Mutations in
pag-3 cause pleiotropic defects including a lethargic phenotype, a reverse kinker uncoordinated phenotype, lineage defects (see abstract by Cameron et al.), and misexpression of touch neuron-specific genes in the BDU interneurons.
pag-3 encodes a zinc finger protein related to the vertebrate proto-oncoprotein Gfi-1. Gfi-1 functions as a transcriptional repressor in transfected cells, and is expressed in lymphoid tissues.
pag-3 is expressed in several neural cell types, including the touch cells, the BDU cells, and cells of the VA, VB and VC motor lineages. We are taking three strategies to identify targets of PAG-3. First, we are trying to identify authentic PAG-3 binding sites in touch neuron genes such as
mec-7 . Second, we are using DNA chip technology to identify genes whose expression is altered in
pag-3 mutants. Third, we are screening for suppressers of the
pag-3 uncoordinated phenotype. To identify PAG-3 binding sites we have focused on the
mec-7 gene.
mec-7 is one of the touch neuron genes misexpressed in the BDU neurons of
pag-3 mutants. PAG-3 expressed in bacteria binds well to the consensus Gfi-1 binding site and to
mec-7 sites containing the core (AAT/GC) of the Gfi-1 consensus site. To correlate the binding of PAG-3 to changes in target gene expression, we transformed worms with
mec-7lacZ mutated at the strongest PAG-3 binding sites. Since PAG-3 may act directly or indirectly on
mec-7 , we have transformed worms with deletions of
mec-7lacZ to identify the element responsible for misexpression in the BDU neurons. For the DNA microarray screen, we have prepared mRNA from
pag-3(
ls64) and N2 L1 larvae. The
ls64 allele contains a nonsense mutation just upstream of the zinc fingers. Northern analysis will be used to confirm that the abundance of specific mRNAs is altered in
pag-3 mutants. Finally, we are looking for suppressers of the uncoordinated phenotype by screening for improved motility in mutagenized
pag-3(
ls20) worms.