Abstract
We have recently shown that a thyroid hormone-responsive transcription stimulatory element exists in the 5'-flanking DNA near the rat growth hormone (rGH) gene (Crew, M. D., and Spindler, S. R. (1986) J. Biol. Chem. 261, 5018-5022). Progressive deletion-transfection analysis of the 5' end of the gene has led to the identification of two genetic elements responsive to thyroid hormone. The first of these is a thyroid hormone-responsive transcription stimulatory element, or TSE. The TSE induced a thyroid hormone-dependent induction-attenuation transcription cycle similar to that of the natural rGH gene. Deletion of sequences between positions -254 and -241 in the rGH 5'-flanking DNA eliminated TSE activity. The second regulatory element is a thyroid hormone-responsive transcription inhibitory element (TIE). When this element was active, thyroid hormone strongly but transiently inhibited rGH promoter utilization. Deletion of sequences between nucleotides -46 and -21 abolished the effects of the TIE. To determine whether the TSE and TIE are enhancer-like, we ligated various regions of rat growth hormone 5'-flanking DNA containing these elements to a chimeric test gene containing the Herpes simplex virus thymidine kinase promoter. Thyroid hormone activated heterologous promoter utilization when a rat growth hormone 5'-flanking DNA fragment containing the TSE (-520 to -115) was linked in cis, regardless of the distance or orientation of the TSE with respect to the promoter. These data suggest that the TSE is a thyroid hormone-dependent enhancer. In contrast, when the TIE was placed immediately 5' to the thymidine kinase promoter, transcription was not effected by 3,5,3'-L-triiodothyronine, suggesting that the TIE is not enhancer-like.
Highlights
We have recently shown that a thyroid hormone- To elucidate the mechanism by which T, regulates ratgrowth hormone (rGH) responsive transcription stimulatory element exists intranscription, we analyzed the effects of T3 onpromoter the 5’-flanking DNA near the rat growth hormone utilization in GC cells stably transfected with chimeric genes gene
We have recently shown that a thyroid hormone- To elucidate the mechanism by which T, regulates rGH
Incontrast,the sponsive transcription inhibitory element (TIEW).hen principal problem associated with stable transfections, “pothis element was active, thyroid hormone strongly bsuittion effects,” can be negated by analysis of pools of transtransiently inhibited rGH promoter utilization
Summary
Its transcrip- ptkneo was constructed by ligating the 180-bpSal[1] to XhoI fragment tion is cell-type-dependent ( 1 , 2 )and regulated by thyroid and of ptkCAT -520tkn were constructed by cleavage of prGH530neo (2) with XbaI, limited digestion with exonuclease Ba131, ligation with HindIII linkers, HindIII digestion, and ligation to HindIII-cleaved, dephosphorylated ptkneo. Consistent with this hypothesis, T3 induces promoter- GH-520/-115tkn with HindIII followedby ligation. Proximal local alterations in chromatin structure which are +lltkn was constructed from prGH134neo by cleavage at theunique temporally coincident with the T3-induced changes in tran- XbaI site, brief digestion with exonuclease Bal[31], ligation of Hind[11] scription (6). TIE, T3inhibitory element; hGH, human growth hormone.
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