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J. Biol. Chem., Vol. 281, Issue 2, 1008-1015, January 13, 2006

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The cis-Regulatory Element Gsl5 Is Indispensable for Proximal Straight Tubule Cell-specific Transcription of Core 2 -1,6-N-Acetylglucosaminyltransferase in the Mouse Kidney^*

Michiko Sekine, Chouji Taya, Hiroshi Shitara, Yoshiaki Kikkawa, Noriko Akamatsu, Masaharu Kotani, Masao Miyazaki¶, Akemi Suzuki¶1, and Hiromichi Yonekawa

From the Department of Laboratory Animal Science and the Department of Clinical Genetics, Tokyo Metropolitan Institute of Medical Science, Tokyo, 113-8613 Japan, and ^¶Sphingolipid Expression Laboratory, RIKEN Frontier Research System, Wako, 351-0198 Japan

Gsl5 regulates the expression of a glycolipid and glycoproteins that contain the Le^X epitope in the mouse kidney through tissue-specific transcriptional regulation of the core 2 -1,6-N-acetylglucosaminyltransferase (core 2 GnT) gene. The core 2 GnT gene has six exons and produces three alternatively spliced transcripts. Gsl5 regulates only the expression of the kidney-type mRNA, which is transcribed from the most 5'-upstream exon. By introducing a 159-kb bacterial artificial chromosome (BAC) clone that carries the mouse core 2 GnT gene and its 5'-upstream region into DBA/2 mice that carry a defective Gsl5 allele, we were able to rescue the deficient phenotype. The BAC clone was subsequently engineered to replace the core 2 GnT gene with the sequence of enhanced green fluorescent protein (EGFP) as a reporter by an inducible homologous recombination system in Escherichia coli. The transgenic mice derived from the modified BAC clone expressed EGFP in the kidney, which suggests that the candidate Gsl5 is in the 5'-upstream region of the core 2 GnT gene. Sequence analysis of the 5'-upstream regions of the BAC clone and DBA/2 genomic DNA revealed a candidate sequence for Gsl5 at about 5.5 kb upstream of exon 1. This sequence consisted of eight repeats of two GT-rich units in the wild-type mice, whereas it consisted of only one pair of GT-rich units with a minor modification in the DBA/2 mice. Transgenic mice produced with the EGFP reporter gene construct that included this candidate sequence expressed EGFP exclusively in the proximal straight tubular cells of the kidney. These results indicated that this unique repeat is indeed the Gsl5, and it is a cis-regulatory element responsible for proximal straight tubule cell-specific transcriptional regulation.

Received for publication, August 23, 2005 , and in revised form, October 27, 2005.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB219560 [GenBank] to AB219563 [GenBank] and AB234879 [GenBank] .

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

¹ To whom correspondence should be addressed: RIKEN Frontier Research System, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan. Tel.: 81-48-467-9615; Fax: 81-48-462-4692; E-mail: aksuzuki{at}riken.jp.

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