This proposal from Dr. Jeffrey Simon is written as an amended version of a proposal submitted three years ago. However, it builds on the significant progress achieved through work supported elsewhere for the past two years. As a result of the criticisms of the previous proposal and new findings, this proposal differs significantly with respect to focus, specific aims and experimental methods. The proposed research concerns the mechanism(s) by which transcription is repressed in the maintenance of developmental decisions and differentiated states. Specifically, the focus of the experiments will be two members of the Polycomb group of genes (PcG genes), best known for their repression of homeotic genes in regions of the body where their expression would lead to inappropriate body patterning. In fact, the genes appear to act as general transcription repressors since their mutant phenotypes extend beyond the spectrum predicted from inappropriate homeotic gene expression. To date, there are some 13 PcG genes. While their protein products are thought to act together to effect stable repression, how they do so is not clear. Individual functions have not been assigned. There are sequences in the upstream regions of each target gene, PRE's (Polycomb Group Repressor Elements), that are required for repression. However, none of the genes yet analyzed seems to have a clear-cut DNA-binding activity. The PREs can be many kilobases removed from the promoters of the genes that they repress and are structurally distinct from those elements required for the initiation of repression mediated by gap and pair-rule gene products. Because the PcG products characterized thus far are uniformly distributed, it is thought that other factors provide the positional information for the interactions between PREs and the PcG proteins. One idea is that the initial repression mediated by gap and pair-rule genes or the decay of those interactions serves to direct PcG products to the relevant genes in appropriate positional context. Two general models for their activity are (1) the proteins generate localized chromatin compaction, rendering genes in that region incapable of being transcribed and (2) the proteins interfere with distal enhancers, thereby blocking the activation of transcription. This proposal, proposes to continue research on the mechanisms of repression by focusing on two genes of this group, extra sex combs (esc) and Sex combs on midleg (Scm). They were chosen for study because their characteristics suggest central roles in repression. Both have loss-of- function phenotypes that are in the most severe class known for PcG genes, the complete transformation of embryonic segments to the identity of the eighth abdominal segment. esc is of interest because it is required very early, between 2 and 4 hrs of embryogenesis, and not later. This is in contrast to most, if not all other PcG genes which must function later in embryogenesis and in post-embryonic stages to maintain repression. Scm was chosen for study in part because the protein has a number of potentially interesting structural motifs, including a set of zinc fingers, raising the possibility that it could have DNA-binding activity. Among the preliminary findings reported are that a 2 hour heat treatment of esc null embryos transformed with hsp 70 controlled esc allow the embryos to survive at least to first instar larval stage, and in some cases, to adults. The window of rescue approximates the time of transition from initiation to maintenance of repression. The esc protein has 5 copies of the WD40 sequence which is thought to have a protein interaction function. Dr. Simon reports that the yeast transcriptional repressor Tup 1 is among the proteins with the most similarity to WD40 repeats, and interestingly, one which acts via contacts with a DNA binding protein, rather than having DNA binding activity itself. Deletions of single repeat elements destroys the repression function. In order to identify other regions of functional interest, Dr. Simon's group has cloned and sequenced esc genes from other species. At 92% identity, the Drosophila virilis sequence is too similar to be helpful; however, the Musca domestica gene and another from a butterfly species which they also cloned and are sequencing are more divergent and are revealing conserved elements. Efforts to isolate esc binding partners via the yeast two-hybrid method, proposed and criticized in the last proposal yielded a component of the ubiquitin- dependent protein degradation system - and the observation that esc can bind to itself. GST-esc fusions confirm the self-binding activity. No evidence was obtained for DNA binding activity. The Scm gene was cloned by members of Dr. Simon's lab by first defining its location by the generation of small deletions from excision of P elements in the 85E region where the gene was known to be located through deletion mapping of lethal Scm alleles. The P elements used proved to be close to Scm, allowing the gene to be cloned by plasmid rescue. The gene was identified by mapping mutant alleles and by rescue of mutant alleles by germ line transformation. The gene was shown to be expressed throughout development. Scm shares some domains with another PcG protein, polyhomeotic, including an HLH-like domain and two putative Zn fingers. The goals of the proposed research are: (1) to define through species comparisons conserved elements of potential functional importance and to perform mutational characterizations of functional domains in the two gene products, (2) to identify the binding partners and biochemical interactions of the two proteins, and (3) to examine the roles and interactions of the proteins in vivo.
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