Transcript Présentation PowerPoint - IGH

Role of the Hsp90 protein chaperone,
and of trxG chromatin regulators, as
capacitors of morphological evolution
a literature seminar held as part of a teaching course in Genetics, University of
Montpellier 2, using as sources the articles:
Rutherford, S. L., and Lindquist, S. (1998). Hsp90 as a capacitor for morphological
evolution [see comments]. Nature 396, 336-342.
And:
Sollars, V., Lu, X., Xiao, L., Wang, X., Garfinkel, M. D., and Ruden, D. M. (2003).
Evidence for an epigenetic mechanism by which Hsp90 acts as a capacitor for
morphological evolution. Nat Genet 33, 70-74.
Giacomo Cavalli, Institute of Human Genetics, CNRS. Montpellier, April 2004
Introductory text to the subject (from Nature 396, 336-42)
Among the major heat-shock proteins, Hsp90 is unique in its functions. It is not required
for the maturation or maintenance of most proteins in vivo. Most of its many identified
cellular targets are signal transducers–cell-cycle and developmental regulators whose
conformational instability is relevant to their roles as molecular switches. Through low
affinity interactions characterized by repeated cycles of binding and release, Hsp90
keeps these unstable signalling proteins poised for activation until they are stabilized by
conformational changes associated with signal transduction. Minor changes in aminoacid sequence can have substantial effects on a protein's conformational stability and
Hsp90 recognizes structural features common to unstable proteins rather than specific
sequence motifs. Thus, individual members of highly homologous protein families, such
as steroid-hormone receptors or cyclin-dependent or Src-family kinases, can vary
greatly in their dependence on Hsp90.
Studies of yeast illustrate the specificity of Hsp90: at normal temperatures, reductions in
Hsp90 levels that have no apparent effects on cell growth or metabolism can completely
abolish signalling through Hsp90-dependent pathways. Conditions that cause general
protein damage can divert Hsp90 from its normal targets to other partially denatured
proteins. Because of its dual involvement with inherently unstable signal transducers on
the one hand, and with the cellular response to stress on the other, Hsp90 may link
developmental programs to environmental contingency.
Illustration from: http://www.picard.ch/DP/research/research.htm
Starting point of the study:
Mutant strains for Hsp90 show
often aberrant phenotypes.
These phenotypes are also seen
in outcrosses between Hsp90
mutant stocks and various
strains
Hsp83
‘Balancer’
X
+
Hsp83
and
+
Hsp83
F1
F2
+
+
1/3
Hsp83
Hsp83
and
+
+
2/3
lethals
Examples of phenotypes
Mutant Hsp83 stocks
Crosses between Hsp83
mutants and laboratory
stocks (phenotypes are
often worse than in
established Hsp83 stocks)
Effect of the Hsp90
inhibitor geldanamycin
« Bad » phenotypes
observed in double Hsp90
mutants (heteroallelic
viable combinations)
Phenotypes are diverse and they are visible in numerous genetic contexts, but they are
characteristic of each context
Origin of the phenotypes?
They depend on Hsp90, since they can be observed in several independent mutant
alleles, they get worse in general in the presence of two mutant alleles (heteroallelism),
and since they are also induced by the Hsp90 inhibitor geldanamycin
Mechanism?
In Hsp90 mutant context, development might become more sensitive to
environmental noise
Alternatively, Hsp90 might induce an increase in the global mutation rate
Finally, Hsp90 might buffer against phenotypic variation dependent on the
accumulation of genetic polymorphism. In case of a decrease of Hsp90 levels,
this cryptic variation might become exposed and thus originate visible
phenotypes. The previous data seem to suggest this third hypothesis, since the
observed phenotypes depend on genetic background of the tested strains. This
seems to exclude a global increase in mutation or an increased sensitivity to
environmentally induced stress, since both conditions should result in a more
random distribution of phenotypes
HE
Eye
Selection of the phenotypes
Isolation of single mutant F1 males
carrying a specific trait, either
affecting the eye or the wing
LE
Wing
HV
Cross with phenotypically
normal females
F2 expressing
the same trait
F2 not expressing
the trait
LV
Selection of « lines »
Positive
Negative
Strong expressors Weak expressors
of the trait
The selection works, and it suggest a multigenic
origin for the observed traits!
HE: High Eye
HV: High Vein
LE: Low Eye
LV: Low Vein
Selection of phenotypes: independent experiments of selection
(« isolates ») show quantitative/qualitative differences within each class
of trait, again suggesting a multigenic origin for them
HE2
HE2
HE2
HE2
HE2
HE3
Effect of growth temperature
Note: in the case of the wing the effect is the reverse, vein phenotypes get worse at
18°C compared to 25°C
Genetic determinants of the observed effects
It is possible to design crosses allowing to isolate fly populations carrying any given chromosome
(in one or two copies) from a specific genetic background, either the background corresponding to
a HE “stock” or a control non-selected background. This allows comparing the percentage of
phenotypes obtained in each genetic background to the control (HE) frequency
The analysis shows that the phenotypes have at least two determinants on chromosome 2, and
at least one on chromosome 3
Genotyping the phenotypically affected flies: does maintenance of the phenotypes
requires continuous impairment of Hsp90?
Since selection was only based on
phenotypes, but not on the
maintenance of the Hsp90 mutant
chromosome, an obvious question was
whether Hsp90 was always mutated in
the individuals carrying the traits
Note that out of the F1 cross, only 2/3
des mouches should carry the
mutation in the F2 and progressively
less in the subsequent generations,
while selection obtains up to 80% of
flies carrying the trait
For this reason they genotyped HE
and LE flies
An example of how this is done is
shown here for the starting strains
All tested flies (i.e. ten to fourty in different
conditions) had lost the mutation !!!
Inactivation of Hsp90 required to initiate, but
not to maintain, development of new traits!
Proposed model to explain the action of Hsp90 (extracted from Ref. 7, see the last slide
of this document). Hsp90 might favour the accumulation of genetic variation that would
normally remain cryptic thanks to the Hsp90-dependent buffering ability. In situations
impairing Hsp90 function (dependent on environmental stress or on mutation of the Hsp90
coding gene), this variation could become exposed and, if not deleterious, might be
subjected to selective pressure. This may ultimately lead to rapid fixation of phenotypic and
genotypic variation that might result in the emergence of novel species
…but there are other data to be considered…
The sensitized Kr-If-1 context:
Krüppel, is an important developmental
transcription factor. Overexpression of Kr
in the eye induces a reduction of the eye
size.
They did a genetic screen using a set of
strains carrying genomic deficiencies,
with the aim of isolating suppressors and
enhancers of this phenotype. Several of
the mutants induced ectopic outgrowths
in the eye
« Deficiency »
‘Balancer
chromosome’
F1
X
Kr-If-1
Kr-If-1
Kr-If-1
+
F1 progeny showed outgrowths
only if the deficiency was inherited
maternally, not paternally
Starting from the deficiencies, by candidate gene approaches they identified genes
responsible of the outgrowths. They were either alleles of the hsp83 gene (coding for
Hsp90), or trxG genes!
Selection of phenotypes
1. Isolation of females carrying an
outgrowth and mutated in a trxG
gene named vtd
Selection of progeny carrying
the outgrowth but not the vtd
mutation
Selection
Accumulation of phenotypes
2.
Isolation of an « isogenic »
population, carrying the Kr-If-1 allele
Geldanamycin
Induction of outgrowth
Selection
Accumulation of phenotypes
Thus, pre-existing genetic variability
is not required for the assimilation of
the phenotype!
The phenotypes might depend
on wingless induction
wt
wg is a crucial regulator of several
developmental processes.
A transgenic line carrying the reporter
gene lacZ inserted downstream of the wg
promoter is available.
This reporter was derepressed by a
mutation in Hsp83, when this mutation
was inherited maternally but not paternally
Hsp83 mut mother
Hsp83 mut father
The phenotypes depend on chromatin effects
Quantification of the Western blot from
a
The histone
deacetylase inhibitor
TSA induced histone
H3 hyperacetylation
and suppressed the
outgrowths, as well as
the overexpression of
wg-lacZ. This is
consistent with a role
of chromatin
modification by trxG
proteins in the
acquisition of the new
phenotypes.
Comment: it is
surprising that
increased histone
acetylation correlates
with reduced wg
expression.
Suppression of the effect by mutants of the trxG? Increase of the effects by PcG mutants? Not
presented in this paper
Mother Nature’s « Tools » contributing to buffering of environmental
variation and, at the same time, to genomic and phenotypic plasticity, might
play an important role for evolution of new species
Chromatin inheritance: the same DNA sequence can adopt different functional states, that are
heritable during cell division and through mitosis (from yeast to mouse, maybe human, see refs
1, 2 and 4).
Stabilisation of homeostasis. Buffering of environmental changes. Since cells are capable of
transmitting the memory of their identity to their progeny via chromatin inheritance in addition
gene regulatory mechanisms based on the relative concentration of regulatory factors, this
redundancy provides robustness to gene regulation and make it relatively insensitive to
environmental changes.
Hsp90. This chaperone protein allows to key regulatory proteins to remain functional even upon
mild mutations, since it favours their correct folding. Thus, mild mutations are tolerated
provided Hsp90 is functional.
Buffers development from genetic or environmental sources of variability
Might induce a strong morphological variability when its activity is reduced by mutations or by
strong stresses that divert this protein from its regular targets (ref 3, 5).
Cooperation between Hsp90 and chromatin factors (ref 6) may contribute to rapid fixation of
new traits in a population
If among the new phenotypes there were favourable ones, natural
selection might fix them in natural populations
Relevant bibliography:
1)
Grewal, S. I. S., and Klar, A. J. S. (1996). Chromosomal inheritance of epigenetic states in fission
yeast during mitosis and meiosis. Cell 86, 95-101.
2)
Cavalli, G., and Paro, R. (1998). The Drosophila Fab-7 chromosomal element conveys
epigenetic inheritance during mitosis and meiosis. Cell 93, 505-518.
3)
Rutherford, S. L., and Lindquist, S. (1998). Hsp90 as a capacitor for morphological evolution [see
comments]. Nature 396, 336-342.
4)
Morgan, H. D., Sutherland, H. G., Martin, D. I., and Whitelaw, E. (1999). Epigenetic inheritance
at the agouti locus in the mouse. Nat Genet 23, 314-318.
5)
Queitsch C, Sangster TA, Lindquist S (2002) Hsp90 as a capacitor of phenotypic variation.
Nature 417, 618-24. Epub 2002 May 12
6)
Sollars, V., Lu, X., Xiao, L., Wang, X., Garfinkel, M. D., and Ruden, D. M. (2003). Evidence for an
epigenetic mechanism by which Hsp90 acts as a capacitor for morphological evolution. Nat
Genet 33, 70-74.
7)
Sangster TA, Lindquist S, Queitsch C (2004). Under cover: causes, effects and implications of
Hsp90-mediated genetic capacitance. Bioessays. 26, 348-62