Identification of Novel Pax-2 Binding Sites by Chromatin Precipitation

doi:10.1074/jbc.271.14.7978

Identification of Novel Pax-2 Binding Sites by Chromatin Precipitation (*)

From the NICMD, National Institutes of Health, Bethesda, Maryland 20892

^¶ To whom correspondence should be addressed. Present address:
Howard Hughes Medical Institute and Dept. of Pathology, University of Michigan, Ann Arbor, MI 48109-0650
. Tel.: 313-764-6490; Fax: 313-763-6640; dressler{at}umich.edu.

↵^§ Performed in partial fulfillment of the Ph.D. requirements in the Graduate Genetics Program at George Washington University. Present address: University of North Carolina, Chapel Hill, NC 27599-3280.

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.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(™)/EMBL Data Bank with accession number(s) U46547 and U46548.
↵¹ The abbreviations used are:

PBS

phosphate-buffered saline

DTT

dithiothreitol

APMSF

4-amidophenylmethanesulfonyl fluoride

BSA

bovine serum albumin

CAT

chloramphenicol acetyltransferase

kb

kilobase pair(s)

PCR

polymerase chain reaction.
- Received September 7, 1995.
- Revision received December 7, 1995.