The human semaphorin 6B gene is down regulated by PPARs

doi:10.1016/j.ygeno.2004.01.002

Genomics

Volume 83, Issue 6, June 2004, Pages 1141-1150

https://doi.org/10.1016/j.ygeno.2004.01.002 Get rights and content

Abstract

The peroxisome proliferator-activated receptors (PPARs) are ligand-inducible transcription factors and belong to the nuclear hormone receptor superfamily. They form heterodimers with the retinoid X receptor and bind to specific peroxisome proliferator-response elements. The latter are direct repeat elements of two hexanucleotides with the consensus sequence TG(A/T)CCT separated by a single nucleotide spacer. Such a sequence, or a similar one, has been found in numerous PPAR-inducible genes. We developed an affinity method to isolate human genomic fragments containing binding sites for PPARs and to identify novel PPAR target genes. For this, an antibody raised against all PPAR subtypes was used. Immunoselected fragments were amplified and sequenced and one of them, ISF5148, was found to bind specifically to PPARs in gel mobility shift, supershift, and competition assays and to exhibit a down transregulation potentiality in transfection experiments under clofibrate (a PPARα agonist) treatment. ISF5148 was mapped by BLAST analysis 8.5 kb upstream of the human semaphorin 6B [(HSA)SEMA6B] gene. The latter encodes a member of the semaphorin family of axon guidance molecules. Expression of this gene in human glioblastoma T98G cells was strongly down regulated after treatment with clofibrate or Wy-14,643, two PPARα agonists. Our study establishes for the first time that PPAR activators diminish the expression of the human (HSA)SEMA6B gene. These data are relevant to the fact that PPARs are implicated in brain development, neuronal differentiation, and lipid metabolism in the central nervous system. In addition, cross talk between the peroxisome proliferator and retinoic acid pathways is suggested.

Section snippets

Results

A PPAR binding site in a DNA fragment is likely to be biologically relevant if it satisfies the following criteria: (i) binding of PPAR should be of high affinity as determined by filter binding, gel mobility shift, and competition assays and (ii) the PPAR binding site should confer PPAR ligand-dependent regulation in transactivation assays.

Discussion

In this study, we have used an immunoprecipitation/PCR amplification protocol to clone human genomic DNA sequences associated with PPARs to identify novel target genes for these transcription factors. Among the different clones obtained, the ISF5148 sequence was located in the upstream region of the human semaphorin 6B gene recently identified on chromosome 19 [17]. This finding led us to focus our investigations on this PPAR-binding DNA fragment.

The semaphorins are secreted or

Conclusions

Our study establishes for the first time that PPAR activators diminish the expression of the human (HSA)SEMA6B gene. These data are relevant to the fact that PPARs are implicated in brain development, neuronal differentiation, and lipid metabolism in the central nervous system. Delineation of the sequence capable of binding PPARs led to the identification of overlapping DR1 and DR6 elements, suggesting a cross talk between peroxisome proliferator and retinoic acid pathways. The biological

Origins of plasmids

Plasmids used for in vitro protein synthesis for PPARs and RXRs were kind gifts from Professor W. Wahli and Dr. A. Pujol, respectively. Reporter plasmids used for transfection experiments were constructed in our laboratory by cloning ISF5148 and the PPRE oligonucleotide of the human acyl CoA oxidase gene upstream of the luciferase reporter gene.

Immunoprecipitation of PPAR-binding human genomic DNA fragments

The immunoselection protocol used (Fig. 1A) was derived from the method established by Rudert and Gronemeyer [32] to isolate retinoic acid-response

Acknowledgements

We thank Professor W. Wahli (University of Lausanne, Switzerland) and Dr. A. Pujol (IGBMC, Illkirch, France) for the kind gifts of the plasmids used in the present study for in vitro PPAR and RXR syntheses, respectively. The A549 and T98G cell lines were generously provided by Dr. N. Frossard (INSERM U425, Illkirch, France) and Dr. A. Pujol, respectively. The authors are grateful to Professor S. Thornton for critical review, to V. Collet for manuscript editing, and to F. Etienne for skillful

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The human semaphorin 6B gene is down regulated by PPARs

Abstract

Section snippets

Results

Discussion

Conclusions

Origins of plasmids

Immunoprecipitation of PPAR-binding human genomic DNA fragments

Acknowledgements

Cell

J. Biol. Chem

Mutat. Res

Biochem. Biophys. Res. Commun

Genomics

Curr. Opin. Neurobiol

Mol. Cell. Neurosci

Mol. Brain Res

Neuron

Biochem. Biophys. Res. Commun

FEBS Lett

J. Steroid Biochem. Mol. Biol

J. Mol. Biol

Cell

Identification of a new member of the steroid hormone receptor superfamily that is activated by a peroxisome proliferator and fatty acids

Mol. Endocrinol

cDNA cloning, chromosomal mapping, and functional characterization of the human peroxisome proliferator activated receptor

Biochemistry

Differential expression and activation of a family of murine peroxisome proliferator-activated receptors

Proc. Natl. Acad. Sci. USA

Isolation of the human peroxisome proliferator activated receptor gamma cDNA: expression in hematopoietic cells and chromosomal mapping

Gene Expression