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.
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