Functional characterization of the rat growth hormone promoter elements required for induction by thyroid hormone with and without a co-transfected β type thyroid hormone receptor

G A Brent,P R Larsen,J W Harney,R J Koenig,D D Moore

doi:10.1016/s0021-9258(17)31240-1

G A Brent, P R Larsen + Show 3 more

Open Access

https://doi.org/10.1016/s0021-9258(17)31240-1

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Abstract

We have extensively characterized the sequences of the rat growth hormone (rGH) promoter required for induction by T3 (thyroid hormone, 3,5,3'-L-triiodothyronine) in a transient transfection system. Oligonucleotides containing portions of the rGH promoter sequence with various deletions and point mutations were placed upstream of the first 137 base pairs of the rGH promoter or the heterologous herpes virus thymidine kinase promoter in chloramphenicol acetyltransferase expression vectors. The rGH137 and thymidine kinase promoters show no or minimal response to T3 in the basal state. The constructs were tested in GH4C1 rat pituitary cells and COS cells (functionally deficient in thyroid hormone receptor) with and without a co-transfected plasmid expressing a beta type c-erbA gene coding for a functional T3 receptor. Oligonucleotides containing the T3 receptor binding site confer hormone-dependent induction in a manner that is independent of either orientation or variation in position on the helix relative to the promoter. Point mutations in the sequence -189 to -173 result in loss of T3 induction, and bases between -173 and -167 were also required for a full T3 response. The minimal length to confer T3 induction to the rGH promoter was 23 base pairs (-190 to -167). Point mutations creating a perfect duplication of 7 base pairs within the receptor binding site conferred 12-fold T3 response to the rGH137 promoter, 3-fold greater than the wild type rGH237 construct. T3 inductibility was also transferred to the thymidine kinase promoter by an oligonucleotide containing the sequence -200 to -157, demonstrating that cell type specific elements located 3' to 157 of the rGH promoter are not required for thyroid hormone responsiveness.

Highlights

From the $Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, the IlHoward Hughes Medical Institute, Br&ham and Women’s Hospital, and the Departmenotfs $Medicine and **Genetics, Haivard Medical School, Boston, Massachusetts02115
GH& rat pituitary cells and COS cells with and without a co-transfected plasmid expressing a @ type c-erbA gene coding for a functional T 3 receptor
Recent studies using T3 receptor purified by an alternate protocol have suggested that this protein binds to additional sitesin the rat growth hormone (rGH) promoter and 5”flanking regions, no functional data were mutations in thesequence -189 to -173 result inloss reported to correlate with these sites [14, 15]

Summary

MATERIALS AND METHODS

The abbreviations used are: T3,3,5,3’-~-triiodothyroninthey,roid Phmids-Standard methods for vector construction were used hormone; rGH, rat growth hormone. The horizontal lines above the wild type rGH sequence (WT) designate the two imperfect direct repeats with 5 of 7 bases in common, which are part of the previously described [10] T3 receptor binding site (-172 to -188). Mutations of the wild type rGH promoter sequence are shown in bold type. 1. The mean T 3 induction ratios for wild type rGH promoter 6’ deletions transiently transfected in GH& cells with and without a co-transfected B type T3 receptor (T3R). A series of transfections with the limits of the deleted bases were:oligo 16, -189 to -173;oligo 17, vector CDM8 [25] alone did not result in any T3 induction. -189 to -183; oligo 18, -179 to -173;oligo 19, -171 to -167.All oligonucleotide constructs were sequenced using the dideoxynucleo-

RESULTS

GH4G without co-transfected T3 receptor prGH137

MutationsWithPreservedOrIncreasedT3Response

TK Promoter Series pUTKAT3

Findings

DISCUSSION