dark yellow
1
ND
A207 homozygote
Asp294His
Dark yellow
2
ND
Coat colour was determined on the first hair cycle and was maintained throughout subsequent hair cycles. Line A207 was bred to homozygosity to allow comparison
of pigmentation between variant MC1R animals containing identical transgene copy numbers.
aFigure 1.
bWhole number range as determined by Southern blot analysis.
cAssayed on neonatal skin RNA by primer extension, mean ± SD.
sensitive to ACTH than the mouse receptor (24). The human
MC1R appears to be relatively insensitive to antagonism by
mouse agouti protein, with the consequence that MC1R signalling is maintained throughout the hair growth cycle, resulting
in an overall black phenotype. This insensitivity to agouti may
be direct (i.e. the human receptor binds the antagonist less
well) but is more likely the result of a higher level of signalling
from the more sensitive human MC1R such that signalling
occurs even in the presence of agouti. Data from the variant
receptors (below) also support the latter. It is worth noting that
in these experiments we are expressing the human receptor
from the mouse promoter and so may be generating a nonphysiological level of MC1R, and consequently a stronger
signalling response. Previous work has shown that mouse
agouti protein is 10 times more effective than the human agouti
protein in inhibition of the melanogenic response from MC1R
on human melanocytes (14). Given the lack of effect of the
more potent mouse agouti protein on human MC1R in our
transgenic assay, it is possible that agouti may not play a
significant role in determination of human hair colour;
however, the consequence of a much lower expression in
human melanocytes remains to be determined.
We used a photometric assay as an objective measure of
melanin content in dorsal hair from age-matched transgenic
mice (3). Phaeomelanin is more soluble than eumelanin at high
pH and so the fraction of alkaline soluble melanin quantifies
the phaeomelanin:eumelanin ratio. Yellow mice lacking Mc1r
have a ratio approximately 0.6, black, non-agouti mice have a
ratio <0.08, whilst in agouti mice it is approximately 0.1. Mice
expressing the human MC1R transgene have an average ratio
of 0.08, equivalent to that in black non-agouti animals (Fig. 2).
Three human MC1R variant alleles, Arg151Cys, Arg160Trp
and Asp294His, together account for >60% of all cases of red
hair and we therefore engineered each of these changes separately into the human coding sequence of the BAC (followed
by the IRES:lacZ sequence). Transgenic lines were generated,
containing each of the variants in recessive yellow mice. The
expression of each of the transgenes is shown in Table 2 and
the effect on the coat in Figure 1D–F. All MC1R variant transgenic lines showed some degree of rescue of eumelanin
synthesis and all had a coat darker than recessive yellow, but
paler than wild-type agouti, although none showed visible
rescue of tail and ear skin. The hair melanin type ratio in the
transgenic mice is intermediate between yellow and agouti
(Fig. 2). Arg151Cys and Arg160Trp result in very similar
ratios, ∼50% that of the Asp294His lines. Interestingly, all
lines showed the agouti hair pattern, unlike those rescued by
the consensus MC1R, indicating that the variant receptors were
antagonized by agouti protein. The phenotype also appears to
be less dosage sensitive. Line A207, carrying one transgene
copy of the Asp294His variant, was bred to make the transgene
homozygous to double the copy number and to compare the
phenotype with the lines carrying equivalent copies of
Arg151Cys and Arg160Trp variants. Homozygote A207 mice
were no different in phenotype from hemizygotes, still much
paler than mice carrying either of the other two variants.
As the variant transgenic MC1R responds to agouti protein,
we asked whether the paler colour was due simply to an
increased sensitivity of the receptor to agouti antagonism or if
they also produced less eumelanin. We crossed the Arg151Cys
and Arg160Trp transgenic lines onto a homozygous nonagouti background. Mice with wild-type Mc1r on a non-agouti
background are black. The variant transgenic mice were dark
grey in colour, indicating that the variant receptors are able to
elicit a eumelanogenic response, although of a lower intensity
than normal. The variant receptors respond normally to agouti
antagonism. The reduced melanogenic response from the
Arg151Cys and Arg160Trp variant MC1Rs is probably not
due to less efficient binding by the hormone but to less effective signalling. Schioth et al. (21) demonstrated that hormone
binding to these variants in transfected cells is indistinguishable from wild-type. The Asp294His variant receptor, in
contrast, binds hormone slightly less well, and the paler coat of
these transgenic mice might in part be accounted for by this. To
try and overcome the poorer binding we administered αMSH
to neonatal transgenic pups by subcutaneous injection four
times over a 7 day period, a protocol which normally darkens
coat colour (25) but which had no effect on the colour of the
two Asp294His lines (data not shown).
2400 Human Molecular Genetics, 2001, Vol. 10, No. 21
Figure 2. Melanin content of hair samples of transgenic and control mice. Bar chart showing ratio of alkali-soluble melanin to total melanin (ASM/TM) in hair
samples from transgenic and control mice. Three mice were sampled from each line and each is shown as a separate bar. Samples of hair from each line are shown
below the baseline.
DISCUSSION
We have demonstrated that Mc1r-containing BAC transgenes
are able to very efficiently rescue the yellow, loss-of-function
phenotype of Mc1re homozygous mice. Furthermore, the transgenic mice show a strong dosage-sensitive phenotype. Addition of only one or two copies in excess of the normal diploid
complement darkens the coat by reduction of the yellow,
agouti-induced band on the hair. A total Mc1r complement of
approximately six copies almost completely suppresses the
effect of agouti. We do not know whether the suppression of
agouti is due to the increase in MC1R on the melanocyte
surface saturating out a limited amount of agouti protein or
whether there is normally a low level of signalling from the
receptors even in the presence of agouti, which has an additive
effect with increased receptor number and overcomes the
antagonism.
Transgenic mice expressing human MC1R show a much
greater suppression of agouti. Indeed, mice expressing only the
normal diploid complement of human MC1R have no detectable phaemelanin and are completely black. This is not due to
a lack of activity of mouse agouti protein on the human
receptor, as others have demonstrated that in cells in culture
the human MC1R is 10-fold more sensitive to mouse agouti
than to the human protein (14). There is evidence to suggest
that human MC1R is more sensitive to stimulation by hormone
than the mouse receptor and this may be sufficient to overcome
the antagonsim by agouti. It also appears that the density of
MC1R on human melanocytes is considerably lower than the
receptor density on mouse melanocytes (24). The transgenic
system we describe here, in which the human receptor is
expressed from the mouse promoter, may result in MC1R
being present at non-physiological, high levels. The variant
human MC1R receptors in transgenic mice do respond to agouti
protein, as shown by the phaeomelanic patterning on their hairs.
These receptors have attenuated signalling (demonstrated by
the pale eumelanin synthesized on a non-agouti background)
which appears to allow agouti to act.
Our data indicate that although the variant MC1R proteins
are clearly defective, in accordance with population and family
data associating them with red hair, none has complete loss of
function when assayed in transgenic mice. Rare patients
mutated in the POMC gene and thus lacking αMSH, have red
hair that is apparently the same as the MC1R-associated red
hair (20). However, POMC-deficient mice do not have the
same phenotype as loss of Mc1r (15). Our finding that red-hair
associated variant human receptors still allow significant
eumelanin synthesis, may indicate that red hair is not the null
phenotype. Although a few MC1R frameshift alleles that
clearly do not produce functional protein have been described
(17,26,27), they are rare and no homozygotes have been found.
Whether MC1R-null individuals have different or additional
phenotypes which means they have not been included in case
control or population studies is unclear at this stage. MC1R has
been suggested to have a role in melanocyte proliferation and
in various non-melanocytic functions such as inflammation.
There is no evidence that MC1R-associated red-haired individuals have any additional phenotype. However, identification
and study of individuals homozygous for MC1R loss of
function might reveal further roles for MC1R in humans.
MATERIALS AND METHODS
Generation of transgenic mice
We isolated a mouse genomic BAC clone (Research Genetics,
CITB-484H6, insert size 120 kb) which contained the Mc1r
coding region approximately central in the cloned segment.
Sequencing of the BAC Mc1r coding region confirmed that it
was identical to the GenBank database sequence X65635 apart
from a 2 bp inversion resulting in alanine rather than valine at
Human Molecular Genetics, 2001, Vol. 10, No. 21 2401
codon 216. We generated transgenic recessive yellow (Mc1re/
Mc1re) mice by pronuclear injection of the circularized modified or unmodified BAC directly into homozygous embryos.
Genotyping was performed by PCR using primers directed at
the BAC vector sequences adjacent to both ends of the insert
(5′-TAACTATGCGGCATCAGAGC-3′ and 5′-GTCGTATTACAATTCACTGGC-3′, product 268 bp; 5′-GGCGTAATCATGGTCATAGC-3′ and 5′-TCATTAATGCAGCTGGCACG-3′,
product 190 bp).
BAC modification
To substitute the murine Mc1r coding region (bases 15–962 in
X65635) with human MC1R wild-type and variant coding
sequences (bases 462–1415 in X65634), homologous recombination based modification of the BAC in recombination-deficient E.coli, was employed as described by Yang et al. (23),
using human MC1R sequences which had been amplified from
human volunteers by PCR and cloned (21). The insert in the
homologously recombined BACs also contained an IRES
appended to a lacZ marker (23). The sequence of the encoded
‘wild-type’ human protein was confirmed by sequencing to be
identical to the Swissprot entry and the three variants
contained only the single variant amino acid stated.
Transgene copy number
Copy number of the Mc1r-encoding BAC was determined by
assay of the ratio of PCR amplified Mc1r (endogenous plus
transgenic) to endogenous Mc5r. PCR was performed on
genomic DNA, extracted from tail tips of 6–8-week-old
transgenic animals, using primers at conserved sequences
(5′-AACCTGCACTCRCCCATGTA-3′ and 32P-radiolabelled 5′-GCATAGAAGATGGWGATGTA-3′), which
amplified Mc1r and Mc5r simultaneously, and the product
digested with AluI prior to 6.5% non-denaturing PAGE. The
transgene copy number was calculated from the area ratios of
the Mc1r (101 bp, endogenous and transgenic) and Mc5r
(209 bp, endogenous) AluI-digested products on a Storm phosphoimager system (Molecular Dynamics). Copy number of
transgenic BACs encoding human MC1R was assayed by
Southern blot hybridization on tail tip DNA digested with
BamHI or with NcoI using mouse Mc1r 3′-untranslated region
as probe. Transgene copy number was calculated from the
peak area ratio of MC1R to Mc1r fragments using a Storm
phosphoimager system.
Copy number was also calculated by single nucleotide
primer extension (SnuPE) using the SnaPshot ddNTP kit
(PE Biosystems) as described below.
Single nucleotide primer extension
The PCR product from amplification of genomic DNA or
random primed cDNA was analysed by primer extension to
determine the content of transgenic versus endogenous Mc1r/
MC1R. Total RNA was isolated from dorsal trunk skin of
4-day-old animals using LiCl/urea (28) and reverse transcription carried out with random primers.
Mouse transgenic and endogenous Mc1r genomic DNA was
amplified using primers 5′-AGCATCGTCTCCAGCACC-3′ and
5′-CATGTGGGCATACAGAATCG-3′, and products of the
transgene (wild-type) and endogenous (Mc1re) distinguished
by extension of the primer, 5′-GGCAGAGCAGAACGGCTGT-3′. The template from the transgenic transcript added
ddGTP, whereas that from endogenous added ddTTP. Transcript levels from the mutant, Mc1re, allele compared to the
wild-type Mc1r were also assessed in heterozygous animals to
control for differential transcription and/or stability.
Human transgene content was assayed following amplification with primers 5′-GGCCCCTTCTTCCTGCATC-3′ and 5′TGCGGAGCTCCTGGCTG-3′, which amplify both the
mouse and human genes, followed by extension of the primer
5′-GTTGAAGTTCTTGAAGATGCAGC-3′, which primes
from both mouse and human sequences, by adding ddTTP
(mouse) or ddCTP (human).
Extension products were separated by capillary electrophoresis on an ABI 310 and peak areas measured using
GeneScan 2.1.
Melanin assays
Total and alkali-soluble melanin was determined by modification of a previous report (3). Briefly, 0.9 ml of Soluene-350
was added to 1 mg of mouse hair in 0.1 ml of sterile water and
heated for 45 min in a boiling water bath. The resulting solutions
were analysed in duplicate for absorbances at 500 nm (A500)
and these values are referred to as total melanin. To determine
alkali-soluble melanin, 800 µl of 8 M urea/1 M NaOH solution
was added to 2 mg of mouse hair in 200 µl of water and
incubated with shaking at 37°C for 30 min. The samples were
vortexed and centrifuged at 10 000 g for 10 min. The supernatants
were analysed for absorbances at 400 nm (A400), the values
being referred to as alkali-soluble melanin. Determination of
melanin and alkali-soluble melanin was performed in duplicate
for each hair sample.
ACKNOWLEDGEMENTS
We thank Brendan Doe and the staff of the Transgenic Unit for
their assistance, N.Heintz for providing the temperature-sensitive
shuttle vector for homologous recombination, and S.Philips for
providing expression vector pCR3.1 (Invitrogen) containing
human wild-type and variant MC1R sequences. E.H. is a
Medical Research Council Clinician Scientist Fellow.
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