Cherbas Lab
Research:
GENETICS
During the last few years we have been using genetics to discover which partners (corepressors, coactivators, neighboring TFs) are involved in different cells. Briefly, we made a mutant EcR that can bind hormone, bind DNA, and dimerize, but can't activate transcription. In flies, it's simple to express this dominant negative EcR (DN-EcR) in various cells using the GAL4 system.
Wherever the DN-EcR is expressed, it
blocks all ecdysone respo
nses.
That's a convenient way to uncover an ecdysone-dependent step in
development. Example: The fly at the right is an adult with a
salivary gland (green because it contains fluorescent glue). Normally
the salivary gland dies during metamorphosis -- but this gland contained
DN-EcR, didn't respond to ecdysone, and didn't commit suicide.
But we can do more with the DN. First, we can flood the same cells with another EcR that may be wildtype or may be altered in some way of interest. Wildtype receptor will rescue the phenotype.
Second, we can use the flies with heterozygous DN as a test environment to look for genes that "interact" with the receptor complex. This approach has provided rich results and has allowed us to demonstrate that the ecdysone receptor complex requires different partners in different cells.
MICROARRAYS
Further progress will require identifying many or all of the responsive genes in single cells, then analyzing each gene to ask which partners are crucial, and finally relating the presence of those factors to the underlying DNA sequence.
To do this we have switched our attention to Drosophila cell lines. The DGRC has collected >100 cell lines of varied developmental origins. All of these cells respond to ecdysone.
We have begun by choosing a panel of 10 lines for initial analysis. Using those cells we are using microarray analysis to identify all the genes that are activated or repressed during the initial few hours of the ecdysone response.
We hope to follow up by using RNAi to eliminate particular "partners" so that we can begin to understand how each cell's complement of regulatory molecules sets the pattern of its immediate ecdysone response.