Limits...
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.

Show MeSH

Related in: MedlinePlus

CRTR-1 co-immunoprecipitates with other CP2 family members.HEK293T cells were co-transfected with pEF-IRES-puro6 expression plasmids encoding CRTR-1 and FLAG-CP2, FLAG-NF2d9 or FLAG-altNF2d9, as indicated. Whole cell lysates were immunoprecipitated (IP) with (A) anti-FLAG (M2) antibody or (B) anti-CRTR-1 antibody and immunoblotted (IB) with anti-CRTR-1 or anti-FLAG antibodies respectively. Western blot analysis of input lysates is shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2908540&req=5

pone-0011702-g003: CRTR-1 co-immunoprecipitates with other CP2 family members.HEK293T cells were co-transfected with pEF-IRES-puro6 expression plasmids encoding CRTR-1 and FLAG-CP2, FLAG-NF2d9 or FLAG-altNF2d9, as indicated. Whole cell lysates were immunoprecipitated (IP) with (A) anti-FLAG (M2) antibody or (B) anti-CRTR-1 antibody and immunoblotted (IB) with anti-CRTR-1 or anti-FLAG antibodies respectively. Western blot analysis of input lysates is shown.

Mentions: CP2 family proteins interact with each other, forming homomers and heteromers [9], with the functional DNA-binding unit thought to be tetrameric [7], [8]. To determine whether CRTR-1 could interact with other CP2 family members, co-immunoprecipitations were performed (Figure 3) using FLAG-tagged CP2, NF2d9 and altNF2d9 co-expressed with CRTR-1 in HEK293T cells. CRTR-1 co-precipitated with all CP2 family proteins (Figure 3A) and this interaction was confirmed with the reciprocal co-immunoprecipitation (Figure 3B). Some background precipitation of FLAG-CP2 and FLAG-NF2d9 was evident (see left-hand panel of Figure 3B). Control experiments performed using pre-immune serum or no antibody (data not shown) demonstrated that this was due to non-specific binding of the FLAG-tagged protein to the agarose beads. However, the greatly enriched pull-down of FLAG-CP2 in the presence of ectopically expressed CRTR-1 indicates that the interaction between CRTR-1 and the other CP2 family members is genuine.


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

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

CRTR-1 co-immunoprecipitates with other CP2 family members.HEK293T cells were co-transfected with pEF-IRES-puro6 expression plasmids encoding CRTR-1 and FLAG-CP2, FLAG-NF2d9 or FLAG-altNF2d9, as indicated. Whole cell lysates were immunoprecipitated (IP) with (A) anti-FLAG (M2) antibody or (B) anti-CRTR-1 antibody and immunoblotted (IB) with anti-CRTR-1 or anti-FLAG antibodies respectively. Western blot analysis of input lysates is shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2908540&req=5

pone-0011702-g003: CRTR-1 co-immunoprecipitates with other CP2 family members.HEK293T cells were co-transfected with pEF-IRES-puro6 expression plasmids encoding CRTR-1 and FLAG-CP2, FLAG-NF2d9 or FLAG-altNF2d9, as indicated. Whole cell lysates were immunoprecipitated (IP) with (A) anti-FLAG (M2) antibody or (B) anti-CRTR-1 antibody and immunoblotted (IB) with anti-CRTR-1 or anti-FLAG antibodies respectively. Western blot analysis of input lysates is shown.
Mentions: CP2 family proteins interact with each other, forming homomers and heteromers [9], with the functional DNA-binding unit thought to be tetrameric [7], [8]. To determine whether CRTR-1 could interact with other CP2 family members, co-immunoprecipitations were performed (Figure 3) using FLAG-tagged CP2, NF2d9 and altNF2d9 co-expressed with CRTR-1 in HEK293T cells. CRTR-1 co-precipitated with all CP2 family proteins (Figure 3A) and this interaction was confirmed with the reciprocal co-immunoprecipitation (Figure 3B). Some background precipitation of FLAG-CP2 and FLAG-NF2d9 was evident (see left-hand panel of Figure 3B). Control experiments performed using pre-immune serum or no antibody (data not shown) demonstrated that this was due to non-specific binding of the FLAG-tagged protein to the agarose beads. However, the greatly enriched pull-down of FLAG-CP2 in the presence of ectopically expressed CRTR-1 indicates that the interaction between CRTR-1 and the other CP2 family members is genuine.

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