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Molecular and Cellular Biology, December 2004, p. 10263-10276, Vol. 24, No. 23
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.23.10263-10276.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Multiple, Distant Gata2 Enhancers Specify Temporally and Tissue-Specific Patterning in the Developing Urogenital System

Melin Khandekar,1 Norio Suzuki,2 Jon Lewton,1 Masayuki Yamamoto,2 and James Douglas Engel1*

Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan,1 Department of Molecular and Developmental Biology, TARA Centre, University of Tsukuba, Tsukuba, Japan2

Received 12 June 2004/ Returned for modification 6 July 2004/ Accepted 9 September 2004

Transcription factor GATA-2 is expressed in a complex temporally and tissue-specific pattern within the developing embryo. Loss-of-function studies in the mouse showed that GATA-2 activity is first required during very early hematopoiesis. We subsequently showed that a 271-kbp yeast artificial chromosome (YAC) transgene could fully complement the loss of Gata2 hematopoietic function but that these YAC-rescued Gata2 null mutant mice die perinatally due to defective urogenital development. The rescuing YAC did not display appropriate urogenital expression of Gata2, implying the existence of a urogenital-specific enhancer(s) lying outside the boundaries of this transgene. Here we outline a coupled general strategy for regulatory sequence discovery, linking bioinformatics to functional genomics based on the bacterial artificial chromosome (BAC) libraries used to generate the mouse genome sequence. Exploiting this strategy, we screened >1 Mbp of genomic DNA surrounding Gata2 for urogenital enhancer activity. We found that the spatially and tissue-specific functions for Gata2 in the developing urogenital system are conferred by at least three separate regionally and temporally specific urogenital enhancer elements, two of which reside far 3' to the Gata2 structural gene. Including the additional enhancers that were discovered using this strategy (called BAC trap) extends the functional realm of the Gata2 locus to greater than 1 Mbp.

* Corresponding author. Mailing address: Department of Cell and Developmental Biology, 4643 Med. Sci. II, 1335 Catherine St., Ann Arbor, MI 48109. Phone: (734) 615-7509. Fax: (734) 763-1166. E-mail: engel{at}umich.edu.

Molecular and Cellular Biology, December 2004, p. 10263-10276, Vol. 24, No. 23
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.23.10263-10276.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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