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Modulation of CP2 family transcriptional activity by CRTR-1 and sumoylation.

To S, Rodda SJ, Rathjen PD, Keough RA - PLoS ONE (2010)

Bottom Line: CRTR-1 was previously shown to be a repressor of transcription.We also demonstrate that CRTR-1 activity is regulated by sumoylation at a single major site, residue K30.These findings imply that functional redundancy with other family members may mask important roles for CRTR-1 in other tissues, including the blastocyst stage embryo and embryonic stem cells.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia.

ABSTRACT
CRTR-1 is a member of the CP2 family of transcription factors. Unlike other members of the family which are widely expressed, CRTR-1 expression shows specific spatio-temporal regulation. Gene targeting demonstrates that CRTR-1 plays a central role in the maturation and function of the salivary glands and the kidney. CRTR-1 has also recently been identified as a component of the complex transcriptional network that maintains pluripotency in embryonic stem (ES) cells. CRTR-1 was previously shown to be a repressor of transcription. We examine the activity of CRTR-1 in ES and other cells and show that CRTR-1 is generally an activator of transcription and that it modulates the activity of other family members, CP2, NF2d9 and altNF2d9, in a cell specific manner. We also demonstrate that CRTR-1 activity is regulated by sumoylation at a single major site, residue K30. These findings imply that functional redundancy with other family members may mask important roles for CRTR-1 in other tissues, including the blastocyst stage embryo and embryonic stem cells.

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CRTR-1 is sumolyated at K30 and sumoylation is enhanced by PIAS1 or Ubc9.A. COS-1 cells were co-transfected with pEF-CRTR-1and pEF-FLAG-SUMO-1 expression plasmids as indicated. Whole cell lysates were immunoprecipitated (IP) with anti-FLAG or anti-CRTR-1 antibody and analysed by immunoblotting (IB) as indicated to detect sumoylated proteins. Bracket identifies higher molecular weight bands as possible multiple or polysumoylated CRTR-1. B. COS-1 cells were co-transfected with pEF-CRTR-1, pEF-FLAG-SUMO-1, pEF-FLAG-Ubc9, and pEF-FLAG-PIAS1 expression plasmids as indicated. Whole cell lysates were immunoprecipitated with anti-FLAG or anti-CRTR-1 antibody and analysed by immunoblotting with anti-CRTR-1 or anti-FLAG antibody respectively to detect sumoylated proteins (bracket). Immunoblotting of input lysates with anti-CRTR-1 antibody detected both sumoylated and unsumoylated CRTR-1. C. COS-1 cells were co-transfected with pEF-CRTR-1, pEF-K30A, pEF-K464A or pEF-2KA together with pEF-FLAG-SUMO-1 expression plasmids. Whole cell lysates were immunoprecipitated with anti-CRTR-1 antibody and analysed by immunoblotting with anti-FLAG antibody respectively to detect sumoylated proteins (bracket). Immunoblotting of input cell lysates with anti-CRTR-1 antibody detected unsumoylated CRTR-1. Molecular weight markers are shown (kD). Also see Figure S3A which is a re-probing of the blot in (C) with anti-CRTR-1 antibody.
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pone-0011702-g006: CRTR-1 is sumolyated at K30 and sumoylation is enhanced by PIAS1 or Ubc9.A. COS-1 cells were co-transfected with pEF-CRTR-1and pEF-FLAG-SUMO-1 expression plasmids as indicated. Whole cell lysates were immunoprecipitated (IP) with anti-FLAG or anti-CRTR-1 antibody and analysed by immunoblotting (IB) as indicated to detect sumoylated proteins. Bracket identifies higher molecular weight bands as possible multiple or polysumoylated CRTR-1. B. COS-1 cells were co-transfected with pEF-CRTR-1, pEF-FLAG-SUMO-1, pEF-FLAG-Ubc9, and pEF-FLAG-PIAS1 expression plasmids as indicated. Whole cell lysates were immunoprecipitated with anti-FLAG or anti-CRTR-1 antibody and analysed by immunoblotting with anti-CRTR-1 or anti-FLAG antibody respectively to detect sumoylated proteins (bracket). Immunoblotting of input lysates with anti-CRTR-1 antibody detected both sumoylated and unsumoylated CRTR-1. C. COS-1 cells were co-transfected with pEF-CRTR-1, pEF-K30A, pEF-K464A or pEF-2KA together with pEF-FLAG-SUMO-1 expression plasmids. Whole cell lysates were immunoprecipitated with anti-CRTR-1 antibody and analysed by immunoblotting with anti-FLAG antibody respectively to detect sumoylated proteins (bracket). Immunoblotting of input cell lysates with anti-CRTR-1 antibody detected unsumoylated CRTR-1. Molecular weight markers are shown (kD). Also see Figure S3A which is a re-probing of the blot in (C) with anti-CRTR-1 antibody.

Mentions: Cell specific behaviour of CRTR-1 is demonstrated by its lower activity in COS-1 cells compared with ES and HEK293T cells and by its ability to enhance, rather than suppress, altNF2d9 activity in this cell type. While this may be due to a number of possible factors, it is well documented that sumoylation of transcription factors can affect their activity and it has also been reported that erythroid-specific activity of CP2 proteins is mediated, in part, by PIAS1, a sumo E3 ligase [2]. We, therefore, investigated the possible role of sumoylation of CRTR-1 on its activity. To determine whether CRTR-1 can be sumoylated, FLAG-SUMO-1 and CRTR-1 expression plasmids were co-transfected into COS-1 cells and whole cell lysates were immunoprecipitated with either FLAG or CRTR-1 antibodies (Figure 6A). Unsumoylated CRTR-1 runs as 2 doublets of approximately 54 and 60 kD. Immunoprecipitated proteins of approximately 75–80 kD are detected with both the CRTR-1 and FLAG antibodies, corresponding to sumoylated forms of CRTR-1. Co-transfection with Ubc9, an E2 sumo conjugating enzyme, or PIAS1 expression plasmids enhanced the level of CRTR-1 sumoylation observed (Figure 6B).


Modulation of CP2 family transcriptional activity by CRTR-1 and sumoylation.

To S, Rodda SJ, Rathjen PD, Keough RA - PLoS ONE (2010)

CRTR-1 is sumolyated at K30 and sumoylation is enhanced by PIAS1 or Ubc9.A. COS-1 cells were co-transfected with pEF-CRTR-1and pEF-FLAG-SUMO-1 expression plasmids as indicated. Whole cell lysates were immunoprecipitated (IP) with anti-FLAG or anti-CRTR-1 antibody and analysed by immunoblotting (IB) as indicated to detect sumoylated proteins. Bracket identifies higher molecular weight bands as possible multiple or polysumoylated CRTR-1. B. COS-1 cells were co-transfected with pEF-CRTR-1, pEF-FLAG-SUMO-1, pEF-FLAG-Ubc9, and pEF-FLAG-PIAS1 expression plasmids as indicated. Whole cell lysates were immunoprecipitated with anti-FLAG or anti-CRTR-1 antibody and analysed by immunoblotting with anti-CRTR-1 or anti-FLAG antibody respectively to detect sumoylated proteins (bracket). Immunoblotting of input lysates with anti-CRTR-1 antibody detected both sumoylated and unsumoylated CRTR-1. C. COS-1 cells were co-transfected with pEF-CRTR-1, pEF-K30A, pEF-K464A or pEF-2KA together with pEF-FLAG-SUMO-1 expression plasmids. Whole cell lysates were immunoprecipitated with anti-CRTR-1 antibody and analysed by immunoblotting with anti-FLAG antibody respectively to detect sumoylated proteins (bracket). Immunoblotting of input cell lysates with anti-CRTR-1 antibody detected unsumoylated CRTR-1. Molecular weight markers are shown (kD). Also see Figure S3A which is a re-probing of the blot in (C) with anti-CRTR-1 antibody.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908540&req=5

pone-0011702-g006: CRTR-1 is sumolyated at K30 and sumoylation is enhanced by PIAS1 or Ubc9.A. COS-1 cells were co-transfected with pEF-CRTR-1and pEF-FLAG-SUMO-1 expression plasmids as indicated. Whole cell lysates were immunoprecipitated (IP) with anti-FLAG or anti-CRTR-1 antibody and analysed by immunoblotting (IB) as indicated to detect sumoylated proteins. Bracket identifies higher molecular weight bands as possible multiple or polysumoylated CRTR-1. B. COS-1 cells were co-transfected with pEF-CRTR-1, pEF-FLAG-SUMO-1, pEF-FLAG-Ubc9, and pEF-FLAG-PIAS1 expression plasmids as indicated. Whole cell lysates were immunoprecipitated with anti-FLAG or anti-CRTR-1 antibody and analysed by immunoblotting with anti-CRTR-1 or anti-FLAG antibody respectively to detect sumoylated proteins (bracket). Immunoblotting of input lysates with anti-CRTR-1 antibody detected both sumoylated and unsumoylated CRTR-1. C. COS-1 cells were co-transfected with pEF-CRTR-1, pEF-K30A, pEF-K464A or pEF-2KA together with pEF-FLAG-SUMO-1 expression plasmids. Whole cell lysates were immunoprecipitated with anti-CRTR-1 antibody and analysed by immunoblotting with anti-FLAG antibody respectively to detect sumoylated proteins (bracket). Immunoblotting of input cell lysates with anti-CRTR-1 antibody detected unsumoylated CRTR-1. Molecular weight markers are shown (kD). Also see Figure S3A which is a re-probing of the blot in (C) with anti-CRTR-1 antibody.
Mentions: Cell specific behaviour of CRTR-1 is demonstrated by its lower activity in COS-1 cells compared with ES and HEK293T cells and by its ability to enhance, rather than suppress, altNF2d9 activity in this cell type. While this may be due to a number of possible factors, it is well documented that sumoylation of transcription factors can affect their activity and it has also been reported that erythroid-specific activity of CP2 proteins is mediated, in part, by PIAS1, a sumo E3 ligase [2]. We, therefore, investigated the possible role of sumoylation of CRTR-1 on its activity. To determine whether CRTR-1 can be sumoylated, FLAG-SUMO-1 and CRTR-1 expression plasmids were co-transfected into COS-1 cells and whole cell lysates were immunoprecipitated with either FLAG or CRTR-1 antibodies (Figure 6A). Unsumoylated CRTR-1 runs as 2 doublets of approximately 54 and 60 kD. Immunoprecipitated proteins of approximately 75–80 kD are detected with both the CRTR-1 and FLAG antibodies, corresponding to sumoylated forms of CRTR-1. Co-transfection with Ubc9, an E2 sumo conjugating enzyme, or PIAS1 expression plasmids enhanced the level of CRTR-1 sumoylation observed (Figure 6B).

Bottom Line: CRTR-1 was previously shown to be a repressor of transcription.We also demonstrate that CRTR-1 activity is regulated by sumoylation at a single major site, residue K30.These findings imply that functional redundancy with other family members may mask important roles for CRTR-1 in other tissues, including the blastocyst stage embryo and embryonic stem cells.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia.

ABSTRACT
CRTR-1 is a member of the CP2 family of transcription factors. Unlike other members of the family which are widely expressed, CRTR-1 expression shows specific spatio-temporal regulation. Gene targeting demonstrates that CRTR-1 plays a central role in the maturation and function of the salivary glands and the kidney. CRTR-1 has also recently been identified as a component of the complex transcriptional network that maintains pluripotency in embryonic stem (ES) cells. CRTR-1 was previously shown to be a repressor of transcription. We examine the activity of CRTR-1 in ES and other cells and show that CRTR-1 is generally an activator of transcription and that it modulates the activity of other family members, CP2, NF2d9 and altNF2d9, in a cell specific manner. We also demonstrate that CRTR-1 activity is regulated by sumoylation at a single major site, residue K30. These findings imply that functional redundancy with other family members may mask important roles for CRTR-1 in other tissues, including the blastocyst stage embryo and embryonic stem cells.

Show MeSH
Related in: MedlinePlus