The c-ets-1 proto-oncogene and the related c-ets-2 gene encode related nuclear chromatin-associated proteins which bind DNA in vitro. To investigate the possibility that Etsl and Ets2 are transcriptional activators, we analyzed the ability of these proteins to trans-activate promoter/enhancer sequences in transient co-transfection experiments. A CAT construct driven by the long terminal repeat of the human T cell leukemia virus, HTLV-1 was found to be trans-activated by both Etsl and Ets2 in NIH3T3 and HeLa cells. The increased levels of CAT activity were paralleled by increased levels of correctly initiated CAT mRNA. Mutant Etsl proteins unable to accumulate in the nucleus were found to be inactive. An ets-responsive sequence between positions -117 and -160 of the LTR was identified by analyses of a series of 5' deletion mutants of the HTLV-1 LTR and of dimerized versions of specific motifs of the LTR enhancer region. Using a gel shift binding assay, Etsl was found to bind specifically to an oligonucleotide corresponding to region -117 to -160. This sequence, which also contributes to Taxl responsiveness of the HTLV-1 LTR, is characterized by the presence of four repeats of a pentanucleotide sequence of the type CC(T/A)CC. Competition

After differential screening of a cDNA library constructed from quail neuroretina cells (QNR) infected with the v-myc-containing avian retrovirus MC29, we have isolated a cDNA clone, Pax-QNR, homologous to the murine Pax-6, which is mutated in the autosomal dominant mutation small eye of mice and in the disorder aniridia in humans. Here we report the characterization of the Pax-QNR proteins expressed in the avian neuroretina. From bacterially expressed Pax-QNR peptides, we obtained rabbit antisera directed against different domains of the protein: paired domain (serum 11), domain between the paired domain and homeodomain (serum 12), homeodomain (serum 13), and carboxyl-terminal part (serum 14). Sera 12, 13, and 14 were able to specifically recognize five proteins (48, 46, 43,33, and 32 kDa) in the neuroretina. In contrast to proteins of 48, 46, and 43 kDa, proteins of 33 and 32 kDa were not recognized by the paired antiserum (serum 11). Paired-less and paired-containing proteins exhibited the same half-life (6 h) and were phosphorylated mostly on seine residues. Immunoprecipitations performed with subcellular fractions of neuroretinas showed that the paired-containing proteins were located in the nucleus, whereas the 33-and 32-kDa proteins were found essentially in the cytoplasmic compartment. However, immunofluorescence experiments performed after transient transfections showed that p46 and p33/32 were also located in vivo into the nucleus. Thus, the Pax-QNR/Pax-6 gene can produce proteins with two DNA-binding domains as well as proteins containing only the DNA-binding homeodomain.Cellular differentiation is the result of differential gene expression, and transcription factors are involved in the regulation of these events. Homeobox-containing genes encode developmentally regulated transcription factors. The homeobox encodes the homeodomain, a conserved DNAbinding domain with a helix-turn-helix motif (23), and several vertebrate homeodomain-containing proteins have been identified as transcription factors required for expression of lineage-specific genes (9,19,20).

The quail Pax-6 gene is expressed from two promoters named P0 and P1. P0 promoter is under the control of a neuroretina-speci®c enhancer (EP). This enhancer activates the P0 promoter speci®cally in neuroretina cells and in a developmental stage-dependent manner. The EP enhancer binds eciently, as revealed by southwestern experiments, to a 110 kDa protein present in neuroretina cells but not in Quail Embryos Cells and Retinal Pigmented Epithelium which do not express the P0-initiated mRNAs. To study the role of p110 in Pax-6 regulation, we have puri®ed the p110 from neuroretina cells extracts. Based on the peptide sequence of the puri®ed protein, we have identi®ed the p110 as the poly(ADP-ribose) polymerase (PARP). Using bandshift experiments and footprinting studies, we present evidence that PARP is a component of protein complexes bound to the EP enhancer that increases the on rate of the protein complex formation to DNA. Using PARP inhibitors (3AB and 6.5 Hphe), we show that these products are able to inhibit EP enhancer activity in neuroretina cells. Finally, we demonstrate that these inhibitors are able to decrease the expression of the P0-initiated mRNA in the MC29-infected RPE cells which, in contrast to the RPE cells, accumulated the PARP in response to v-myc expression. Our results suggest that PARP is involved in the Pax-6 regulation.