During development and differentiation, genes either become competent to be expressed or are stably silenced in an epigenetically heritable manner. This selective activation/repression of genes leads to the differentiation of tissue types. Our group is interested in the molecular mechanisms that lead to the heritable transmission of the silenced state. In particular, we are studying the mechanism of gene silencing by the Polycomb group genes (PcG) in Drosophila. Drosophila is an excellent system in which to study the process because of its short generation time, our ability to generate and study developmental mutants, and the availability of molecular and genetic tools. During development in organisms as diverse as Drosophila and man, the PcG genes encode a diverse group of proteins known to be important for silencing homeotic and segmentation genes. Many PcG genes encode chromatin-associated proteins, and it has been proposed that they silence transcription by forming protein complexes that inactivate chromatin. At least three different PcG protein complexes have been characterized to date, and they may act by distinct biochemical mechanisms. PcG proteins exert their effect through poorly defined cis-acting DNA sequences called Polycomb group Response Elements (PREs). PREs are thought to recruit PcG protein complexes to the DNA. Given the different types of PcG protein complexes, there may be different types of PREs. One focus of our laboratory is to characterize fully the sequences and DNA binding proteins required for PRE function.

Polycomb Group Response Elements Require the Activity of Many DNA Binding Proteins
Americo, Brown, Grau, Kassis
We have completed a detailed study of one PRE from the segmentation gene engrailed. Within a 139-bp minimal PRE, there are eight protein-binding sites. Our functional data have shown that at least five of these sites are required for PRE activity. One of the proteins that binds to this PRE is the product of the pleiohomeotic (pho) gene, a known PcG gene. Pleiohomeotic encodes a Drosophila homolog of the mammalian zinc-finger transcription factor Yin Yang 1 (YY1). Another site is the sequence GAGAG, known to bind two Drosophila proteins, GAGA factor, and pipsqueak. There are two Pho binding sites and two GAGAG sequences within the engrailed PRE. The proteins that bind to the other four sites have not been identified. Isolation of these proteins is a major focus of our laboratory. We are using a yeast one-hybrid screen and biochemical methods to isolate the other DNA binding proteins involved in this important process.

The Role of pho-like in Polycomb Group Repression
Brown, Kassis
PHO binding sites have been identified in many different PREs, suggesting that PHO is a key component for the recruitment of PcG protein complexes to the DNA. If this is the case, then the phenotype of pho mutants should be severe derepression of homeotic genes. Curiously, homeotic genes are only mildly derepressed in pho mutants. The sequence of the Drosophila genome permits a possible explanation of this apparent paradox; in particular, another YY1 homolog exists in Drosophila. We are currently studying the function of this gene, which we call pho-like. First, we generated mutants in the pho-like gene. We found that flies mutant for pho-like are viable but that the females are sterile. Homozygous pho-like mothers lay eggs that are fertilized yet fail to undergo nuclear divisions. Thus, pho-like must be deposited in the egg by the mother for development to occur. Organisms that are double mutant for pho and pho-like (from mothers and fathers heterozygous for these two mutations) develop into larvae and have much more severe derepression of homeotic genes than pho mutants alone, showing that pho-like enhances the pho phenotype. Interestingly, homeotic genes are not completely derepressed in pho/pho-like double mutants, suggesting that the two genes are only partially responsible for the activity of PREs. Thus, some PREs may not require pho and pho-like function. Alternatively, the Pho and pho-like proteins deposited in the egg by the mother, in conjunction with other DNA binding proteins, may be able to recruit PcG proteins to the PRE. Loss of pho and pho-like proteins later in development may only partially destabilize PcG protein/PRE interactions and lead to dramatic but incomplete derepression of homeotic genes. Other experiments in the laboratory are designed to test for other types of PREs.