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Regulation of rDNA transcription by proto-oncogene PELP1.

Gonugunta VK, Nair BC, Rajhans R, Sareddy GR, Nair SS, Vadlamudi RK - PLoS ONE (2011)

Bottom Line: Using pharmacological compounds and CDK site mutants of PELP1, we found that CDK's activity plays an important role on PELP1 nucleolar localization.Depletion of PELP1 by siRNA decreased the expression of pre-rRNA.Collectively, our results suggest that proto-oncogene PELP1 plays a vital role in rDNA transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, and Cancer Therapy and Research Center, University of Texas Health Science Center, San Antonio, Texas, United States of America.

ABSTRACT

Background: Proline-, glutamic acid-, and leucine-rich protein (PELP1) is a novel nuclear receptor coregulator with a multitude of functions. PELP1 serves as a scaffolding protein that couples various signaling complexes with nuclear receptors and participates as a transcriptional coregulator. Recent data suggest that PELP1 expression is deregulated in hormonal cancers, and that PELP1 functions as a proto-oncogene; however, the mechanism by which PELP1 promotes oncogenesis remains elusive.

Methodology/principal findings: Using pharmacological inhibitors, confocal microscopy and biochemical assays, we demonstrated that PELP1 is localized in the nucleolus and that PELP1 is associated with the active ribosomal RNA transcription. Cell synchronization studies showed that PELP1 nucleolar localization varies and the greatest amount of nucleolar localization was observed during S and G2 phases. Using pharmacological compounds and CDK site mutants of PELP1, we found that CDK's activity plays an important role on PELP1 nucleolar localization. Depletion of PELP1 by siRNA decreased the expression of pre-rRNA. Reporter gene assays using ribosomal DNA (pHrD) luc-reporter revealed that PELP1WT but not PELP1MT enhanced the expression of reporter. Deletion of nucleolar domains abolished PELP1-mediated activation of the pHrD reporter. ChIP analysis revealed that PELP1 is recruited to the promoter regions of rDNA and is needed for optimal transcription of ribosomal RNA.

Conclusions/significance: Collectively, our results suggest that proto-oncogene PELP1 plays a vital role in rDNA transcription. PELP1 modulation of rRNA transcription, a key step in ribosomal biogenesis may have implications in PELP1-mediated oncogenic functions.

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Related in: MedlinePlus

PELP1 associates with the rDNA promoter and regulates rDNA transcription.(A) Schematic representation of the rDNA gene and primer pairs used in the ChIP assay. (B) ChIP assay was done using antibodies specific for PELP1 or isotype rabbit IgG control in ZR-75 cells. DNA recovered from ChIP or input controls was subjected to conventional PCR using the indicated primers spanning the promoter and coding regions. (C) Total RNA was isolated from ZR-75 overexpressing PELP1 and HeLa, ZR-75 cells expressing control or PELP1 siRNA analyzed for the status of pre-rRNA transcription by RT-PCR.
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pone-0021095-g006: PELP1 associates with the rDNA promoter and regulates rDNA transcription.(A) Schematic representation of the rDNA gene and primer pairs used in the ChIP assay. (B) ChIP assay was done using antibodies specific for PELP1 or isotype rabbit IgG control in ZR-75 cells. DNA recovered from ChIP or input controls was subjected to conventional PCR using the indicated primers spanning the promoter and coding regions. (C) Total RNA was isolated from ZR-75 overexpressing PELP1 and HeLa, ZR-75 cells expressing control or PELP1 siRNA analyzed for the status of pre-rRNA transcription by RT-PCR.

Mentions: Earlier studies showed that PELP1 facilitate chromatin modifications by recruiting to target gene promoters [22]. We therefore examined whether PELP1 was directly involved in the rDNA transcription by recruiting to the rDNA promoter (Figure 6A). Previous studies identified several regulatory regions in the rDNA promoter [23], which are schematically represented in Figure 6A. Chromatin immunoprecipitation studies revealed that PELP1 was efficiently recruited to the promoter region of the rDNA (Figure 6B). Further analysis using additional primers showed efficient recruitment of PELP1 at various known regulatory sites in the rDNA promoter (Figure 6B) with no or weak recruitment to the coding regions. Accordingly, overexpression of PELP1 in ZR-75 cells had increased rRNA transcription as measured by RT-PCR analysis. On the other hand, PELP1 knock-down in ZR-75 and HeLa cells resulted in substantially reduced rRNA transcript levels (Figure 6C). Collectively, these results suggest that PELP1 has potential to recruit to the rDNA promoter and can modulate pre-rRNA transcript levels.


Regulation of rDNA transcription by proto-oncogene PELP1.

Gonugunta VK, Nair BC, Rajhans R, Sareddy GR, Nair SS, Vadlamudi RK - PLoS ONE (2011)

PELP1 associates with the rDNA promoter and regulates rDNA transcription.(A) Schematic representation of the rDNA gene and primer pairs used in the ChIP assay. (B) ChIP assay was done using antibodies specific for PELP1 or isotype rabbit IgG control in ZR-75 cells. DNA recovered from ChIP or input controls was subjected to conventional PCR using the indicated primers spanning the promoter and coding regions. (C) Total RNA was isolated from ZR-75 overexpressing PELP1 and HeLa, ZR-75 cells expressing control or PELP1 siRNA analyzed for the status of pre-rRNA transcription by RT-PCR.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113909&req=5

pone-0021095-g006: PELP1 associates with the rDNA promoter and regulates rDNA transcription.(A) Schematic representation of the rDNA gene and primer pairs used in the ChIP assay. (B) ChIP assay was done using antibodies specific for PELP1 or isotype rabbit IgG control in ZR-75 cells. DNA recovered from ChIP or input controls was subjected to conventional PCR using the indicated primers spanning the promoter and coding regions. (C) Total RNA was isolated from ZR-75 overexpressing PELP1 and HeLa, ZR-75 cells expressing control or PELP1 siRNA analyzed for the status of pre-rRNA transcription by RT-PCR.
Mentions: Earlier studies showed that PELP1 facilitate chromatin modifications by recruiting to target gene promoters [22]. We therefore examined whether PELP1 was directly involved in the rDNA transcription by recruiting to the rDNA promoter (Figure 6A). Previous studies identified several regulatory regions in the rDNA promoter [23], which are schematically represented in Figure 6A. Chromatin immunoprecipitation studies revealed that PELP1 was efficiently recruited to the promoter region of the rDNA (Figure 6B). Further analysis using additional primers showed efficient recruitment of PELP1 at various known regulatory sites in the rDNA promoter (Figure 6B) with no or weak recruitment to the coding regions. Accordingly, overexpression of PELP1 in ZR-75 cells had increased rRNA transcription as measured by RT-PCR analysis. On the other hand, PELP1 knock-down in ZR-75 and HeLa cells resulted in substantially reduced rRNA transcript levels (Figure 6C). Collectively, these results suggest that PELP1 has potential to recruit to the rDNA promoter and can modulate pre-rRNA transcript levels.

Bottom Line: Using pharmacological compounds and CDK site mutants of PELP1, we found that CDK's activity plays an important role on PELP1 nucleolar localization.Depletion of PELP1 by siRNA decreased the expression of pre-rRNA.Collectively, our results suggest that proto-oncogene PELP1 plays a vital role in rDNA transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, and Cancer Therapy and Research Center, University of Texas Health Science Center, San Antonio, Texas, United States of America.

ABSTRACT

Background: Proline-, glutamic acid-, and leucine-rich protein (PELP1) is a novel nuclear receptor coregulator with a multitude of functions. PELP1 serves as a scaffolding protein that couples various signaling complexes with nuclear receptors and participates as a transcriptional coregulator. Recent data suggest that PELP1 expression is deregulated in hormonal cancers, and that PELP1 functions as a proto-oncogene; however, the mechanism by which PELP1 promotes oncogenesis remains elusive.

Methodology/principal findings: Using pharmacological inhibitors, confocal microscopy and biochemical assays, we demonstrated that PELP1 is localized in the nucleolus and that PELP1 is associated with the active ribosomal RNA transcription. Cell synchronization studies showed that PELP1 nucleolar localization varies and the greatest amount of nucleolar localization was observed during S and G2 phases. Using pharmacological compounds and CDK site mutants of PELP1, we found that CDK's activity plays an important role on PELP1 nucleolar localization. Depletion of PELP1 by siRNA decreased the expression of pre-rRNA. Reporter gene assays using ribosomal DNA (pHrD) luc-reporter revealed that PELP1WT but not PELP1MT enhanced the expression of reporter. Deletion of nucleolar domains abolished PELP1-mediated activation of the pHrD reporter. ChIP analysis revealed that PELP1 is recruited to the promoter regions of rDNA and is needed for optimal transcription of ribosomal RNA.

Conclusions/significance: Collectively, our results suggest that proto-oncogene PELP1 plays a vital role in rDNA transcription. PELP1 modulation of rRNA transcription, a key step in ribosomal biogenesis may have implications in PELP1-mediated oncogenic functions.

Show MeSH
Related in: MedlinePlus