Members of the steroid/thyroid hormone nuclear receptor superfamily play an important role in complex processes such as differentiation, development and oncogenesis. Receptors function by linking cellular signals directly to the nucleus where they alter the rate of transcription. Orphan nuclear receptors are transcription factors which share sequence homology with the steroid/thyroid receptor superfamily but have no known ligand and/or specific function. Nur77 and Nurr1 are ligand-independent receptors. Their mRNAs increase in response to a variety of stimulants, such as serum growth factors, epidermal growth factor, nerve growth factor and membrane depolarization factors. Although Nur77 and Nurr1 are structurally similar, differences have been reported in their tissue distribution, responses to stimuli and developmental expression. Our functional data using transient transfection assays and Nurr1 and Nur77 mutants indicates that the last 15 amino acids of the Nurr1 carboxy-terminal region are required for full transactivation. In contrast, a 15 amino acid truncation of Nur77 retained wild-type like activity. Thus, even though Nurr1 and Nur77 have similar structural characteristics, they might function through carboxy-terminal dependent mechanisms of transactivation differently. The situ hybridization analysis showed that Nurr1 and Nur77 mRNAs have overlapping and different distribution patterns within brain suggesting that they might regulate different sets of responsive genes. However, a function of Nurr1 in vivo remains unknown. Thus, we have isolated and characterized the Nurr1 gene. Five kilobases of genomic DNA located upstream of the two starts of transcription were sequenced and studied for promoter activity using 5' and 3' deletion mutants. The promoter region contains AP1, AP2 and one cAMP response elements. The latter resides in the first untranslated exon and functions as a strong enhancer. Genomic sequence of Nurr1 was also inserted between the neomycin resistance gene and the herpes simplex virus thymidine kinase gene in targeting vector. Thus, the neomycin gene replaces Nurr1 exon 2 which contains the translation initiation codon and the DNA binding domain. This targeting vector was introduced into the J1 line of embryonic stem cells. Selected embryonic/stem cells for three targeted cell lines were used for microinjection into blastocoel mouse embryos. Chimeric mice have been bred and transmission of the knockout allele and was identified by southern analysis. These heterozygous mice are presently used for production of null offspring.
