Analysis of the rat lactate dehydrogenase A subunit gene promoter/regulatory region

M. L. Short, D. Huang, D. M. Milkowski, S. Short, K. Kunstman, C. J. Soong, K. C. Chung, R. A. Jungmann

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Abstract

The rat lactate dehydrogenase (LDH) A subunit gene promoter contains a putative AP-1 binding site at -295/-289 bp, two consensus Sp1 binding sites at -141/-136 bp and -103/-98 bp, and a single copy of a consensus cyclic AMP-responsive element (CRE) at -48 to -41 bp upstream of the transcription initiation site. Additionally, an as yet unidentified silencer element is located within the -1173/-830 bp 5'-flanking region. Transient transfection analyses of a -1173/+25 bp LDH A-chloramphenicol acetyltransferase fusion gene has indicated a complete inability of the promoter fragment to direct basal or forskolin-induced transcription. Deletion of the -1173/-830 bp sequence restored basal and cyclic AMP (cAMP)-inducible activity. Point mutations in the Sp1 binding sites of a -830/+25 bp promoter fragment reduced basal but not the relative degree of cAMP-inducible activity. cAMP-regulated transcriptional activity was dependent upon an 8 bp CRE, -TGACGTCA-, located at the -48/-41 bp upstream region. Mutations in the CRE abolished cAMP-mediated induction and reduced basal activity by about 65%. The CRE binds a 47 kDa protein which has previously been identified as CRE binding protein (CREB)-327, an isoform of the activating transcription factor/CREB transcription factor gene family. Co-transfection of a vector that expresses the catalytic subunit of cAMP-dependent protein kinase stimulates LDH A subunit promoter activity suggesting that cAMP induces LDH A subunit gene expression through phosphorylative modification of CREB-327. This study emphasizes a fundamental role of several modules including Sp1 and CREB binding sites in regulating basal and cAMP-mediated transcriptional activity of the LDH A gene.

Original languageEnglish
Pages (from-to)391-398
Number of pages8
JournalBiochemical Journal
Volume304
Issue number2
DOIs
Publication statusPublished - 1994 Jan 1

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Short, M. L., Huang, D., Milkowski, D. M., Short, S., Kunstman, K., Soong, C. J., Chung, K. C., & Jungmann, R. A. (1994). Analysis of the rat lactate dehydrogenase A subunit gene promoter/regulatory region. Biochemical Journal, 304(2), 391-398. https://doi.org/10.1042/bj3040391