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A neuronal-specific differentiation protein that directly modulates retinoid receptor transcriptional activation.

Henry II KW, Spencer ML, Theodosiou M, Lou D, Noonan DJ - Nucl. Recept. (2003)

Bottom Line: Expression of NPDC-1 was also observed to repress transcription mediated by retinoid receptors as well as by several other nuclear receptor family members, although not in a universal manner.CONCLUSIONS: These results suggest that NPDC-1, through direct interaction with retinoid receptors, functions to enhance the transcription complex formation and DNA binding function of retinoid receptors, but ultimately repress retinoid receptor-mediated gene expression.As with NPDC-1, retinoids and their receptors have been implicated in brain development and these data provide a point of convergence for NPDC-1 and retinoid mediation of neuronal differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, 800 Rose Street, Lexington, KY 40536, USA. dnoonan@pop.uky.edu

ABSTRACT
BACKGROUND: The specificity of a nuclear receptor's ability to modulate gene expression resides in its ability to bind a specific lipophilic ligand, associate with specific dimerization partners and bind specific DNA sequences in the promoter regions of genes. This sequence of events appears to be the basis for targeting an additional regulatory complex composed of a variety of protein and RNA components that deliver signals for facilitation or inhibition of the RNA polymerase complex. Characterization of the tissue and cell-specific components of these coregulatory complexes appear to be integral to our understanding of nuclear receptor regulation of transcription. RESULTS: A novel yeast screen sensitive to retinoid-X receptor (RXR) transcriptional activation resulted in the isolation of the rat homologue of the mouse NPDC-1 gene. NPDC-1 has been shown to be involved in the control of neural cell proliferation and differentiation, possibly through interactions with the cell cycle promoting transcription factor E2F-1. Although the amino acid sequence of NPDC-1 is highly conserved between mouse, rat and human homologues, their tissue specific expression was seen to vary. A potential for direct protein:protein interaction between NPDC-1, RXR and retinoic acid receptor beta (RARbeta) was observed in vitro and NPDC-1 facilitated RXR homodimer and RAR-RXR heterodimer DNA binding in vitro. Expression of NPDC-1 was also observed to repress transcription mediated by retinoid receptors as well as by several other nuclear receptor family members, although not in a universal manner. CONCLUSIONS: These results suggest that NPDC-1, through direct interaction with retinoid receptors, functions to enhance the transcription complex formation and DNA binding function of retinoid receptors, but ultimately repress retinoid receptor-mediated gene expression. As with NPDC-1, retinoids and their receptors have been implicated in brain development and these data provide a point of convergence for NPDC-1 and retinoid mediation of neuronal differentiation.

No MeSH data available.


Related in: MedlinePlus

Rat brain cDNA expression library screening in yeast identifies a plasmid containing an approximately 1.4 kb cDNA fragment capable of enhancing RXR transcriptional activation. (A) Yeast cells transformed with the β-galactosidase reporter vector YRpβRE and the yeast expression vector YEpRXRα were exposed to increasing concentrations of 9-cis retinoic acid (9cRA). The βGal activity present in yeast extracts was measured as the colorimetric change of the βGal substrate ONPG (A415) normalized for cell number approximations (A600). The dose response of YRpβRE transformed yeast is also shown (Reporter Alone). Values reported are averages of duplicate data points. (B) An outline of the screening strategy used to identify a RXR transcriptional enhancing cDNA product. Transformed yeast expressing rat brain cDNA products that elevate transcription of the βGal reporter gene result in the formation of blue colonies. (C) BJ5409 yeast cells were transformed with the transcription reporter plasmid YRpβRE(2), co-transformed with either YEpRXRα or pcRB1.4 expression plasmids and YRpβRE(2), or co-transformed with YEpRXRα, pcRB1.4 and YRpβRE(2). The βGal activity present in yeast extracts was measured as in (A). Values reported are averages of multiple data points and error bars reflect standard deviations, n = 3.
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Figure 1: Rat brain cDNA expression library screening in yeast identifies a plasmid containing an approximately 1.4 kb cDNA fragment capable of enhancing RXR transcriptional activation. (A) Yeast cells transformed with the β-galactosidase reporter vector YRpβRE and the yeast expression vector YEpRXRα were exposed to increasing concentrations of 9-cis retinoic acid (9cRA). The βGal activity present in yeast extracts was measured as the colorimetric change of the βGal substrate ONPG (A415) normalized for cell number approximations (A600). The dose response of YRpβRE transformed yeast is also shown (Reporter Alone). Values reported are averages of duplicate data points. (B) An outline of the screening strategy used to identify a RXR transcriptional enhancing cDNA product. Transformed yeast expressing rat brain cDNA products that elevate transcription of the βGal reporter gene result in the formation of blue colonies. (C) BJ5409 yeast cells were transformed with the transcription reporter plasmid YRpβRE(2), co-transformed with either YEpRXRα or pcRB1.4 expression plasmids and YRpβRE(2), or co-transformed with YEpRXRα, pcRB1.4 and YRpβRE(2). The βGal activity present in yeast extracts was measured as in (A). Values reported are averages of multiple data points and error bars reflect standard deviations, n = 3.

Mentions: RXR transcriptional activation was reconstituted in yeast by manipulation of the S. cerevisiae auxotrophic mutant cell line BJ5409. Yeast transcription reporter plasmids incorporate a HRE into an enhancerless gal 1 promoter controlling the conditional expression of a βGal gene. A RXR expressing, βRE-βGal reporter plasmid yeast transformant was isolated and maintained by nutritional selection. Confirmation of the maintenance of both plasmids was demonstrated by ligand (9cRA) dose dependent induction of βGal expression (Fig. 1A). A third nutritionally selectable plasmid expressing a rat brain cDNA library (pcRB) [31] was then transformed into these yeast and plated onto minimal media plates containing the βGal substrate X-GAL (Fig. 1B). Approximately 150,000 transformants were analyzed for the presence of βGal activity (blue colonies).


A neuronal-specific differentiation protein that directly modulates retinoid receptor transcriptional activation.

Henry II KW, Spencer ML, Theodosiou M, Lou D, Noonan DJ - Nucl. Recept. (2003)

Rat brain cDNA expression library screening in yeast identifies a plasmid containing an approximately 1.4 kb cDNA fragment capable of enhancing RXR transcriptional activation. (A) Yeast cells transformed with the β-galactosidase reporter vector YRpβRE and the yeast expression vector YEpRXRα were exposed to increasing concentrations of 9-cis retinoic acid (9cRA). The βGal activity present in yeast extracts was measured as the colorimetric change of the βGal substrate ONPG (A415) normalized for cell number approximations (A600). The dose response of YRpβRE transformed yeast is also shown (Reporter Alone). Values reported are averages of duplicate data points. (B) An outline of the screening strategy used to identify a RXR transcriptional enhancing cDNA product. Transformed yeast expressing rat brain cDNA products that elevate transcription of the βGal reporter gene result in the formation of blue colonies. (C) BJ5409 yeast cells were transformed with the transcription reporter plasmid YRpβRE(2), co-transformed with either YEpRXRα or pcRB1.4 expression plasmids and YRpβRE(2), or co-transformed with YEpRXRα, pcRB1.4 and YRpβRE(2). The βGal activity present in yeast extracts was measured as in (A). Values reported are averages of multiple data points and error bars reflect standard deviations, n = 3.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC222963&req=5

Figure 1: Rat brain cDNA expression library screening in yeast identifies a plasmid containing an approximately 1.4 kb cDNA fragment capable of enhancing RXR transcriptional activation. (A) Yeast cells transformed with the β-galactosidase reporter vector YRpβRE and the yeast expression vector YEpRXRα were exposed to increasing concentrations of 9-cis retinoic acid (9cRA). The βGal activity present in yeast extracts was measured as the colorimetric change of the βGal substrate ONPG (A415) normalized for cell number approximations (A600). The dose response of YRpβRE transformed yeast is also shown (Reporter Alone). Values reported are averages of duplicate data points. (B) An outline of the screening strategy used to identify a RXR transcriptional enhancing cDNA product. Transformed yeast expressing rat brain cDNA products that elevate transcription of the βGal reporter gene result in the formation of blue colonies. (C) BJ5409 yeast cells were transformed with the transcription reporter plasmid YRpβRE(2), co-transformed with either YEpRXRα or pcRB1.4 expression plasmids and YRpβRE(2), or co-transformed with YEpRXRα, pcRB1.4 and YRpβRE(2). The βGal activity present in yeast extracts was measured as in (A). Values reported are averages of multiple data points and error bars reflect standard deviations, n = 3.
Mentions: RXR transcriptional activation was reconstituted in yeast by manipulation of the S. cerevisiae auxotrophic mutant cell line BJ5409. Yeast transcription reporter plasmids incorporate a HRE into an enhancerless gal 1 promoter controlling the conditional expression of a βGal gene. A RXR expressing, βRE-βGal reporter plasmid yeast transformant was isolated and maintained by nutritional selection. Confirmation of the maintenance of both plasmids was demonstrated by ligand (9cRA) dose dependent induction of βGal expression (Fig. 1A). A third nutritionally selectable plasmid expressing a rat brain cDNA library (pcRB) [31] was then transformed into these yeast and plated onto minimal media plates containing the βGal substrate X-GAL (Fig. 1B). Approximately 150,000 transformants were analyzed for the presence of βGal activity (blue colonies).

Bottom Line: Expression of NPDC-1 was also observed to repress transcription mediated by retinoid receptors as well as by several other nuclear receptor family members, although not in a universal manner.CONCLUSIONS: These results suggest that NPDC-1, through direct interaction with retinoid receptors, functions to enhance the transcription complex formation and DNA binding function of retinoid receptors, but ultimately repress retinoid receptor-mediated gene expression.As with NPDC-1, retinoids and their receptors have been implicated in brain development and these data provide a point of convergence for NPDC-1 and retinoid mediation of neuronal differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, 800 Rose Street, Lexington, KY 40536, USA. dnoonan@pop.uky.edu

ABSTRACT
BACKGROUND: The specificity of a nuclear receptor's ability to modulate gene expression resides in its ability to bind a specific lipophilic ligand, associate with specific dimerization partners and bind specific DNA sequences in the promoter regions of genes. This sequence of events appears to be the basis for targeting an additional regulatory complex composed of a variety of protein and RNA components that deliver signals for facilitation or inhibition of the RNA polymerase complex. Characterization of the tissue and cell-specific components of these coregulatory complexes appear to be integral to our understanding of nuclear receptor regulation of transcription. RESULTS: A novel yeast screen sensitive to retinoid-X receptor (RXR) transcriptional activation resulted in the isolation of the rat homologue of the mouse NPDC-1 gene. NPDC-1 has been shown to be involved in the control of neural cell proliferation and differentiation, possibly through interactions with the cell cycle promoting transcription factor E2F-1. Although the amino acid sequence of NPDC-1 is highly conserved between mouse, rat and human homologues, their tissue specific expression was seen to vary. A potential for direct protein:protein interaction between NPDC-1, RXR and retinoic acid receptor beta (RARbeta) was observed in vitro and NPDC-1 facilitated RXR homodimer and RAR-RXR heterodimer DNA binding in vitro. Expression of NPDC-1 was also observed to repress transcription mediated by retinoid receptors as well as by several other nuclear receptor family members, although not in a universal manner. CONCLUSIONS: These results suggest that NPDC-1, through direct interaction with retinoid receptors, functions to enhance the transcription complex formation and DNA binding function of retinoid receptors, but ultimately repress retinoid receptor-mediated gene expression. As with NPDC-1, retinoids and their receptors have been implicated in brain development and these data provide a point of convergence for NPDC-1 and retinoid mediation of neuronal differentiation.

No MeSH data available.


Related in: MedlinePlus