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.

What are heat-shock proteins?

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?

Current projects

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Current projects:

With funding from the National Science Foundation, we are elucidating the role of transposons in the evolution of heat-shock gene expression:

A. INTRODUCTION AND HYPOTHESES

The promoters of heat-shock genes are distinctive.

Heat-shock genes are poised for massive and rapid expression. As the work of others has shown, the chromatin is constitutively decondensed, the polymerase apparatus is pre-assembled but paused, and the heat-shock response elements are exposed and awaiting the binding of transcription factors.

We have hypothesized that these distinctive features make heat-shock promoters vulnerable to the insertion of mobile genetic elements:

Because the mobile element (red) and its transposition machinery (blue) can more readily gain access to the chromatin, insertion should be easier into a heat-shock promoter than into a typical promoter. Promoters with transposable elements inserted are thereafter alleles of the wild-type promoter, and segregate in natural populations (see previous page).

In turn, these insertions ought to disrupt the interaction of the heat-shock transcription factor (HSF) with the polymerase, thereby decreasing heat-shock gene expression. In the cartoon, because the transposon insertion (red double arrow, bottom) changes their distance from the polymerase, two HSF complexes no longer interact with the polymerase and the rate of initiation is lower.

As we have shown,
--in environments in which heat shock is common, decreased heat-shock gene expression should be harmful and transposon-bearing alleles should be decreased by natural selection.
--in environments in which heat shock is rare, decreased heat-shock gene expression should have no benefit or even be harmful, and transposon-bearing alleles should be increased by natural selection.

B. TESTING THE HYPOTHESES

We screened 48 Drosophila populations from around the world and in each characterized all transposable elements in the proximal promoters of the following genes:

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Our findings are:

Every colored square on the map corresponds to the discovery of a transposable element in the proximal promoter region of a gene. More than 200 transposable elements were eventually discovered, of which 96% are P elements – which invaded the Drosophila genome during the last century. As shown, almost all have inserted into heat-shock promoters.

Clearly transposon insertions are not restricted to Hsp70 promoters, but are abundant in many Hsp promoters. They are most numerous for Hsr-omega, which encodes a heat-inducible RNA. In the cartoon, insertions are indicated by black lines, with each F corresponding to a natural population with an insertion at that point.

In a second experiment, we experimentally created new insertions into the Hsp70 genes in the laboratory. Of 45 independent transpositions into Hsp70 (below, indicated by red arrows, with the number of transpostions below the arrow if >1), 23 were into just two nucleotides. Interestingly, this insertion preference mirrors natural insertions (above, indicated by black lines, with each F corresponding to a natural population with an insertion at that point).

C. ONGOING WORK

We are presently examining the consequences of transposable element insertions for gene expression, protein abundance, growth, development, and fecundity, with a view towards understanding the fitness consequences of this substantial genetic variation that is segregating in natural populations.

Acknowledgements:

Aspects of this work have been supported by the Howard Hughes Medical Institute, National Institutes of Health, the Sloan Foundation, the Binational Science Foundation of Israel and the USA, but to the largest extent by the National Science Foundation.

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For a complete list of laboratory publications on the above topics, go to

LIST OF PUBLICATIONS