Two upstream elements activate transcription of a major histocompatibility complex class I gene<i>in vitro</i>

The thyroid hormone and retinoid X receptors form a heterodimer with each other and mediate thyroid hormone (T3)-dependent transcription. Retinoid X receptor, in addition, forms a homodimer and mediates 9-cis-retinoic aciddependent transcription. Here, recombinant thyroid hormone receptor and recombinant retinoid X receptor .8 expressed from baculovirus vectors have been studied for ligandmediated activation of transcription in vitro. We show that the two recombinant receptors, most likely as a heterodimer, cooperatively enhance transcription in vitro from a template containing functional T3 responsive elements. The enhancement was specific for the T3 responsive element and was greatest when T3 was added to the reaction (-14-fold increase). Albeit to a lesser degree, the two receptors also directed transcription in the absence of T3. Template competition experiments suggest that the two receptors enhance formation of the preinitiation complex and that activation by T3 occurs when the ligand binds the receptor prior to (or during), but not after, the formation of the preinitiation complex. Although 9-cis-retinoic acid had no effect on the T3-dependent transcription, this ligand activated transcription in vitro directed by recombinant retinoic X receptor 1A, most likely as a homodimer. This activation was observed when using nuclear extracts from embryonal carcinoma cells as a source of basal transcription factors, but not those from B lymphocytes. These results demonstrate that transcriptional activation mediated by T3 and 9-cis-retinoic acid can be reconstituted in vitro with the respective recombinant receptors.

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“…pLd4OGF was constructed from pLd6OGF (31) by overlap amplification. The ME-TRE and TREp templates ( Fig.…”

Section: Methodsmentioning

confidence: 99%

“…The ME-TRE and TREp templates ( Fig. 1) (31). To some reaction mixtures, 0.05% sarkosyl was added 15 sec before addition of NTPs to allow a single round of transcription (35).…”

Section: Methodsmentioning

confidence: 99%

Recombinant thyroid hormone receptor and retinoid Xreceptor stimulate ligand-dependent transcription in vitro.

Lee

Driggers

Medin

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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“…NT2 cells (1,2) were obtained from L. Staudt (National Institutes of Health) and were maintained in Dul-becco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), gentamicin (50 ,ug/ml), and glutamine (20 mM) Transfection and reporter assays. MHC class I chloramphenicol acetyltransferase (CAT) reporter constructs pLdl400.CAT, pLd237.CAT, pLdl23.CAT, and pLd6O.CAT have been described (23). Mutant pLdl400.CAT constructs were prepared by the oligonucleotide-directed mutagenesis procedure, using polymerase chain reaction (see Fig.…”

Section: Methodsmentioning

confidence: 99%

“…pLdl23.CAT gave slightly higher CAT activity than pLd237.CAT and pLdl400. CAT, presumably because of modest activity of a downstream regulatory element(s) present in pLdl23.CAT (23) or lack of negative control by an upstream repressor element. Negative regulation of pLdl23.CAT was observed in F9 EC cells (25).…”

Section: Methodsmentioning

confidence: 99%

Retinoic acid induction of major histocompatibility complex class I genes in NTera-2 embryonal carcinoma cells involves induction of NF-kappa B (p50-p65) and retinoic acid receptor beta-retinoid X receptor beta heterodimers.

Segars¹,

Nagata²,

Bours³

et al. 1993

Mol. Cell. Biol.

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Retinoic acid (RA) treatment of human embryonal carcinoma (EC) NTera-2 (NT2) cells induces expression of major histocompatibility complex (MHC) class I and 13-2 microglobulin surface molecules. We found that this induction was accompanied by increased levels of MHC class I mRNA, which was attributable to the activation of the two conserved upstream enhancers, region I (NF-KB like) and region H. This activation coincided with the induction of nuclear factor binding activities specific for the two enhancers. Region I binding activity was not present in undifferentiated NT2 cells, but binding of an NF-KB heterodimer, p50-p65, was induced following RA treatment. The p50-p65 heterodimer was produced as a result of de novo induction of p50 and p65 mRNAs. Region H binding activity was present in undifferentiated cells at low levels but was greatly augmented by RA treatment because of activation of a nuclear hormone receptor heterodimer composed of the retinoid X receptor (RXR3) and the RA receptor (RARI3). The RXR,-RARj3 heterodimer also bound RA responsive elements present in other genes which are likely to be involved in RA triggering of EC cell differentiation. Furthermore, transfection of p50 and p65 into undifferentiated NT2 cells synergistically activated region I-dependent MHC class I reporter activity. A similar increase in MHC class I reporter activity was demonstrated by cotransfection of RXRI3 and RAR(3. These data show that following RA treatment, heterodimers of two transcription factor families are induced to bind to the MHC enhancers, which at least partly accounts for RA induction of MHC class I expression in NT2 EC cells.

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“…Most reported regulatory factors have a relatively small effect on MHC class I gene transcription, depending on the cell type and the specific MHC class I locus (25,(72)(73)(74). However, even small changes in MHC class I gene expression (i.e.…”

Section: Discussionmentioning

confidence: 99%

Identification of CCAAT Displacement Protein (CDP/cut) as a Locus-specific Repressor of Major Histocompatibility Complex Gene Expression in Human Tumor Cells

Snyder¹,

Wang²,

Waring³

et al. 2001

Journal of Biological Chemistry

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The human MHC 1 class I genes encode the highly polymorphic HLA class I antigen heavy chains, membrane-spanning proteins that combine with the invariant ␤ 2 -microglobulin to form the HLA class I surface antigen (1). Classical MHC class I (HLA-A, -B, and -C) antigen expression is essential for processing and presentation of peptide antigens to cytotoxic CD8ϩ lymphocytes (2). In addition, recent evidence suggests that certain classical and nonclassical class I antigens play an important role in inhibition of natural killer (NK) cell function (3-6) and certain subsets of cytotoxic T-cells (7). Therefore, changes in MHC class I gene expression can have profound effects on the overall susceptibility to NK cell and cytotoxic T-cell-mediated lysis, the net effect depending on the specific MHC Class I genes involved. Down-regulation of MHC class I gene expression is observed in many human tumors and transformed cell lines, resulting in decreased susceptibility to cytotoxic T-cell-mediated lysis (8 -13). Global down-regulation of surface class I expression can arise by several mechanisms, including loss of ␤ 2 -microglobulin or peptide transporter gene expression (9, 14). More frequently, loss of MHC class I antigen expression in tumor cells occurs at a single locus. For example, selective down-regulation of HLA-B locus gene expression has been observed in many cell lines derived from patients with metastatic melanoma (15) and colon cancer (16). The specific MHC class I alleles in a particular tumor that activate cytotoxic T-cells may be different from those that inhibit NK cell function. Therefore, understanding how MHC class I antigen expression is controlled at the level of specific loci and alleles is an important goal with possible implications for the development of vaccine-based cancer therapeutics.The mechanisms that mediate locus-specific down-regulation of MHC class I gene expression are incompletely understood. Transcriptional mechanisms appear to be involved in many cases (17-19) but have not been well characterized. The promoter and 5Ј-flanking regions of most MHC class I genes contain several highly conserved, well characterized DNA sequence elements involved in maintenance of constitutive expression (20 -22). Down-regulation of MHC class I expression due to decreased binding of members of the rel family of transcription factors to the highly conserved enhancer A element has been reported in human tumor cell lines (23) and in adenovirus-12-transformed cell lines (24). Our laboratory has previously shown that another highly conserved element, the interferon-stimulated response element, functions as a locusspecific activator of HLA-A gene expression in several

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Two upstream elements activate transcription of a major histocompatibility complex class I genein vitro

Cited by 32 publications

References 69 publications

Recombinant thyroid hormone receptor and retinoid Xreceptor stimulate ligand-dependent transcription in vitro.

Recombinant thyroid hormone receptor and retinoid Xreceptor stimulate ligand-dependent transcription in vitro.

Retinoic acid induction of major histocompatibility complex class I genes in NTera-2 embryonal carcinoma cells involves induction of NF-kappa B (p50-p65) and retinoic acid receptor beta-retinoid X receptor beta heterodimers.

Identification of CCAAT Displacement Protein (CDP/cut) as a Locus-specific Repressor of Major Histocompatibility Complex Gene Expression in Human Tumor Cells

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