Tissue-specific Regulation of the Ecto-5′-nucleotidase Promoter: ROLE OF THE cAMP RESPONSE ELEMENT SITE IN MEDIATING REPRESSION BY THE UPSTREAM REGULATORY REGION

Jozef Spychala,Albert G Zimmermann,Beverly S Mitchell

doi:10.1074/jbc.274.32.22705

Jozef Spychala, Albert G Zimmermann + Show 1 more

Open Access

https://doi.org/10.1074/jbc.274.32.22705

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Abstract

We have isolated the 5' region of the ecto-5'-nucleotidase (low K(m) 5'-NT) gene and established that a 969-base pair (bp) fragment confers cell-specific expression of a CAT reporter gene that correlates with the expression of endogenous ecto-5'-NT mRNA and enzymatic activity. A 768-bp upstream negative regulatory region has been identified that conferred lymphocyte-specific negative regulation in a heterologous system with a 244-bp deoxycytidine kinase core promoter. DNase I footprinting identified several protected areas including Sp1, Sp1/AP-2, and cAMP response element (CRE) binding sites within the 201-bp core promoter region and Sp1, NRE-2a, TCF-1/LEF-1, and Sp1/NF-AT binding sites in the upstream regulatory region. Whereas the CRE site was essential in mediating the negative activity of the upstream regulatory region in Jurkat but not in HeLa cells, mutation of the Sp1/AP-2 site decreased promoter activity in both cell lines. Electrophoretic mobility shift assay analysis of proteins binding to the CRE site identified both ATF-1 and ATF-2 in Jurkat cells. Finally, phorbol 12-myristate 13-acetate increased the activity of both the core and the 969-bp promoter fragments, and this increase was abrogated by mutations at the CRE site. In summary, we have identified a tissue-specific regulatory region 5' of the ecto-5'-NT core promoter that requires the presence of a functional CRE site within the basal promoter for its suppressive activity.

Highlights

We have isolated the 5؅ region of the ecto-5؅-nucleotidase gene and established that a 969-base pair fragment confers cell-specific expression of a chloramphenicol acetyltransferase (CAT) reporter gene that correlates with the expression of endogenous ecto-5؅-NT mRNA and enzymatic activity
Cell-specific Expression of Ecto-5Ј-nucleotidase mRNA—To determine the level of ecto-5Ј-NT mRNA in human cell lines, Northern blot analysis was performed (Fig. 1)
Cell-specific Activity of the Ecto-5Ј-nucleotidase Promoter—In order to determine the relative levels of ecto-5Ј-NT promoter activity, we transiently transfected several cell lines with the pCAT-Basic reporter construct containing 969, 371, or 201 bp of the promoter

Summary

EXPERIMENTAL PROCEDURES

Materials—Fetal calf serum was obtained from HiClone and Sigma. TRI Reagent, a high efficiency hybridization system, and Northern/ Southern transfer solution were obtained from Molecular Research Center Inc. (Cincinnati, OH). Nuclear factors were extracted in buffer C (20 mM HEPES, pH 7.9, 0.2 mM EDTA, 0.2 mM EGTA, 2 mM DTT, 20% glycerol, 0.15 mM spermine, 0.75 mM spermidine, 1 mM phenylmethylsulfonyl fluoride, 0.4 M NaCl, 100 nM ocadaic acid), followed by centrifugation at 30,000 ϫ g for 45 min. Ten ng of 32P-labeled DNA were incubated with 120 and 240 ␮g of Jurkat and HeLa cell nuclear extracts in the presence of 15 ␮g of poly(dI-dC), 6.1% glycerol, 0.07 mM EDTA, 0.07 mM EGTA, 7.2 mM HEPES, pH 7.9, 39 mM KCl, 7.5 mM MgCl2, and 0.7 mM DTT. One ␮l of probe (approximately 0.1 ng of DNA) was incubated with 8 ␮g of nuclear extract in the presence of 20 mM HEPES, pH 7.5, 0.5 mM DTT, 1 mM EDTA, 2 mM MgCl2, 50 mM KCl, 12% glycerol, and 2 ␮g of poly(dI-dC) (Sigma).

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