Isolation of genomic DNA fragments corresponding to genes modulatedin vivoby a transcription factor

Abstract

A new methodology for the identification of genes modulated by transcription factors in vivo is described. Mouse genomic DNA fragments bound by the thyroid hormone receptor (T3R) were selected and amplified in vitro. Subsequent hybridisation with biotinylated cDNA allowed the selection of those DNA fragments containing binding sites for T3R that corresponded to transcribed DNA. Expression analysis of the corresponding genes showed that more than 80% are indeed modulated by thyroid hormones in vivo in the liver. Together with the presence of consensus binding sites for T3R this result suggests that the selected DNA fragments may contain T3R transcriptional regulatory elements. This method, extensive to other ligand-modulated transcription factors, might be useful to all transcription factors with slight modifications.

Thyroid hormone-dependent transcriptional repression of neural cell adhesion molecule during brain maturation.

“…Isolation and identification of genomic fragments corresponding to genes transcriptionally regulated by T3 in the cerebral cortex We performed WGPCR basically as described (Kinzler and Vogelstein, 1989;Caubin et al, 1994). Rat genomic DNA was partially digested with Sau3A1 and ligated to adaptors containing an EcoRI site and appropriate sequence for amplification.…”

“…This is a reflection of the crucial role of T3 in CNS development. Also, we characterized recently a dozen genes regulated by T3 in the mouse liver (Caubin et al, 1994). Some of them will probably be primary transcriptional targets, while many others will be indirectly affected or regulated at levels other than transcription, i.e.…”

“…As described for mouse liver genes using the same approach (Caubin et al, 1994), the fact that most of the genes found are upregulated in the hypothyroid state could reflect a preferential binding of the in vitro-translated T3Rf3-RXRoc proteins to negative elements under the experimental conditions used or, alternatively, to the presence of negative elements in intragenic (intron, exon) sequences which are preferentially selected by the hybridization step used. Both are upregulated in the hypothyroid rat brain.…”

“…For T3R/c-erbA, Eisenman (1994, 1995) have used a partially different protocol, immunoprecipitating DNA-protein complexes obtained from a pituitary cell line, and found two novel T3regulated genes. Also, we characterized recently a dozen genes regulated by T3 in the mouse liver (Caubin et al, 1994).…”

“…The emerging picture is that we have effectively characterized new T3-regulated genes expressed in the cerebral cortex such as NCAM, cx-tubulin and the unknown RBB3, and that definitive demonstration for the remaining genes needs additional studies. As described for mouse liver genes using the same approach (Caubin et al, 1994), the fact that most of the genes found are upregulated in the hypothyroid state could reflect a preferential binding of the in vitro-translated T3Rf3-RXRoc proteins to negative elements under the experimental conditions used or, alternatively, to the presence of negative elements in intragenic (intron, exon) sequences which are preferentially selected by the hybridization step used. In the literature there is at least one case of T3 regulation through a (positive) element located in an intron: the rat growth hormone gene (Sap et al, 1990).…”