Research Description
HEAT-SHOCK PROTEINS AND GENES
Summary: My laboratory investigates the heat-shock protein Hsp70, its encoding genes, and its regulation in Drosophila as a model system for understanding evolutionary adaptation. Hsp70 is a molecular chaperone that deters stress-induced protein aggregation, but has numerous other functions. Hsp70 is necessary for full-strength tolerance (in terms of survival, normal development, normal function) of high temperature. Such tolerance is critical in nature, where non-adult Drosophila undergo harmful to lethal high temperatures. In nature, Drosophila populations vary in stress tolerance and Hsp70 levels. Our current major focus is on understanding the genomic basis for this variation. The number of hsp70 gene copies and evolution of the hsp70 coding sequence are partial or inadequate explanations. Evidently cis-regulatory regions such as proximal promoters underlie intraspecific variation in Hsp70 levels. Repeated insertion of mobile genetic elements into these promoters is a recurrent mechanism of evolution.
Why are heat-shock proteins important?
Are heat-shock proteins important in nature?
How is natural variation in Hsp70 expression encoded at the genomic level?
How is natural variation in Hsp70 expression encoded at the
genomic level?
A. hsp70 copy number
In the ancestors of Drosophila, two hsp70 genes were arranged in inverted orientation. These copies have proliferated via a combination of
duplication of the entire gene cluster, presumably by retrotransposition, and
tandem duplication of individual genes. Thus, for example, D. virilis and D. melanogaster have evolved similar copy numbers but in entirely
different ways. Once evolved, hsp70 copies can also degenerate, as in D. lummei.
These patterns cannot,
however, explain the differing Hsp70 expression among natural populations of Drosophila
melanogaster. No natural population has yet
been found to have other than 5 hsp70 copies.
B. hsp70 coding sequence
5 genes encode hsp70 in natural populations of Drosophila melanogaster. Despite
the fact that these have been separate genes for as much as 100,000,000 years
or more, locus-mates have evolved zero fixed differences at the level of the
nucleotide! This
extraordinarily conservation of coding sequence is likely due to extensive gene
conversion. Indeed, as shown
below, several shared polymorphisms implicate gene conversion as an effective
mechanism for both eliminating sequence variation and distributing novel
sequence among all hsp70 copies.
c. hsp70 flanking sequence
The hsp70 genes of Drosophila melanogaster vary in their promoters, 5' and 3' untranslated
regions, and upstream of the their promoters; any, some, or all of this
variation could be the mechanistic basis for the variation in Hsp70 protein
levels among natural populations.
Because the hsp70 promoter
has been characterized in detail, we have chosen to focus on it.
In essence, hsp70 transcription results when stress results in
proteins in non-native conformation, which titrate pre-existing heat-shock
protein away from heat shock factor [a transcription factor], which can then
trimerize, localize to the nucleus, and bind HSEs [heat-shock response
elements] in the proximal promoter, which results in release of the
pre-assembled but paused polymerase apparatus, which results in hsp70 transcription.
Importantly, the number and spacing of HSEs is critical for
full-strength transcription.
As shown above, we first
discovered four independent instances in which transposable or mobile genetic
elements have inserted in the hsp70
proximal promoter in nature, thereby disrupting the spacing of HSEs. In each case, alleles with or without a
transposon segregate in the source population. At least 4 other transposons
have inserted elsewhere in the gene clusters.
The frequency of
transposon-bearing alleles varies along geographic/climatic gradients. For example, in Evolution Canyon,
transposon-bearing alleles are more frequent on the north-facing slope.
We hypothesize that
transposition into the hsp70
promoter reduces hsp70 transcription by altering the spacing of the HSEs, and
that transposon-bearing alleles are maintained by natural selection when
decreased Hsp70 is desirable but purged by selection when increased Hsp70 is
advantageous.
We have gathered diverse
data consistent with this hypothesis:
Ribonuclease protection
assay: Compare the hsp70Ba gene product to that of the hsp70Bb and hsp70Bc genes for 5 independent lines each where a Jockey element is absent (left) or present (right) in the hsp70Ba proximal promoter.
Expression constructs: Compare luciferase luminescence where a
transposon was absent (white) or present (red) in the proximal promoter of a hsp70B-luciferase fusion.
Compare Hsp70 protein levels
in whole Drosophila matched for
population and sex where a transposon was absent (white) or present (orange) in
the proximal promoter of the hsp70Ba gene.
The hsp70 genes are distinctive in that their chromatin is
constitutively decondensed. We
hypothesize that this distinction renders them vulnerable to transposable
element insertion.
For a complete list of
laboratory publications on the above topics, go to