During development and differentiation, genes 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. Accordingly, we are studying the mechanism of gene silencing by the Polycomb group genes (PcG) in Drosophila. The PcG genes encode a diverse group of proteins known to be important for silencing of homeotic genes during development in organisms as diverse as Drosophila and man. 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 that may act by distinct biochemical mechanisms. PcG proteins act 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 that there are different types of PcG protein complexes, different types of PREs may also exist. A major focus of our laboratory is to characterize fully the sequences and DNA-binding proteins required for PRE function.

The Role of pho-like in Polycomb Group Repression
Brown, Kassis; in collaboration with Fritsch, Müller
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 were the case, then the phenotype of pho mutants should be severe derepression of homeotic genes. Curiously, in pho mutants, homeotic genes are only mildly derepressed, but the sequence of the Drosophila genome has yielded a possible explanation for this apparent paradox. Another YY1 homolog exists in Drosophila. We are studying the function of this gene that we call pho-like. We generated mutants in the pho-like gene. Flies mutant for pho-like are viable, but the females are sterile. Homozygous pho-like mothers lay eggs that are fertilized but undergo no 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. In fact, the derepression of homeotic genes seen in imaginal disks from pho-like, pho double mutants is comparable to that seen in many other PcG mutants. This suggests that Pho and Pho-like play redundant but highly important roles in PcG repression.

Pho-like binds to Pho binding sites in vitro and pho-like, pho double mutants show more severe misexpression of homeotic genes than the single mutants. But how do these two proteins function? Given that Pho and Pho-like are DNA-proteins, one hypothesis is that they may recruit or anchor PcG protein complexes to the DNA. To address this possible function, we examined the distribution of five PcG proteins on polytene chromosomes from pho-like, pho double mutants. These five PcG proteins are not DNA-binding proteins, but they are associated with specific sites on wild-type polytene chromosomes. We reasoned that if Pho and Pho-like are required for anchoring PcG protein complexes to the DNA, then association of PcG chromatin proteins with chromosomal sites might be lost. Our results showed that the PcG proteins Pc, Psc, Scm, E(z), and Ph remain bound to polytene chromosomes at most sites in the absence of Pho and Pho-like. At a few chromosomal locations, however, some of the PcG proteins were no longer present in the absence of Pho and Pho-like. The data suggest the presence of both Pho-like/Pho-dependent and Pho-like/Pho-independent PREs. We submit that, at most genes, both types of PREs exist. Thus, in polytene chromosomes, only a subset of PcG protein complexes may be lost at each genomic location. Alternatively, Pho and Pho-like may act in concert with other PcG proteins but not be required for their association with the DNA.