Dissecting the cis-regulatory DNA that controls the POU-domain transcription factor genes, pdm-1 & pdm-2. Jermaine Ross^1,2, Thomas Brody¹, Ward F. Odenwald¹. 1) Neural Cell-Fate Determinants Section, NINDS, NIH, Bethesda, MD; 2) Brown University, Providence, RI.

   While neuroblast (NB) lineage studies have identified transcription factors (TFs) important to cell identity decisions, we currently have only an incomplete understanding of the cis-regulatory elements that control their expression. The comparative genomic programs, EvoPrinter and cis-Decoder (also see Brody et al. abstract), along with transgenic reporter assays, were used to identify, compare, and functionally test these regulatory sequences. Here, we describe the enhancers that regulate the pdm-1 & -2 genes, which encode two POU-domain TF paralogs that perform overlapping functions during CNS lineage development. Thus far, we have identified over 70 enhancers located within a 125 kb region spanning the pdm loci, and have catalogued these enhancers in an online database, cisPatterns, that documents their expression and DNA conservation. These enhancers are functionally autonomous and control different subsets of pdm expression patterns during embryonic, larval and/or adult development, including expression in overlapping but nonidentical patterns during intermediate stages of embryonic and larval NB lineage development. Further, cis-Decoder analysis of the conserved DNA within the NB enhancers identifies shared and unique conserved elements. Site-directed mutagenesis of these sequences reveals that they are important for enhancer function. One of the tested sequences includes the highly conserved 9-mer TAAAAATTG identified in both the pdm-1 & -2 NB enhancers. Based on previous work, this sequence corresponds to the DNA binding site of Castor, a zinc-finger TF that is required for proper pdm expression. We found that deletion of the 9-mer sequence triggers ectopic reporter expression in the cephalic lobes. Currently, we are testing the functional significance of other putative pdm enhancers.