Identification of Novel Pax-2 Binding Sites by Chromatin Precipitation

Abstract

The Pax genes encode a family of developmental transcription factors that bind to specific DNA sequences via the paired domain and are necessary for the morphogenesis of a variety of tissues. The murine Pax-2 gene, through alternative splicing, encodes two nuclear proteins, Pax-2A and Pax-2B, which are transiently expressed during the differentiation of specific neural cell types and early kidney formation. In order to identify potential in vivo Pax-2 target sequences, chromatin from embryonic neural tube was immunoprecipitated with Pax-2 specific antibodies and cloned. Two unique immunoprecipitated clones containing three specific Pax-2 binding sites were identified by functional binding assays using Pax-2 proteins produced in both Escherichia coli and eukaryotic cells. In vitro DNA binding assays, using Pax-5 and Pax-8 DNA recognition sequences as well as the three immunopurified Pax-2 binding sites, demonstrated that both forms of the Pax-2 protein bind DNA with a similar specificity and that this binding is mediated by the paired domain. The binding sites identified in this report share significant homology among themselves and with previously defined consensus sequences for Pax-5 and Pax-2. The genomic clones can now be used as sequence tags to identify potential target loci.

The Paired Homeodomain Transcription Factor Pax-2 Is Expressed in the Endocrine Pancreas and Transactivates the Glucagon Gene Promoter

“…Consistent with the similar binding affinity of Pax-2A and Pax-2B for the isolated G3 and G1 elements, both isoforms transactivated G3Ϫ31Glu and G1Ϫ31Glu to a similar extent. Our data are consistent with previous reports observing similar binding and transactivation of Pax-2A and Pax-2B on a Pax-2 consensus element and on three recognition sites identified by chromatin precipitation (6,43). The role of the 23-amino acid insertion in Pax-2A versus Pax-2B ( Fig.…”

“…Pax-2 transcriptionally activates the Wilms tumor suppressor gene WT1, Engrailed 2, and, cooperatively with homeodomain proteins, the Pax-5 enhancer (45)(46)(47). In addition, two Pax-2 target genes with yet unknown function have been identified by chromatin precipitation using mouse embryonic spinal cord (43). Here we demonstrate that Pax-2 is expressed in the endocrine pancreas and that it transactivates the glucagon gene promoter through two cis-acting sequences, G1 and G3.…”

N-terminal PAX8 polyclonal antibody shows cross-reactivity with N-terminal region of PAX5 and is responsible for reports of PAX8 positivity in malignant lymphomas

“…Morgenstern et al 13 showed cross-reactivity between commercially available antibodies raised against PAX5 and PAX2. Phelps and Dressler 18 and Gilmore and Dewar 19 also reported cross-reactivity between PAX2 and PAX5 antibodies.…”

The C-terminal Subdomain Makes an Important Contribution to the DNA Binding Activity of the Pax-3 Paired Domain

“…However, many naturally occurring paired domain recognition sequences identified within candidate target promoters are longer than the consensus sequences defined in vitro, and require a contribution from both subdomains for recognition (14). A clear example of this is seen with Pax-2, where selection from a pool of random oligonucleotides defined a 13-bp consensus lacking a C-terminal subdomain recognition motif (25), whereas the purification of Pax-2-bound complexes from native chromatin defined a 23-bp consensus with recognition motifs for both subdomains (31). Therefore, in some instances, the role of the C-terminal subdomain in DNA recognition by Pax proteins may have been masked by the tendency of the N-terminal subdomain to dominate over the C-terminal subdomain in standard in vitro selection protocols.…”