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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.


NPDC-1 LXXLL Motifs are not implicated in NPDC-1 corepression of RAR-mediated transcription Site directed mutagenesis was used to change critical leucines in the pCMV-hNPDC-1 LLRLLL motif (see insert in figure for specifics) to alanines. These expression constructs were cotransfected with the RAR/RXR-specific βRE reporter construct into human embryonic kidney cells (HEK) and analyzed for transcriptional activation of the reporter in the presence of 10-6 M all trans retinoic acid (atRA). Transfections were normalized to a β-galactosidase expression construct included in all transfections, and data was calculated as described in Fig. 7. Data was expressed as a percent of the reporter alone transfection and statistics were performed on the results of 3 independent experiments. Error bars represent the value of standard deviations n = 3.
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Figure 8: NPDC-1 LXXLL Motifs are not implicated in NPDC-1 corepression of RAR-mediated transcription Site directed mutagenesis was used to change critical leucines in the pCMV-hNPDC-1 LLRLLL motif (see insert in figure for specifics) to alanines. These expression constructs were cotransfected with the RAR/RXR-specific βRE reporter construct into human embryonic kidney cells (HEK) and analyzed for transcriptional activation of the reporter in the presence of 10-6 M all trans retinoic acid (atRA). Transfections were normalized to a β-galactosidase expression construct included in all transfections, and data was calculated as described in Fig. 7. Data was expressed as a percent of the reporter alone transfection and statistics were performed on the results of 3 independent experiments. Error bars represent the value of standard deviations n = 3.

Mentions: The above data suggest NPDC-1 functions as a steroid/hormone nuclear receptor corepressor protein [17-21]. Many of the corepressors identified thus far have been shown to mediate their activities through specific interactions between receptors and a short conserved LXXLL amino acid sequence motif found on the repressor proteins [22,23]. Within the amino terminus of the NPDC protein there exists a highly conserved LLRLLL motif (see Fig. 2). To analyze the functional role of this LLRLLL motif in transcriptional repression mediated by NPDC-1, a series of mutations in the LLRLLL motif were made that sequentially converted key leucines into alanines. These mutated NPDC-1 constructs were analyzed in the mammalian transcription assay for their ability to repress RAR-mediated transcription (Fig. 8). As seen here, none of the mutations were able to impact the repression associated with NPDC-1 cotransfection. These data suggest the LXXLL motifs of NPDC-1 do not play a key role in its ability to repress transcription.


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)

NPDC-1 LXXLL Motifs are not implicated in NPDC-1 corepression of RAR-mediated transcription Site directed mutagenesis was used to change critical leucines in the pCMV-hNPDC-1 LLRLLL motif (see insert in figure for specifics) to alanines. These expression constructs were cotransfected with the RAR/RXR-specific βRE reporter construct into human embryonic kidney cells (HEK) and analyzed for transcriptional activation of the reporter in the presence of 10-6 M all trans retinoic acid (atRA). Transfections were normalized to a β-galactosidase expression construct included in all transfections, and data was calculated as described in Fig. 7. Data was expressed as a percent of the reporter alone transfection and statistics were performed on the results of 3 independent experiments. Error bars represent the value of standard deviations n = 3.
© Copyright Policy
Related In: Results  -  Collection

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Figure 8: NPDC-1 LXXLL Motifs are not implicated in NPDC-1 corepression of RAR-mediated transcription Site directed mutagenesis was used to change critical leucines in the pCMV-hNPDC-1 LLRLLL motif (see insert in figure for specifics) to alanines. These expression constructs were cotransfected with the RAR/RXR-specific βRE reporter construct into human embryonic kidney cells (HEK) and analyzed for transcriptional activation of the reporter in the presence of 10-6 M all trans retinoic acid (atRA). Transfections were normalized to a β-galactosidase expression construct included in all transfections, and data was calculated as described in Fig. 7. Data was expressed as a percent of the reporter alone transfection and statistics were performed on the results of 3 independent experiments. Error bars represent the value of standard deviations n = 3.
Mentions: The above data suggest NPDC-1 functions as a steroid/hormone nuclear receptor corepressor protein [17-21]. Many of the corepressors identified thus far have been shown to mediate their activities through specific interactions between receptors and a short conserved LXXLL amino acid sequence motif found on the repressor proteins [22,23]. Within the amino terminus of the NPDC protein there exists a highly conserved LLRLLL motif (see Fig. 2). To analyze the functional role of this LLRLLL motif in transcriptional repression mediated by NPDC-1, a series of mutations in the LLRLLL motif were made that sequentially converted key leucines into alanines. These mutated NPDC-1 constructs were analyzed in the mammalian transcription assay for their ability to repress RAR-mediated transcription (Fig. 8). As seen here, none of the mutations were able to impact the repression associated with NPDC-1 cotransfection. These data suggest the LXXLL motifs of NPDC-1 do not play a key role in its ability to repress transcription.

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.