Functional role of TTF‐1 binding sites in bovine thyroglobulin promoter

Abstract

Wehav~?stt&d the bindingofpurifkd~-1 on thebotine thyroglobulin gencpromotcr. DNasc I foolprintingcxperiments revealed three binding siw which auresponded in location to the A. B and C sites found in the rat thyroglobulin promoter. hlutants in the A and C regions showing r&u& binding of IXF-1, also exhibited largely decrtzzd promoter activity in transient exprcssiop experiments in primary-cultured dog thyrocyts TWY mutants in the B site that exhibited a reduced capaaty to hind ITF-I also displayed a drastically affcctcd transcriptional activity in tmnsient assays As in the rat sites A and C only are critical for promoter activity. these results suggest :hat fuli occupa~lcy of the T. site is required for thyroglobulin ptcrmntn activity in the cow only. INTR0DUCTiONThe expression of t floglobulin (Tg) gene is restricted to the follicular cells of the thyroid gland. The expression of the gene is under the positive control of thyrotropin which acts mainly via the intracellular messenger cAMP [I]. This control occurs. at least partly, at the transcriptional level [2,3]. It has been shown that the specific expression of a reporter-gene, the chloramphenicol acetyltran&rase (CAT) gcnc. rcquircd only the first 250 bp of the bovine Tg promoter when transfccted in thyroid cells in primary culture [4]. Likewise. 170 bp of the rat Tg promoter wcrc suficicnt lo direct the cell-type-specific expression of the same reporter gent when transfmed in FRTL-5 cells [SJ The contra! of gene expression depends largely on the binding of transcription factors on promoter sequences. A DNA sequence repeated three times in the rat T'g promoter has been sho&m to interact in footprinting experiments with a thyroid-specific nuclear factor called TTF-1 (s]. The lTF-1 protein hzs bccu purified and Cbdracterized by cDNA cloning i7]_In this study. we compare the binding of TTF-1 on both rat and bovine promoters and its functional role in ?hc control of the cxpmsion of ihe corresponding gcna. Although ITF-l WCO@ZCS ilrra s~qu~na in bolh promotcrz in DIQ.sc I footprinting assays, o&y two caqmccs appear functionally rclcvaot in the rat promoter while occupancy of the three sites seems n_ scntial to sustain normal activity of the bovine promoter. MATERIALS AND METWDS

Thyroid transcription factor 1 and Pax8 synergistically activate the promoter of the human thyroglobulin gene

“…Two sites, corresponding to the sites A and C in the rat Tg promoter, were found to bind TTF-1, whereas the middle site B did not bind. This result is in contrast to those from studies performed on rat and bovine Tg promoters, in which TTF-1 recognized three sites (A, B and C) in both promoters (Javaux et al 1992). Another difference between human and rat is that Pax8 did not bind to any of the three sites investigated in the present study, whereas in the rat both TTF-1 and Pax8 bind to the same site C (Zannini et al 1992).…”

“…Similar analyses have been performed in the rat (Zannini et al 1992, Fabbro et al 1998) and bovine (Javaux et al 1992), but not in the human, Tg gene. With respect to the  5.…”

“…The functional roles of TTF-1 and Pax8 binding sites have been studied in rat and bovine Tg gene promoters (Zannini et al 1992, Fabbro et al 1998, Javaux et al 1992, but the roles of both human transcription factors have not been characterized in human Tg promoter, even though the cDNA sequences from human TTF-1 and Pax8 are known (Poleev et al 1995, Saiardi et al 1995.…”

Pendrin Is a Novel In Vivo Downstream Target Gene of the TTF-1/Nkx-2.1 Homeodomain Transcription Factor in Differentiated Thyroid Cells

“…We next examined the effect of the reduced TTF-1 activity induced by N188K on the levels of thyroglobulin, whose promoter is a well-established downstream target of TTF-1 in vitro (6,25,34,50). We measured Tg mRNA levels in FRTL-5/ N188K clones 1 and 3 versus mock-transfected cells (pcDNA).…”

The dog thyroid primary culture system: a model of the regulation of function, growth and differentiation expression by cAMP and other well-defined signaling cascades

“…Tg promoters from different mammalian species, including dog, cattle, man and rat, are equally active when transfected into dog thyrocytes, which suggests that some basic molecular mechanisms involved in the control of Tg gene transcription have been conserved among these species (119). Mutagenesis experiments revealed that thyroid transcription factor-1 (TTF-1) (121) plays a major role in the transcriptional activity of the Tg promoter (122). However, whether TTF-1 is only required for basal promoter activity or whether it mediates the effect of cAMP remained an open question (123,124), as the very low level of transcriptional activity of the promoters when transfected in unstimulated cells precluded a clear demonstration of the role of TTF-1 in this basal activity.…”