Download Report

London Research Institute Milestone 11
Why can’t a woman be more like a man? The race for the male sex-determining gene
•
1990: Peter Goodfellow’s lab show that the testis-determining factor, which specifies
maleness, is encoded by the SRY gene.
Background
As is well known, humans normally have 46 chromosomes,
comprising 22 pairs of autosomes and 1 pair of sex chromosomes,
XX for females, and XY for males. It seems hard to believe
now, but the Y chromosome’s function as a determinant of
maleness has only been known since 1959, with the discovery
of men with chromosome complement 47XXY, and women
who were 45XO. The presence of a Y chromosome overrides
any number of X chromosomes, and is necessary and sufficient
for testis development and maleness. But what was it on the
Y chromosome that specified maleness? By painstaking analysis
of patients with jumbled sex chromosomes lacking pieces of Y,
or where the X and Y had managed to mix together, scientists
were able to narrow the region containing the so-called Testis
Determining Factor (TDF) to a chunk of the Y-specific part of the
Y chromosome, near the boundary with the pseudoautosomal
region (the part of the Y chromosome which is able to pair with
the X). But this still meant that many hundreds of thousands of
base pairs of DNA had to be searched for the TDF gene.
Peter Goodfellow, fanatical Arsenal fan, sometime poet, and all
round eccentric, was recruited by Walter Bodmer in 1979, in his
drive to bring human genetics to the ICRF. Peter’s expertise lay
in gene mapping, pinpointing genes in the vast human genome, at
that time a complicated and esoteric procedure. He had started
his career mapping components of the immune system as a PhD
student with Walter Bodmer in Oxford, and, after a postdoc at
Stanford (during which he attended the last Sex Pistols concert
and had the honour of being spat on by Sid Vicious), he started a
lab at the ICRF to do the same thing for genes
encoding tumour-specific antigens.
By coincidence, one of these
mapped very near the
putative TDF locus.
The unsolved
problem of
male sex
determination sparked Peter’s interest, and, equipped as he was
with the expertise to find genetic needles in genomic haystacks,
he decided to identify and clone the elusive TDF gene.
The research
Things progressed very well to begin with. The Goodfellow lab’s
advanced methodologies allowed them to produce a very good
genetic roadmap of the TDF region, and in 1987, they published a
paper in Nature which narrowed down the region to be searched
to a manageable 50 - 150 kilobase (50,000 - 150,000 basepair)
piece of DNA abutting the pseudoautosomal boundary, containing
a candidate locus for the TDF gene. Then, what looked like disaster
struck; the nightmare of every scientist working on a hot problem
is to be scooped by the competition, and, in a Cell paper in 1987,
David Page, of the Massachusetts Institute of Technology in Boston,
announced the cloning and sequencing of the gene corresponding
to Peter’s candidate locus. This gene, which Page called ZFY, was
claimed, with much trumpeting, to be the long-awaited TDF.
After some time spent sitting under his desk writing poetry and
drinking too much coffee, Peter emerged to continue working.
The Page paper had not truly nailed the problem, and unequivocal
evidence that ZFY was indeed the TDF was still lacking. Rumours
began to circulate in the sex determination field that Page had got
it wrong.
To be the TDF, ZFY had to fulfil a number of criteria. The first,
that it should be on the Y-specific part of the Y chromosome,
was clearly fine, and that it appeared to be the only gene in a
rather empty area of DNA was also in its favour. However, one
significant problem was that the X chromosome carried a highly
homologous gene, called ZFX. This was rather odd, as the two
genes were so closely related that it would be hard for them
to have different functions, as they must if ZFY, but not ZFX,
determined maleness. Secondly, both Page and Goodfellow
contacted an Australian expert on marsupial genetics, Jenny
Graves, and asked her to probe the marsupial genome to
check that marsupial Zfy was also on the Y chromosome. To
everyone’s surprise, it was not; it lay on an autosome, which was
very unexpected, as maleness in marsupials is also specified by
the presence of a Y chromosome. This was a significant nail in
the coffin, but ZFY’s short life as the TDF truly ended after the
Christmas 1989 edition of Nature, which carried two papers, one
from Peter’s lab together with the Fellous lab in Paris, and one
from Peter’s collaborator Robin Lovell-Badge; the first described a
number of sex-reversed XX men whose genomes did not contain
Peter Goodfellow
38
London Research Institute 1902-2014
ZFY, and the second showed that murine Zfy was not found in the
cells specifying maleness.
The race was back on again, and this time, the Goodfellow lab
won. Andrew Sinclair, the PhD student in Jenny Graves’s lab who
had shown that marsupial Zfy was autosomal, moved to London
to do a postdoc with Peter, and in 1990, was first author on
a Nature paper describing the positional cloning and correct
identification of SRY as the gene for determining maleness. The
SRY gene was 150kb away from ZFY, and had been missed before
because it was tiny, a mere 1000 bases long.
SRY was subjected to the same scrutiny as ZFY, but passed with
flying colours. The Goodfellow lab published another paper using
the Fellous lab’s clinical material, showing that the three XX men
described in the 1989 paper had picked up the part of the Y
chromosome containing SRY; and as final proof that SRY was the
real deal, Peter published yet another paper in Nature in 1991
with Robin Lovell-Badge, showing that female mice engineered to
carry the SRY gene developed as males. This latter paper, with its
memorable front cover image of Randy the sex-reversed mouse,
caused a media storm at the time, and with its appearance, signalled
an end to one of the highest profile scientific races ever.
The consequences
SRY proved to be a difficult protein to study. After many years of
effort, we now know that it is a rather weedy transcription factor,
whose sole purpose as a testis determining factor is to switch
on a second gene, SOX9 (also cloned in Peter’s laboratory),
which then does all the rest of the work in establishing maleness.
Interestingly, SOX9 is an autosomal gene, and appears to be a
much more ancient specifier of maleness than SRY; it exists in
multiple non-mammalian species, in contrast to SRY, which is solely
mammalian.
In a recent twist to the story, the long-held dogma that femaleness
was a default state, and to be male one simply had to activate
SRY and SOX9, was overturned in 2009 by Robin Lovell-Badge
in a collaborative study which showed that in mice the autosomal
gene FoxL2 specifies femaleness, and when present, overrides
Sox9 and prevents ovaries changing into testes. Loss of FoxL2 in
adult female mice upregulates Sox9, causing reprogramming of
some ovarian cell types to those found in testes. Remarkably, as
in life, the Sox9/FoxL2 story shows that maleness and femaleness
appear to be established by what some might view as a balancing
act, and others as a war.
What happened next?
In 1992, the flamboyance quotient of the ICRF was reduced to
a depressingly normal level when Peter Goodfellow moved to
the University of Cambridge to become the Balfour Professor of
Genetics. He still carries the distinction of being the only head of
the Cambridge University Science Department to have a ponytail.
He left academia to become Senior Vice President at Smith Kline
Beecham in 1996, and became Senior VP, Discovery Research
upon Smith Kline’s merger with Glaxo in 2001. He remained at
Glaxo Smith Kline until his retirement in 2006, and now acts as a
biotech consultant. His work on sex determination won him the
1995 Louis-Jeantet Prize, which he shared with Robin LovellBadge and three others, and, with Lovell-Badge and David Page,
London Research Institute 1902-2014
39
the 1997 Francis Amory Prize of the American Academy of Arts
and Sciences. He was elected to the Royal Society in 1992, and
in 1998, became a founding member of the Academy of Medical
Sciences.
Key references*
The sex-determining region of the human Y chromosome
encodes a finger protein. Page DC, Mosher R, Simpson EM, Fisher
EM, Mardon G, Pollack J, McGillivray B, de la Chapelle A, Brown LG.
Cell 1987 51:1092-1104.
Sequences homologous to ZFY, a candidate human sexdetermining gene, are autosomal in marsupials. Sinclair AH, Foster
JW, Spencer JA, Page DC, Palmer M, Goodfellow PN, Graves JA.
Nature. 1988 336:780-3.
A gene from the human sex-determining region encodes a
protein with homology to a conserved DNA-binding motif.
Sinclair AH, Berta P, Palmer MS, Hawkins JR, Griffiths BL, Smith
MJ, Foster JW, Frischauf AM, Lovell-Badge R, Goodfellow PN.
Nature. 1990 346:240-4.
Genetic evidence equating SRY and the testis-determining
factor. Berta P, Hawkins JR, Sinclair AH, Taylor A, Griffiths BL,
Goodfellow PN, Fellous M. Nature.1990 348:448-50.
Male development of chromosomally female mice transgenic for
Sry. Koopman P, Gubbay J, Vivian N, Goodfellow P, Lovell-Badge R.
Nature. 1991 351:117-21.
Somatic sex reprogramming of adult ovaries to testes by FOXL2
ablation. Uhlenhaut NH, Jakob S, Anlag K, Eisenberger T, Sekido R,
Kress J, Treier AC, Klugmann C, Klasen C, Holter NI, Riethmacher
D, Schütz G, Cooney AJ, Lovell-Badge R, Treier M. Cell. 2009
139:1130-42.
The Exception That Proves the Rule: An Interview with Jenny
Graves. Jane Gitschier. PLoS Genet. 2008 4: e1000063.
*LRI scientists shown in bold
Sex reversal in mice: a chromosomally-female (XX) mouse carrying the Sry gene grows as a
male with testes. This mouse is a product of research led jointly by Robin Lovell-Badge of the
Medical Research Council & Peter Goodfellow of the Imperial Cancer Research Fund.