Molecular characterization of the CP2-related transcription factor, CRTR-1
A thesis submitted to The University of Adelaide for the degree of Doctor of Philosophy
Sarah To, B.Sc.(Biomed.Sc.)(Hons)
School of Molecular and Biomedical Sciences Discipline of Biochemistry The University of Adelaide Adelaide, South Australia
February 2009 CHAPTER 6 Analysis of CRTR-1 subcellular localization 6. ANALYSIS OF CRTR-1 SUBCELLULAR LOCALIZATION
6.1. Introduction
CP2 family members display a varied subcellular localization pattern that is cell type specific. Endogenous CP2 is detected in the nucleus in lens cells (Murata et al., 1998) and in the I.29 μ B cell line (Drouin et al., 2002). However, in differentiated rat B103 neuroblastoma cells, endogenous CP2 is detected predominantly in the cytoplasm
(Kashour et al., 2003). Endogenous CP2 is also detected in the nucleus in COS-7 cells
(Zambrano et al., 1998), but ectopically expressed CP2, as well as NF2d9, was shown to localize to the cytoplasm of COS-7, HepG2, and HeLa cells (Sato et al., 2005). Unlike other CP2 family members, ectopically expressed altNF2d9 localizes diffusely throughout the nucleus in HepG2 and HeLa cells (Sato et al., 2005), but when expressed in COS-7 cells, altNF2d9 localizes to nuclear speckles, some of which overlap with PML nuclear bodies (Sato et al., 2005). Moreover, altNF2d9 appears to regulate the localization of other family members, as co-expression with CP2 or NF2d9 resulted in nuclear accumulation of CP2 and NF2d9 in HepG2 and HeLa cells (Sato et al., 2005), and CP2 and NF2d9 localized to nuclear speckles when co-expressed with altNF2d9 in COS-7 cells (Sato et al., 2005).
Subcellular localization is a potential mechanism for regulating the cell-type specific activity of CP2 family proteins. This chapter examines the subcellular localization of
CRTR-1 in different cell types and the effect of CRTR-1 on the localization of other CP2 family members.
72 6.2. Results
6.2.1. Subcellular localization of CRTR-1
The subcellular localization of CRTR-1 was investigated by confocal microscopy using indirect immunofluorescence and an anti-CRTR-1 antibody. In ES cells, both endogenous and ectopically expressed CRTR-1 were localized diffusely throughout the nucleus, but excluded from nucleoli (Figure 6. 1). In contrast, CRTR-1 expressed ectopically in
HEK293T cells showed a predominantly cytoplasmic localization pattern (Figure 6. 2).
CRTR-1 ectopically expressed in COS-1 cells appeared to exhibit a mixed subcellular localization pattern (Figure 6. 3) with 27% of transfected cells showing cytoplasmic fluorescence, 46% showing nuclear fluorescence, and 27% showing both nuclear and cytoplasmic fluorescence (see Figure 6. 10 for cell counts). These localization patterns appeared to correlate with the level of CRTR-1 expression in a given cell. Those cells expressing high levels of CRTR-1 showed a predominantly cytoplasmic localization pattern as well as localization to distinct nuclear dot-like structures (Figure 6. 3). (Note:
The relative expression/fluorescence levels are not apparent in these figures due to compensation by the camera. The correlation was clear when observed down the microscope. However, no quantitative data on expression levels was obtained in these studies). Those cells expressing a low level of CRTR-1 exhibited a predominantly nuclear localization pattern while those expressing a moderate level of CRTR-1 showed equal distribution throughout both cellular compartments (Figure 6. 3). In all cases, CRTR-1 also localized to nuclear speckles, whether cells were expressing high or low levels of
CRTR-1 (Figure 6. 3). These speckles may correspond to PML bodies as has been observed previously with altNF2d9 (Sato et al., 2005). Unfortunately, this was unable to be confirmed as immunostaining with an anti-PML antibody was unsuccessful due to high background staining by the antibody. Overall, CRTR-1 is localized in the nucleus of ES cells, is predominantly cytoplasmic in HEK293T cells and has a varied distribution in COS-
1 cells that may be dependent on expression level.
73 CRTR-1 DAPI Merge
Endogenous
CRTR-1
Figure 6. 1. CRTR-1 localizes to the nucleus in ES cells.
Subcellular localization of endogenous CRTR-1 or transiently transfected CRTR-1 (2 )g
pEF.CRTR-1 expression plasmid) in ES cells was detected by indirect
immunofluorescence. Cells were fixed with 4% PFA 48 h post-transfection. Endogenous
and ectopically expressed CRTR-1 were detected with affinity purified anti-CRTR-1
antibody and Alexa-488-labeled goat anti-rabbit antibody (green). Nuclei are stained with
DAPI (blue) and the overlapping staining pattern is shown in the merged image. Images
were taken using confocal microscopy. Scale bar = 25 )m. CRTR-1 DAPI Merge
Figure 6. 2. Localization of CRTR-1 in HEK293T cells.
HEK293T cells were transiently transfected with pEF.CRTR-1 or pCAG.CRTR-1 expression plasmid (2 µg). Cells were fixed with 4% PFA 48 h post-transfection.
Ectopically expressed CRTR-1 was detected with affinity purified anti-CRTR-1 antibody and Alexa-488-labeled goat anti-rabbit antibody (green). Nuclei are stained with DAPI
(blue) and the overlapping staining pattern is shown in the merged image. Images were taken using confocal microscopy. Scale bar = 25 )m. CRTR-1 DAPI Merge
nuclear speckle
Figure 6. 3. Localization of CRTR-1 in COS-1 cells.
COS-1 cells were transiently transfected with pEF.CRTR-1 expression plasmid (2 µg).
Cells were fixed with 4% PFA 48 h post-transfection. Ectopically expressed CRTR-1 was detected with affinity purified anti-CRTR-1 antibody and Alexa-488-labeled goat anti-rabbit antibody (green). Nuclei are stained with DAPI (blue) and the overlapping staining pattern is shown in the merged image. Images were taken using confocal microscopy. Scale bar
= 25 )m. 6.2.2. Localization of CRTR-1 when co-expressed with CP2 family members
To determine the localization of CRTR-1 on coexpression with CP2 family members in ES cells, it was necessary to first characterize the subcellular localization of CP2 family members in ES cells. Constructs expressing FLAG-tagged CP2 family members were transiently transfected into ES cells and protein localization analysed by indirect immunofluorescence using an anti-FLAG antibody. Ectopically expressed FLAG-tagged
CP2 and NF2d9 were detected exclusively in the cytoplasm (Figure 6. 4 and Figure 6. 5 respectively), whereas FLAG-tagged altNF2d9 showed a diffuse nuclear staining pattern in ES cells (Figure 6. 6). To investigate whether CP2 family members, including CRTR-1, showed altered localization when coexpressed, ES cells were co-transfected with expression plasmids encoding CRTR-1 and FLAG-tagged CP2, NF2d9 or altNF2d9 and double-stained using anti-CRTR-1 and anti-FLAG antibodies. Both endogenous and exogenous CRTR-1 relocalized to the cytoplasm when coexpressed with CP2 in ES cells
(Figure 6. 4). Coexpression of CRTR-1 and NF2d9 in ES cells resulted in the redistribution of CRTR-1 throughout the cell, while NF2d9 maintained cytoplasmic localization (Figure 6. 5). In contrast, when CRTR-1 and altNF2d9 were co-expressed in
ES cells, both proteins maintained their nuclear localization (Figure 6. 6). It has previously been reported that altNF2d9 localized to nuclear speckles in COS-7 cells (Sato et al.,
2005). Therefore, the localization of CRTR-1 on coexpression with altNF2d9 was also investigated in COS-1 cells. All cells expressing high, moderate, or low levels of CRTR-1 showed accumulation in nuclear speckles on coexpression with altNF2d9 in COS-1 cells
(Figure 6. 7). Overall, these results demonstrate that the subcellular localization of CP2 family members can be altered by other family members.
74 CP2CRTR-1 DAPI Merge
CRTR-1
Endogenous
CP2
CRTR-1 + CP2
Figure 6. 4. CRTR-1 localizes to the cytoplasm when co-expressed with CP2 in ES
cells.
ES cells were transiently transfected with pEF.CRTR-1 and pEF.FLAG-CP2 expression
plasmids (2 µg of each). Cells were fixed with 4% PFA 48 h post-transfection.
Endogenous and ectopically expressed CRTR-1 were detected with affinity purified anti-
CRTR-1 antibody and Alexa-488-labeled goat anti-rabbit antibody (green). FLAG-tagged
CP2 was detected with anti-FLAG M2 antibody and Texas red-labeled donkey anti-mouse
antibody (red). Nuclei are stained with DAPI (blue) and the overlapping staining pattern is
shown in the merged image. Images were taken using confocal microscopy. Scale bar =
25 )m. CRTR-1 NF2d9 DAPI Merge
NF2d9
CRTR-1 + NF2d9
Figure 6. 5. CRTR-1 is distributed throughout the cell when co-expressed with
NF2d9 in ES cells.
ES cells were transiently transfected with pEF.CRTR-1 and pEF.FLAG-NF2d9 expression
plasmids (2 µg of each). Cells were fixed with 4% PFA 48 h post-transfection. CRTR-1
was detected with affinity purified anti-CRTR-1 antibody and Alexa-488-labeled goat anti-
rabbit antibody (green). FLAG-tagged NF2d9 was detected with anti-FLAG M2 antibody
and Texas red-labeled donkey anti-mouse antibody (red). Nuclei are stained with DAPI
(blue) and the overlapping staining pattern is shown in the merged image. Images were
taken using confocal microscopy. Scale bar = 25 )m. CRTR-1 altNF2d9 DAPI Merge
altNF2d9
CRTR-1 + altNF2d9
Figure 6. 6. CRTR-1 is localized in the nucleus when co-expressed with altNF2d9 in
ES cells.
ES cells were transiently transfected with pEF.CRTR-1 and pEF.FLAG-altNF2d9
expression plasmids (2 µg of each). Cells were fixed with 4% PFA 48 h post-transfection.
CRTR-1 was detected with affinity purified anti-CRTR-1 antibody and Alexa-488-labeled
goat anti-rabbit antibody (green). FLAG-tagged altNF2d9 was detected with anti-FLAG
M2 antibody and Texas red-labeled donkey anti-mouse antibody (red). Nuclei are stained
with DAPI (blue) and the overlapping staining pattern is shown in the merged image.
Images were taken using confocal microscopy. Scale bar = 25 )m. CRTR-1 altNF2d9 DAPI Merge
altNF2d9
CRTR-1 + altNF2d9
Figure 6. 7. CRTR-1 localizes to the nucleus when co-expressed with altNF2d9 in
COS-1 cells.
COS-1 cells were transiently transfected with pEF.CRTR-1 and pEF.FLAG-altNF2d9
expression plasmids (1 µg of each). Cells were fixed with 4% PFA 48 h post-transfection.
CRTR-1 was detected with affinity purified anti-CRTR-1 antibody and Alexa-488-labeled
goat anti-rabbit antibody (green). FLAG-tagged altNF2d9 was detected with anti-FLAG
M2 antibody and Texas red-labeled donkey anti-mouse antibody (red). Nuclei are stained
with DAPI (blue) and the overlapping staining pattern is shown in the merged image.
Images were taken using confocal microscopy. Scale bar = 25 )m. 6.2.3. Nucleocytoplasmic shuttling of CRTR-1 and other CP2 family proteins
The subcellular localization pattern of CRTR-1 appears to be cell-type specific and its localization is altered on coexpression with CP2 family members. However, CRTR-1 and other CP2 family members are able to activate transcription regardless of their localization pattern. Therefore, the possibility that these proteins shuttle between the nucleus and cytoplasm was investigated by treating cells with Leptomycin B (LMB), an inhibitor of chromosome region maintenance 1 (CRM1)-dependent nuclear export (Nishi et al., 1994).
In the absence of LMB, CP2 localized in the cytoplasm in ES cells (Figure 6. 4). Addition of LMB resulted in nuclear accumulation of CP2 (Figure 6. 8), suggesting that CP2 undergoes LMB-sensitive nuclear export. Treatment of ES cells coexpressing CRTR-1 and CP2 with LMB resulted in the accumulation of both CRTR-1 and CP2 in the nucleus, suggesting that both proteins shuttle between the nucleus and cytoplasm (Figure 6. 8).
Similarly, NF2d9 localized in the cytoplasm in ES cells in the absence of LMB (Figure 6. 5) and accumulated in the nucleus on treatment with LMB (Figure 6. 9). Treatment of ES cells coexpressing CRTR-1 and NF2d9 with LMB resulted in the accumulation of both
CRTR-1 and NF2d9 in the nucleus (Figure 6. 9). These results demonstrate that CP2,
NF2d9, and CRTR-1 shuttle between the nucleus and cytoplasm via a CRM1-dependent pathway.
The mixed subcellular localization pattern of CRTR-1 in COS-1 cells suggested that
CRTR-1 may also shuttle between the nucleus and cytoplasm in this cell type. Treatment with LMB resulted in the nuclear accumulation of CRTR-1 in nearly 75% of cells compared with 46% in untreated cells (Figure 6. 10), suggesting that CRTR-1 shuttles between the nucleus and cytoplasm via a CRM1-dependent pathway in COS-1 cells.
75 Figure 6. 8. CP2 shuttles between the nucleus and cytoplasm via a CRM1- dependent pathway.
ES cells, transiently transfected with pEF.CRTR-1 and pEF.FLAG-CP2 expression plasmids (2 µg of each), were treated with leptomycin B (LMB) at 10 ng/ml for 3 h. Cells were fixed with 4% PFA 48 h post-transfection. Endogenous and ectopically expressed
CRTR-1 were detected with affinity purified anti-CRTR-1 antibody and Alexa-488-labeled goat anti-rabbit antibody (green). FLAG-tagged CP2 was detected with anti-FLAG M2 antibody and Texas red-labeled donkey anti-mouse antibody (red). Nuclei are stained with DAPI (blue) and the overlapping staining pattern is shown in the merged image.
Images were taken using confocal microscopy. Scale bar = 25 )m. CRTR-1 CP2 DAPI Merge Endogenous
-LMB
CP2 Endogenous
+ LMB
-LMB
CRTR-1 + CP2
+ LMB Figure 6. 9. NF2d9 shuttles between the nucleus and cytoplasm via a CRM1- dependent pathway.
ES cells, transiently transfected with pEF.CRTR-1 and pEF.FLAG-NF2d9 expression plasmids (2 µg of each), were treated with LMB at 10 ng/ml for 3 h. Cells were fixed with
4% PFA 48 h post-transfection. Endogenous and ectopically expressed CRTR-1 were detected with affinity purified anti-CRTR-1 antibody and Alexa-488-labeled goat anti-rabbit antibody (green). FLAG-tagged NF2d9 was detected with anti-FLAG M2 antibody and
Texas red-labeled donkey anti-mouse antibody (red). Nuclei are stained with DAPI (blue) and the overlapping staining pattern is shown in the merged image. Images were taken using confocal microscopy. Scale bar = 25 )m. CRTR-1 NF2d9 DAPI Merge Endogenous
-LMB
NF2d9 Endogenous
+ LMB
-LMB
CRTR-1 + NF2d9
+ LMB 100
90
80
70
60
- LMB 50 + LMB
40
Percentage of cells (%) 30
20
10
0 CNC + N
Figure 6. 10. CRTR-1 shuttles between the nucleus and cytoplasm via a CRM1- dependent pathway.
COS-1 cells, transiently transfected with pEF.CRTR-1 expression plasmid (2 µg), were treated with leptomycin B at 10 ng/ml for 4 h. Transfected cells were counted and the localization of CRTR-1 was classified as cytoplasmic (C), nuclear (N), or both cytoplasmic and nuclear (C+N). The data presented are the mean ± SEM of two independent experiments [n=116 (-LMB), n=140 (+LMB) and n=101 (-LMB), n=121 (+LMB) respectively]. 6.3. Discussion
6.3.1. Cell-type specific localization pattern of CRTR-1
Differences in the subcellular localization of CRTR-1 in ES, HEK293T, and COS-1 cells may be responsible for the different levels of CRTR-1 mediated activation in these cells.
This chapter shows the cell-type specific subcellular localization pattern of CRTR-1, as has been observed previously for CP2 (see section 1.6.5). Whilst CRTR-1 exhibits a nuclear localization pattern in ES cells, it is predominantly cytoplasmic in HEK293T cells and shows a mixed localization pattern in COS-1 cells that appeared to correlate with the level of CRTR-1 expression in a given cell. The observed correlation between expression level and localization could be verified by fluorescent intensity quantitation. Endogenous
CRTR-1 protein localization was not able to be examined in HEK293T and COS-1 cells and overexpression of proteins can result in artefactual cytoplasmic localization due to saturation of the nuclear import pathway (Cazeneuve et al., 2004; Kremmidiotis et al.,
1999). However, the fact that CRTR-1 activates transcription, most likely in a DNA- binding dependent manner, regardless of its apparent localization pattern, suggested that it may shuttle between the nucleus and cytoplasm (see section 6.3.3).
6.3.2. CRTR-1 localizes to nuclear speckles in COS-1 cells
Nuclear speckle localization has been observed previously for altNF2d9 in COS-1 cells, most of which was shown to overlap with PML nuclear bodies (Sato et al., 2005). The fact that coexpression with altNF2d9 resulted in an increase in accumulation of CRTR-1 in nuclear speckles raises the possibility that CRTR-1 may also colocalize to PML bodies.
However, the present study was unable to confirm the colocalization of CRTR-1 to PML bodies due to the difficulty in the detection of endogenous PML using the anti-PML (A-20) antibody. Although this antibody was generated against an epitope of human PML, it has been reported to cross-react with mouse and rabbit PML and has also been successful in
76 the detection of endogenous PML in COS-1 cells (Kindle et al., 2005). It is unclear why these experiments failed, but high background staining was observed throughout the nucleus. Therefore, an alternative approach to confirm the localization of CRTR-1 to PML bodies could be to perform immunofluorescence studies on COS-1 cells transiently co- transfected with vectors expressing CRTR-1 and PML to increase staining above background. Furthermore, co-immunoprecipitation experiments could be performed to determine if there is a physical interaction between CRTR-1 and PML.
Although the biological function of these nuclear structures has not yet been elucidated, several models propose a role for PML nuclear bodies in transcriptional regulation. One model proposes that these nuclear bodies act as depots for the storage and titration of transcription factors (Zhong et al., 2000). Alternatively, it has been proposed that PML bodies sequester transcription factors to be post-translationally modified or that they act as compartmentalization centres in which cofactor complexes are assembled to be utilized by transcription factors (Bernardi and Pandolfi, 2007; Borden, 2002). Since CRTR-1 does not activate transcription in COS-1 cells to the same extent as in ES and HEK293T cells
(see chapter 4), it is possible that its activity is regulated by recruitment into PML bodies.
6.3.3. CP2 family members undergo nucleocytoplasmic shuttling
Unlike CRTR-1 and altNF2d9, CP2 and NF2d9 localized in the cytoplasm of ES cells.
The re-localisation of CRTR-1 to the cytoplasm on co-expression with CP2 or NF2d9 suggests that CP2 and NF2d9 may mediate the nuclear export or cytoplasmic retention of
CRTR-1. Treatment with LMB showed that CRTR-1 and other family members shuttle between the nucleus and cytoplasm via a CRM1-dependent nuclear export pathway, providing a possible mechanism for regulating CP2 family proteins. In the absence of
LMB, it is likely that the net steady state cytoplasmic localization of CP2 and NF2d9 observed is a result of the rate of nuclear export exceeding the rate of import.
77 Interestingly, however, CP2 and NF2d9 were sensitive to LMB in ES cells but were apparently not responsive to LMB in COS-7 cells (Sato et al., 2005). This may suggest a cell-type specific nucleocytoplasmic shuttling activity.
CRM1 is a nuclear export receptor that actively transports proteins containing a nuclear export sequence (NES) into the cytoplasm (Fukuda et al., 1997). The most common NES are characterized by small, hydrophobic, leucine rich regions with the consensus sequence Lx(2-3)Lx(2-3)LxL (Bogerd et al., 1996), although some variations exist. Scanning of the amino acid sequences of the CP2 family proteins identified a putative leucine rich
NES-like motif between amino acids 432 and 440 of CP2 and between amino acids 406 and 414 of CRTR-1 (Figure 6. 11). AltNF2d9 does not appear to contain an NES motif but contains a 36 amino acid insert that functions as a nuclear localization sequence (NLS)
(Sato et al., 2005). In contrast, NF2d9 appears to lack both an NLS and an NES motif.
Therefore, LMB may be indirectly affecting the localization of NF2d9 through an NES- containing NF2d9-interacting partner protein. Similarly, the affect of LMB on CRTR-1 in the presence of ectopically expressed CP2 or NF2d9 may be indirectly mediated through these proteins. Therefore, mutational inactivation of the putative NES within CRTR-1 and other CP2 family members will be necessary to confirm the presence of a functional NES and the direct effect of LMB on the localization of these proteins.
In summary, this chapter has presented data showing cell-type specific localization patterns of CRTR-1 protein and the ability of CP2 family members to regulate the subcellular localization of other family members. This is also the first demonstration that
CRTR-1 and other family members undergo nucleocytoplasmic shuttling.
78 CRTR-1 L E E L T T L E L aa 406-414
CP2 L E E L T A V E L aa 432-440
NES consensus L X(2-3) L X(2-3) L X L
Figure 6. 11. Putative NES in the CRTR-1 and CP2 amino acid sequence.
Numbers indicate the positions of the amino acids (aa) within the protein. Conserved hydrophobic residues are shown in boldface. In the NES consensus sequence, X is any amino acid, and L (leucine) can be substituted by other large hydrophobic residues. CHAPTER 7 Investigating the sumoylation state of CRTR-1 7. INVESTIGATING THE SUMOYLATION STATE OF CRTR-1
7.1. Introduction
Sumoylation has been shown to regulate the function of many transcription factors by affecting their transcriptional activity, altering their subcellular localization, controlling their stability, or modulating their DNA-binding activity (for review see Melchior, 2000).
Previously, a yeast-2-hybrid screen, using amino acids 47-478 of mutant CRTR-1 as bait and a cDNA library prepared from adult mouse testis found that CRTR-1 interacted with multiple components of the sumoylation machinery; the E2 SUMO conjugating enzyme
Ubc9 and the E3 SUMO ligase PIAS1 (Rodda, 2003). These sumoylation enzymes, as well as SUMO-1, have also been proposed to be CP2-interacting proteins (Kang et al.,
2005a), suggesting that CP2 family members may be a target for SUMO modification.
This chapter investigates the possibility that CRTR-1 is regulated by sumoylation. Two potential sumoylation sequences are identified in CRTR-1 and their sumoylation status determined. The effect of SUMO modification on CRTR-1 transcriptional activity and subcellular localization is examined and the effect of Ubc9 and PIAS1 on CRTR-1 activity is also investigated.
7.2. Results
7.2.1. CRTR-1 is sumoylated
To determine whether CRTR-1 undergoes sumoylation, COS-1 cells were co-transfected with expression vectors encoding CRTR-1 and FLAG-tagged SUMO-1. FLAG-SUMO-1- conjugated proteins were immunoprecipitated with anti-FLAG affinity beads and the immunocomplexes were analysed for the presence of CRTR-1 by immunoblotting with
79 anti-CRTR-1 antibody. CRTR-1 runs as two doublets at 54 kD and 60 kD (Figure 4.11 A).
However, it is detected as a doublet at ~80 kD in the anti-FLAG immunoprecipitate (Figure
7. 1). This is consistent with sumoylated CRTR-1 as SUMO modification increases the apparent molecular weight of a target protein by ~20 kD (Johnson, 2004). A similar ~80 kD band was also detected when CRTR-1 was immunoprecipitated with the anti-CRTR-1 antibody and immunoblotted with anti-FLAG antibody, demonstrating that CRTR-1 can be sumoylated (Figure 7. 1). The ~80 kD form of CRTR-1 was not detected when cell lysates were prepared in the absence of iodoacetamide, a SUMO isopeptidase inhibitor, and N- ethylmaleimide (NEM), a potent inhibitor of deubiquitinating enzymes which also inhibits
SUMO-1 hydrolase activity (Suzuki et al., 1999) (data not shown), demonstrating that
SUMO-1 conjugation to proteins is readily hydrolysed by endogenous isopeptidases, consistent with other studies (Shao et al., 2004; Spengler et al., 2002). These results demonstrate that CRTR-1 is a target for SUMO-1 modification.
7.2.2. Mapping of the SUMO-1 modification sites in CRTR-1
SUMO conjugation is known to occur on the lysine residue within the consensus sequence ψKXE, where ψ represents a hydrophobic amino acid and X represents any amino acid (Rodriguez et al., 2001). Examination of the CRTR-1 amino acid sequence revealed the presence of two lysine residues that fall within a consensus sumoylation motif: FK30QE and LK464AE. To determine which lysine residues were subject to sumoylation, constructs containing lysine to alanine substitutions at positions 30, 464 or at both lysines (K30A, K464A, and 2KA, respectively) were generated and the ability of these CRTR-1 mutants to be sumoylated was analysed. COS-1 cells were co-transfected with expression plasmids encoding FLAG-SUMO-1 and CRTR-1 mutants K30A, K464A, or 2KA. CRTR-1 sumoylation site mutants were immunoprecipitated with the anti-CRTR-1 antibody and FLAG-SUMO-1 conjugated CRTR-1 mutants were then detected by immunoblotting with the anti-FLAG antibody. The ~80 kD sumoylated form was detected
80 anti-FLAG IP anti-CRTR-1 IP
CRTR-1 --++ --++ + FLAG-SUMO-1 - - + + - - + + + kD 180
115
82 Sumoylated CRTR-1
64 Non-sumoylated CRTR-1 IgG
anti-CRTR-1 WB anti-FLAG WB anti-CRTR-1 WB
Figure 7. 1. CRTR-1 is post-translationally modified by SUMO-1 in COS-1 cells
COS-1 cells, seeded at 5 x 105 cells per 10 cm dish, were co-transfected with 3 )g pEF.CRTR-1 and 3 )g pEF.FLAG-SUMO-1 expression plasmids. Total DNA transfected was made up to 6 )g with pEF-IRES-puro6 empty vector. 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. Bands were detected using ECF substrate. for the K464A mutation but was no longer detectable for the K30A mutation (Figure 7. 2
A). Similarly, mutation of both lysine residues resulted in loss of the ~80 kD band (Figure
7. 2 A). A reciprocal experiment, in which FLAG-SUMO-1 conjugated proteins were immunoprecipitated with the anti-FLAG antibody and CRTR-1 proteins were detected by immunoblotting with the anti-CRTR-1 antibody (Figure 7. 2 B), confirmed these results.
Re-probing the anti-CRTR-1 or anti-FLAG immunoprecipitates with anti-CRTR-1 or anti-
FLAG antibodies, respectively, demonstrated that CRTR-1 and FLAG-SUMO-1 had been successfully immunoprecipitated (Figure 7. 2 C and D). These data indicate that lysine 30 is the major sumoylation site of CRTR-1.
7.2.3. Direct and indirect effects of SUMO-1 on CRTR-1 transactivation
SUMO modification often regulates transcription factor function by modulating transcriptional activity (for review see Lyst and Stancheva (2007)). To determine the effect of SUMO-1 expression on CRTR-1 activity, COS-1 and ES cells were co- transfected with expression vectors encoding CRTR-1 and increasing amounts of FLAG- tagged SUMO-1, together with the reporter plasmid pTK-4xWT-CP2-LUC.
Overexpression of SUMO-1 resulted in an enhancement of CRTR-1-mediated transactivation of up to 2.5- and 4.4-fold in COS-1 and ES cells respectively (Figure 7. 3 A and B). To determine whether SUMO-1 directly regulates CRTR-1 activity, ES and COS-
1 cells were co-transfected with expression vectors encoding the CRTR-1 sumoylation site mutants and the reporter plasmid pTK-4xWT-CP2-LUC. Mutation of lysine 464 had little effect on CRTR-1 transcriptional activity, but mutation of lysine 30 significantly increased CRTR-1 transactivation by up to 6-fold in both ES and COS-1 cells (Figure 7.
4), suggesting that SUMO-1 conjugation to CRTR-1 blocks maximal CRTR-1 activity.
Mutation of both lysine 464 and lysine 30 in CRTR-1 resulted in slightly higher activity than the single mutation at lysine 30 in both ES and COS-1 cells (Figure 7. 4), suggesting
81 AB
anti-CRTR-1 IP anti-FLAG IP
- WT K30A K464A 2KA - WT K30A K464A 2KA
FLAG-SUMO-1 - - +++++------+++++ kD 180 115
82 Sumoylated CRTR-1
64 49
37
26
19 anti-FLAG WB anti-CRTR-1 WB
CD
Sumoylated CRTR-1 Non-sumoylated CRTR-1 IgG IgG
IgG Free SUMO-1 anti-CRTR-1 WB anti-FLAG WB
Figure 7. 2. SUMO-1 is conjugated to lysine 30 of CRTR-1.
COS-1 cells, seeded at 5 x 105 cells per 10 cm dish, were co-transfected with pEF.CRTR-
1, pEF.K30A, pEF.K464A or pEF.2KA together with pEF.FLAG-SUMO-1 expression plasmids (3 )g of each). Total DNA transfected was made up to 6 )g with pEF-IRES- puro6 empty vector. Whole cell lysates were immunoprecipitated with (A) anti-CRTR-1 or
(B) anti-FLAG antibody and analysed by immunoblotting with anti-FLAG or anti-CRTR-1 antibody respectively to detect sumoylated proteins (bracket). The membrane from (A) and (B) were re-probed with (C) anti-CRTR-1 or (D) anti-FLAG antibody respectively.
Bands were detected using ECF substrate. Figure 7. 3. SUMO-1 enhances CRTR-1 transcriptional activity in COS-1 and ES cells.
COS-1 (A) or ES (B) cells were co-transfected with the indicated amounts of pEF.CRTR-1 and pEF.FLAG-SUMO-1 expression plasmids together with 200 ng pTK-4xWT-CP2-LUC.
Firefly luciferase activity was normalised against renilla activity from the co-transfected pRL-SV40 plasmid (5 ng). Total DNA transfected was made up to 605 ng with pEF-IRES- puro6 empty vector. Luciferase activity was measured 40 h post-transfection. The data presented are a representative experiment of three independent experiments, each of which was conducted in triplicate. Error bars represent ±SEM calculated for each triplicate. A
0.006 COS-1 cells
0.005
0.004
0.003
0.002
Relative Luciferase Activity 0.001
0
CRTR-1 (ng) 0 50 100 150 200 0 000 200200200200
SUMO-1 (ng) 0 000050 100 150 200 50 100 150 200
B
0.25 ES cells
0.2
0.15
0.1
Relative Luciferase Activity 0.05
0 CRTR-1 (ng) 0 50 100 150 200 0 000 200200200200
SUMO-1 (ng) 0 000050 100 150 200 50 100 150 200 Figure 7. 4. Mutation of lysine 30 in CRTR-1 enhances CRTR-1 transcriptional activity in COS-1 and ES cells.
ES (A) or COS-1 (B) cells were transfected with the indicated amounts of pEF.CRTR-1, pEF.K30A, pEF.K464A or pEF.2KA expression plasmids together with 200 ng pTK-4xWT-
CP2-LUC. Firefly luciferase activity was normalised against renilla activity from the co- transfected pRL-SV40 plasmid (5 ng). Total DNA transfected was made up to 605 ng with pEF-IRES-puro6 empty vector. Luciferase activity was measured 40 h post-transfection.
The data presented are a representative experiment of three independent experiments, each of which was conducted in triplicate. Error bars represent ±SEM calculated for each triplicate. A 0.6 ES cells
0.5
0.4
0.3
0.2 Relative Luciferase Activity 0.1
0
CRTR-1 (ng) 0 50 100 150 200 000000000000
K30A (ng) 0000050 100 150 200 00000000
K464A (ng) 00000000050 100 150 200 000 0
2KA (ng) 000000000 000 050 100 150 200
B 0.014 COS-1 cells
0.012
0.010
0.008
0.006
0.004 Relative Luciferase Activity
0.002
0.000
CRTR-1 (ng) 0 50 200 000000
K30A (ng) 00050 200 0000
K464A (ng) 0000050 200 00
2KA (ng) 00000 0 050 200 that lysine 464 may also be a target for sumoylation but requires sumoylation at lysine 30 first.
The possibility that SUMO-1 overexpression enhances CRTR-1 transactivation indirectly, via sumoylation of other proteins that interact with CRTR-1, was examined by co- transfecting ES cells with expression vectors encoding the CRTR-1 sumoylation site mutants and increasing amounts of FLAG-tagged SUMO-1 together with the reporter plasmid pTK-4xWT-CP2-LUC. Transactivation by K30A, K464A, and 2KA was enhanced up to 2-, 3.7, and 1.8-fold respectively compared to the 4.2-fold enhancement with wild type CRTR-1 on overexpression of SUMO-1 (Figure 7. 5). These results suggest that
SUMO-1 overexpression enhances CRTR-1 transactivation both directly through lysine 30 of CRTR-1 as well as indirectly through endogenous CRTR-1-interacting proteins.
7.2.4. Effect of CRTR-1 sumoylation on regulation of the activities of CP2 family
members
To determine whether sumoylation of CRTR-1 affects its ability to regulate the activities of other CP2 family members, COS-1 and ES cells were co-transfected with increasing amounts of CRTR-1 sumoylation site mutants and a fixed amount of FLAG-tagged CP2,
NF2d9, or altNF2d9 (200 ng), together with the reporter plasmid pTK-4xWT-CP2-LUC. In
COS-1 cells, CP2-, NF2d9-, and altNF2d9-mediated activation was enhanced up to 1.6-,
3-, and 4.4-fold respectively with 50 ng K30A (Figure 7. 6 A). However, the difference in these levels compared to 50 ng wild type CRTR-1 was not statistically significant (1.9-,
2.2-, and 3.3-fold respectively) (Figure 7. 6 A). K464A and 2KA also enhanced CP2- mediated activation at 50 ng at levels that were similar to 50 ng wild type CRTR-1 (Figure
7. 6 A). However, at 200 ng of 2KA and K30A, enhanced CP2- and NF2d9-mediated activation was observed at levels greater than that of 200 ng wild type CRTR-1 (P =
0.0058 and P = 0.0234 respectively). A statistically significant difference in the level of
82 Figure 7. 5. Effect of SUMO-1 on the transcriptional activity of CRTR-1 sumoylation site mutants in ES cells.
ES cells were co-transfected with the indicated amounts of (A) pEF.CRTR-1 or pEF.K30A or (B) pEF.K464A or pEF.2KA expression plasmids together with pEF.FLAG-SUMO-1 and
200 ng pTK-4xWT-CP2-LUC. Firefly luciferase activity was normalised against renilla activity from the co-transfected pRL-SV40 plasmid (5 ng). Total DNA transfected was made up to 605 ng with pEF-IRES-puro6 empty vector. Luciferase activity was measured
40 h post-transfection. The data presented are a representative experiment of three independent experiments, each of which was conducted in triplicate. Error bars represent
±SEM calculated for each triplicate. A 0.6 ES cells 0.5
0.4
0.3
0.2
Relative Luciferase Activity Luciferase Relative 0.1
0
K30A (ng) 0 200 200 200 200 200 0 0 0 0 000
CRTR-1 (ng) 0 000 0 0 200 200 200 0 000
SUMO-1 (ng)0 0 50 100 150 200 0 50 100 50 100 150 200
B 0.6 ES cells 0.5
0.4
0.3
0.2 Relative Luciferase Activity Luciferase Relative 0.1
0
K464A (ng) 0 200 200 200 200 200 0 0 0 000 000
2KA (ng) 0 000 0 0 200 200 200 200 200 0 000
SUMO-1 (ng)0 0 50 100 150 200 0 50 100 150 200 50 100 150 200 Figure 7. 6. Transcriptional regulation of CP2 family members by CRTR-1 sumoylation site mutants in COS-1 and ES cells.
COS-1 (A) and ES (B and C) cells were co-transfected with the indicated amounts of pEF.CRTR-1, pEF.K30A, pEF.K464A or pEF.2KA expression plasmids together with pEF.FLAG-CP2, pEF.FLAG-NF2d9 or pEF.FLAG-altNF2d9 and 200 ng pTK-4xWT-CP2-
LUC. Firefly luciferase activity was normalised against renilla activity from the co- transfected pRL-SV40 plasmid (5 ng). Total DNA transfected was made up to 605 ng with pEF-IRES-puro6 empty vector. Luciferase activity was measured 40 h post-transfection.
The data presented are the mean ± SEM of three independent experiments, each of which was conducted in triplicate. Statistical significance was determined by one-tailed paired t-test using GraphPad Prism (version 5.01). * denotes statistical significance with
P<0.05, ** denotes statistical significance with P<0.01, and ns denotes no significance. COS-1 cells ns A ns 0.021 ns ns
P=0.0234 *ns 0.018 ns ns P=0.0498 P=0.0058 * 0.015 **
P=0.0061 0.012 **
0.009 Relative Luciferase Activity Luciferase Relative 0.006
0.003
0
CRTR-1 (ng) 00000000000 0 000 0 0 0 0 0 20050 20050 20050 20050
K30A (ng) 0 50 200 0 50 2000 50 200 0 50 200 00 0 00000 00 000000
K464A (ng) 00000000 000050200 50 200 00 00 0000 00 0 0
2KA (ng) 0000000000000000 50 200 20050 00000 00 0
CP2 (ng) 0 0 0 200 200 200 0 0 0 0 0 000200 200 0 0 200 200 00 200 200 0000
NF2d9 (ng) 0000 0 0 200 200 2000 0 000000 0 00 0000200 200 0 0
altNF2d9 (ng) 000000000 200 200 200 000 000 00000000 200 200 B C 1.8 1.8 ES cells ES cells 1.6 1.6
1.4 1.4
1.2 1.2
1.0 1.0
0.8 0.8
0.6 0.6 Relative Luciferase Activity Luciferase Relative Relative Luciferase Activity Luciferase Relative
0.4 0.4
0.2 0.2
0.0 0.0
K30A (ng) 0 50 200 0 50 2000 50 200 0 50 200 K464A (ng) 0 0 20050 20050 00 00
CP2 (ng) 0 0 0 200 200 200 0 0 0000 2KA (ng) 0 0 000 0 50 200 20050
NF2d9 (ng)0000 0 0 200 200 2000 0 0 CP2 (ng) 0 200 00200 200 0 0 200 200 altNF2d9 (ng)0000000 0 0 200 200 200 CP2-mediated activation was also observed with 200 ng K464A (1.2-fold) compared to
200 ng wild type CRTR-1 (1.7-fold) (P = 0.0498) (Figure 7. 6 A). These results show that sumoylation of CRTR-1 can affect its ability to regulate the activities of CP2 family members in COS-1 cells. In general, at high concentrations, the CRTR-1 sumoylation site mutants appear to differ from that of wildtype CRTR-1 in their effect on the activities of
CP2 family members.
In ES cells, K30A, K464A, and 2KA (50 ng) enhanced CP2-mediated activation 6.5-, 8-, and 10-fold respectively (Figure 7. 6 B and C). These fold changes are consistent with the 10-fold enhancement with 50 ng wild type CRTR-1 (Figure 4.7 A). However, a 4-, 2-, and 5-fold enhancement of CP2-mediated activation was observed with 200 ng K30A,
K464A, and 2KA respectively compared to the 1.5-fold enhancement with 200 ng wild type CRTR-1 (Figure 4. 7 A). NF2d9-mediated activation was also enhanced 10- and 3.5- fold with 50 ng and 200 ng K30A respectively (Figure 7. 6 B), compared to the 3- and 1- fold enhancement with 50 ng and 200 ng wild type CRTR-1 respectively (Figure 4. 8 A).
AltNF2d9-mediated activation was suppressed 1.3-fold with 50 ng K30A, a similar level to that attained with 50 ng wild type CRTR-1 (1.7-fold). However, 200 ng K30A suppressed altNF2d9-mediated 2-fold compared to the 4.7-fold suppression with 200 ng wild type
CRTR-1 (Figure 4. 9 A). Similar to the effect observed in COS-1 cells, these results show that at high concentrations, the CRTR-1 sumoylation site mutants differ from that of wild type CRTR-1 in their ability to modulate the activities of CP2 family complexes in ES cells.
7.2.5. Sumoylation does not alter CRTR-1 subcellular localization
Sumoylation has been shown to alter the localization of several proteins including ATF7,
HIPK2, Pdx1, and Mdm2 (Comerford et al., 2003; Hamard et al., 2007; Kim et al., 1999;
Kishi et al., 2003; Miyauchi et al., 2002). To determine the effect of SUMO-1 overexpression on CRTR-1 subcellular localization, ES and COS-1 cells were co-
83 transfected with expression plasmids encoding CRTR-1 and FLAG-tagged SUMO-1. The localization of CRTR-1 and FLAG-SUMO-1 proteins was examined by indirect immunofluorescence using anti-CRTR-1 and anti-FLAG antibodies respectively. SUMO-1 protein was detected diffusely throughout the nucleus in ES cells but displayed a predominantly cytoplasmic localization pattern in COS-1 cells (Figure 7. 7). CRTR-1 protein remained localized in the nucleus of ES cells and in both the cytoplasm and nuclear speckles in COS-1 cells when coexpressed with SUMO-1 (Figure 7. 7), demonstrating that overexpression of SUMO-1 did not affect CRTR-1 localization. The failure to detect FLAG-SUMO-1 in the nuclear speckles in COS-1 cells also suggests that sumoylated CRTR-1 does not accumulate in these speckles.
The localization of the CRTR-1 sumoylation site mutants was examined. ES cells were transiently transfected with expression plasmids encoding the CRTR-1 sumoylation site mutants, K30A, K464A, or 2KA and indirect immunofluorescence was performed using the anti-CRTR-1 antibody. K30A, K464A, and 2KA were detected exclusively in the nucleus in ES cells, a localization pattern similar to that of wild type CRTR-1 (Figure 7. 8 A). In
COS-1 cells, these mutants were detected predominantly in the cytoplasm as well as in distinct nuclear dot-like structures (Figure 7. 8 B). This appears similar to the localization pattern of wild type CRTR-1 in COS-1 cells. However, cell counts are required to determine whether the cytoplasmic and nuclear distribution pattern is altered.
7.2.6. Ubc9 and PIAS1 enhance CRTR-1 sumoylation
Ubc9 and PIAS1 were previously identified as interacting partners of the mutant form of
CRTR-1 (Rodda, 2003). Therefore, to confirm this interaction using wild type CRTR-1, co- immunoprecipitation experiments were performed in COS-1 cells co-transfected with wild type CRTR-1 and FLAG-tagged Ubc9 and PIAS1. Unfortunately, standard co-
84 Figure 7. 7. SUMO-1 does not alter the subcellular localization of CRTR-1 in COS-1 or ES cells.
COS-1 (A) and ES (B) cells were transiently transfected with pEF.CRTR-1 and pEF.FLAG-SUMO-1 expression plasmids (1 µg or 2 µg of each into COS-1 or ES cells respectively). Cells were fixed with 4% PFA 48 h post-transfection. CRTR-1 proteins were detected using anti-CRTR-1 antibody and Alexa-488-labeled goat anti-rabbit antibody (green). FLAG-tagged SUMO-1 proteins were detected using anti-FLAG M2 antibody and Texas red-labeled donkey anti-mouse antibody (red). Overlapping of the staining patterns from both signals can be seen in yellow in the merged image. Nuclei are stained with DAPI (blue). Images were taken using confocal microscopy. Scale bar = 25
)m. A. COS-1 cells
CRTR-1 SUMO-1 DAPI Merge
SUMO-1
CRTR-1 + SUMO-1
B. ES cells
CRTR-1 SUMO-1 DAPI Merge
SUMO-1
CRTR-1 + SUMO-1 Figure 7. 8. Mutation of the CRTR-1 sumoylation sites does not alter CRTR-1 subcellular localization in COS-1 or ES cells.
ES (A) or COS-1 (B) cells were transiently transfected with pEF.CRTR-1 (WT), pEF.K30A, pEF.K464A or pEF.2KA expression plasmids (1 µg or 2 µg into COS-1 or ES cells respectively). Cells were fixed with 4% PFA 48 h post-transfection. CRTR-1 proteins were detected using anti-CRTR-1 antibody and Alexa-488-labeled goat anti-rabbit antibody (green). Overlapping of the staining patterns from both signals can be seen in yellow in the merged image. Nuclei are stained with DAPI (blue). Images were taken using confocal microscopy. Scale bar = 25 )m. A. ES cells CRTR-1 DAPI Merge
WT
K30A
K464A
2KA B. COS-1 cells
CRTR-1 DAPI Merge
WT
K30A
K464A
2KA immunoprecipitation conditions (2.3.4) failed to confirm an interaction between wild type
CRTR-1 and FLAG-tagged Ubc9 or PIAS1 (Figure 7. 9).
To determine whether Ubc9 and PIAS1 were capable of enhancing SUMO-1 ligation to
CRTR-1, COS-1 cells were co-transfected with expression plasmids encoding CRTR-1,
FLAG-SUMO-1 , and FLAG-Ubc9 or FLAG-PIAS1. CRTR-1 proteins were immunoprecipitated with the anti-CRTR-1 antibody and FLAG-SUMO-1 conjugated
CRTR-1 was detected by immunoblotting the samples with anti-FLAG antibody. A greater amount of FLAG-SUMO-1-conjugated CRTR-1 was detected on coexpression with PIAS1 compared to Ubc9 (Figure 7. 10 A). Immunoblotting of the lysates with the anti-CRTR-1 antibody suggested that Ubc9 and PIAS1 enhanced SUMO conjugation to CRTR-1
(Figure 7. 10 A). This effect is more clearly shown in a repeat experiment (Figure 7. 10
B), supporting a role for Ubc9 and PIAS1 in enhancing CRTR-1 sumoylation.
7.2.7. Ubc9 and PIAS1 overexpression enhances CRTR-1-mediated activation
To determine the effect of Ubc9 and PIAS1 on CRTR-1 transcriptional activity, COS-1 cells were transiently co-transfected with an expression vector encoding CRTR-1 and increasing amounts of vector expressing FLAG-tagged Ubc9 or PIAS1, together with the reporter plasmid pTK-4xWT-CP2-LUC. Although luciferase activity was increased by up to 3.8-fold with 200 ng Ubc9 alone in COS-1 cells (Figure 7. 11 A), Ubc9 enhanced
CRTR-1-mediated activation up to 10-fold in a dose-dependent manner (Figure 7. 11 A).
Preliminary results also show enhancement of CRTR-1-mediated activation by PIAS1 in a dose-dependent manner (up to 2.7-fold) in COS-1 cells (Figure 7. 11 B). Therefore, these studies demonstrate that the activity of CRTR-1 is affected by overexpression of Ubc9 or
PIAS1 in COS-1 cells.
85 AB anti-FLAG IP anti-CRTR-1 IP CRTR-1 - - - ++ + - - - ++ + FLAG-Ubc9 - - + - + - - - + - + - FLAG-PIAS1 - --+ - + - --+ - + kD 115
82
NS 64
NS 49
37
26
19
anti-CRTR-1 WB anti-FLAG WB
CD115
82 PIAS1 64 IgG CRTR-1 49 IgG
37
26 IgG
Ubc9 19 anti-FLAG WB anti-CRTR-1 WB
Figure 7. 9. Interaction between wild type CRTR-1 and Ubc9 or PIAS1.
CRTR-1 was expressed in COS-1 cells in combination with FLAG-tagged Ubc9 or PIAS1.
Whole cell lysates were immunoprecipitated with anti-FLAG (A) or anti-CRTR-1 (B) antibody and analysed by immunoblotting with anti-FLAG or anti-CRTR-1 antibody respectively. The membrane from (A) and (B) were re-probed with anti-FLAG (C) or anti-
CRTR-1 antibody (D) respectively. Bands were detected using ECF substrate. A
anti-CRTR-1 IP anti-FLAG IP lysates
CRTR-1 - + + + + - - +++ + - - +++ + - FLAG-SUMO-1 - - + + + + - - + + + + - - + + + + FLAG-Ubc9 - - - + - + - --+ - + - - - + - + FLAG-PIAS1 - - - - + + - ---+ + - - --+ + kD 180 180
115 115 82 82 Sumoylated CRTR-1 64 Non-sumoylated 64 CRTR-1
anti-FLAG WB anti-CRTR-1 WB anti-CRTR-1 WB
B lysates CRTR-1 - +++ + - - - FLAG-SUMO-1 - - + + + + + + FLAG-Ubc9 - - - + - - + - FLAG-PIAS1 - ---+ - - +
kD
115
82 Sumoylated CRTR-1 64 Non-sumoylated CRTR-1
anti-CRTR-1 WB
Figure 7. 10. Ubc9 and PIAS1 enhance CRTR-1 sumoylation in COS-1 cells.
COS-1 cells, seeded at 3 x 105 cells per 10 cm dish, were co-transfected with pEF.CRTR-
1, pEF.FLAG-SUMO-1, and pEF.FLAG-Ubc9 or pEF.FLAG-PIAS1 expression plasmids
(1.5 )g of each). Total DNA transfected was made up to 6 )g with pEF-IRES-puro6
empty vector. 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). Lysate lanes represent 8% of the
input for co-immunoprecipitation. Bands were detected using ECF substrate. Figure 7. 11. Effect of Ubc9 and PIAS1 on CRTR-1 transcriptional activity in COS-1 cells.
COS-1 cells were co-transfected with the indicated amounts of pEF.CRTR-1 and (A) pXMT2.6xHis-Ubc9-FLAG or (B) pXMT2.6xHis-PIAS1-FLAG expression plasmids together with 200 ng pTK-4xWT-CP2-LUC. Firefly luciferase activity was normalised against renilla activity from the co-transfected pRL-SV40 plasmid (5 ng). Total DNA transfected was made up to 605 ng with the indicated amounts of pXMT2 and pEF-IRES- puro6 empty vector. Luciferase activity was measured 40 h post-transfection. The data presented are a representative experiment of three (A) and two (B) independent experiments, each of which was conducted in triplicate. Error bars represent ±SEM calculated for each triplicate. A 0.025 COS-1 cells
0.02
0.015
0.01
Relative Luciferase Activity Luciferase Relative 0.005
0
CRTR-1 (ng) 0 50 100 200 0 000 200200200200
Ubc9 (ng) 0 00050 100 150 200 50 100 150 200
pXMT2 (ng) 0 200 200 200 150 100 50 00150 100 50 pEF-IRES-puro6 (ng)0 150 100 0 200200200200 0 000
B 0.035 COS-1 cells 0.03
0.025
0.02
0.015
0.01
Relative Luciferase Activity Luciferase Relative 0.005
0
CRTR-1 (ng) 0 2000 000 200200200200
PIAS1 (ng) 00 50 100 150 200 50 100 150 200
pXMT2 (ng) 0 200 150 100 50 00150 100 50
pEF-IRES-puro6 (ng) 0 0 200200200200 0 00 0 To determine whether Ubc9 enhances CRTR-1 transactivation via sumoylation of CRTR-
1, COS-1 cells were co-transfected with an expression vector encoding the CRTR-1 sumoylation site mutant K30A and increasing amounts of FLAG-tagged Ubc9, together with the reporter plasmid pTK-4xWT-CP2-LUC. Enhancement of transactivation by the
K30A CRTR-1 mutant was observed on co-expression with Ubc9 (up to 4.5-fold) (Figure
7. 12). This suggests that Ubc9 may be functioning as a CRTR-1 coactivator in addition to its role as an E2 SUMO conjugating enzyme. Alternatively, Ubc9 overexpression may result in the sumoylation of other proteins that interact with CRTR-1 leading to the observed increase in activity.
7.3. Discussion
7.3.1. Sumoylation of CRTR-1
Data presented in this chapter have identified sumoylation as a potential mechanism for regulating CRTR-1 activity. Immunoprecipitation experiments demonstrated that CRTR-1 is a target for SUMO-1 conjugation and mapped the major site of SUMO-1 conjugation to lysine 30 of CRTR-1. To confirm that CRTR-1 is a physiological SUMO substrate, sumoylation of endogenous CRTR-1 needs to be demonstrated. As less than 1% of endogenous substrates have been found to be sumoylated (Johnson, 2004), it is likely to be difficult to detect sumoylated CRTR-1 in ES cells. Transfection of ES cells with FLAG-
SUMO-1 expression vector and immunoprecipitation of endogenous CRTR-1 with the anti-CRTR-1 antibody may facilitate detection of endogenous sumoylated CRTR-1.
In addition to the predominant ~80 kD sumoylated form of CRTR-1, several higher molecular weight bands of lower intensity were also detected in the anti-CRTR-1 immunoprecipitate when immunoblotted with anti-FLAG antibody (Figure 7. 1 and Figure
86 0.1 COS-1 cells
0.08
0.06
0.04
Relative Luciferase Activity Luciferase Relative 0.02
0
CRTR-1 (ng) 0 200 0 0 0 0 0 0 0 0 0 0
K30A (ng) 0 0 50 100 150 200 0 0 200 200 200 200
Ubc9 (ng) 0 0000 0 50 200 50 100 150 200
pXMT2 (ng) 0 0 200 200 200 200 150 00150 100 50
pEF-IRES-puro6 (ng)0 0 150 100 50 0 200200 0 000
Figure 7. 12. Ubc9 enhances K30A transcriptional activity in COS-1 cells.
COS-1 cells were co-transfected with the indicated amounts of pEF.K30A and pXMT2.6xHis-Ubc9-FLAG expression plasmids together with 200 ng pTK-4xWT-CP2-
LUC. Firefly luciferase activity was normalised against renilla activity from the co- transfected pRL-SV40 plasmid (5 ng). Total DNA transfected was made up to 605 ng with the indicated amounts of pXMT2 and pEF-IRES-puro6 empty vector. Luciferase activity was measured 40 h post-transfection. The data presented are a representative experiment of three independent experiments, each of which was conducted in triplicate.
Error bars represent ±SEM calculated for each triplicate. 7. 10). Although the SUMO-1 protein lacks a consensus sumoylation sequence, poly-
SUMO-1 chains have been reported to form on RanBP2 and human topoisomerase I
(Pichler et al., 2002; Yang et al., 2006). Therefore, these higher molecular weight bands may represent poly-SUMO-1 conjugated forms of CRTR-1. Alternatively, they may represent CRTR-1 protein that is monosumoylated at multiple sites. As sumoylation at non-consensus sites has been demonstrated for PML and Smad4 (Kamitani et al., 1998;
Long et al., 2004), it is possible that in addition to lysine 464, SUMO-1 may conjugate to sites within CRTR-1 that do not conform to the consensus motif ψKXE. The fact that these higher molecular weight bands were not detected on mutation of lysine 30 suggests that SUMO-1 conjugation to these sites may occur sequentially beginning with lysine 30.
Similar findings have been reported for the progesterone receptor B (PRB) and the nuclear factor of activated T cells (NFAT1) proteins (Man et al., 2006; Terui et al., 2004).
7.3.2. Regulation of CRTR-1 activity by SUMO modification
Sumoylation has been shown to regulate the activity of many transcription factors. CRTR-
1 sumoylation site mutants K30A and 2KA elicited significantly greater luciferase expression than wild type CRTR-1 in COS-1 cells, suggesting that SUMO modification of
CRTR-1 blocks its maximal activation capacity. The fact that K30A CRTR-1 activity in
COS-1 cells was significantly greater than wild type CRTR-1, which had little activity in this cell type, suggests that the limited activity may be due to higher level sumoylation in
COS-1 cells compared to HEK293T or ES cells. This could be examined by analysing the transcriptional activity of wild type CRTR-1 in COS-1 cells on overexpression of a SUMO isopeptidase which cleaves the isopeptide bond between SUMO and its target (reviewed in Melchior et al., 2003). Although SUMO-1 conjugation to CRTR-1 in ES cells has not been demonstrated, CRTR-1 sumoylation site mutants K30A and 2KA also exhibited increased transactivation capacity in ES cells, suggesting sumoylation of CRTR-1 in this cell line.
87 Mutation of both lysine 464 and lysine 30 in K2A CRTR-1 resulted in slightly higher activity than the single mutation at lysine 30 in both ES and COS-1 cells, suggesting that lysine
464 may be sumoylated but requires SUMO conjugation to lysine 30 first. CP2-mediated activation was also affected differently with 200 ng K464A CRTR-1 compared to 200 ng wild type CRTR-1, further suggesting that lysine 464 may be sumoylated. Although several studies have identified SUMO conjugation sites by substituting the critical lysine residue with alanine (Chang et al., 2004; Park et al., 2007; Perdomo et al., 2005), arginine substitutions have also been used to conserve the positively charged residue (Lee et al.,
2003b; Wasylyk et al., 2005; Wei et al., 2007). As such, the activities of CRTR-1 sumoylation site mutants with lysine to arginine substitutions should also be analysed to confirm that substitution with an uncharged residue does not interfere with the binding of specific interacting partners or other properties.
Overexpression of SUMO-1 in ES and COS-1 cells resulted in enhancement of CRTR-1- mediated transactivation. This was unexpected given that sumoylation is generally associated with transcriptional repression and mutation of the sumoylation sites results in increased activity. This effect may be an artifact of overexpression where corepressors are be sequestered by endogenous SUMO-modified proteins, thereby reducing the number of free corepressors available for recruitment by SUMO-modified CRTR-1. This has been described as a possible model by which SUMO-1 overexpression enhances glucocorticoid receptor (GR) activity (Gill, 2003): mutation of a sumoylation site within GR resulted in enhancement of GR activity (Tian et al., 2002) and overexpression of SUMO-1 also enhanced GR activity (Le Drean et al., 2002). Alternatively, since the other SUMO family members, SUMO-2 and SUMO-3, have been shown to exert effects distinct from that of SUMO-1 (Ayaydin and Dasso, 2004; Holmstrom et al., 2003), it cannot be ruled out that enhancement of CRTR-1 transactivation on mutation of lysine 30, and on
88 overexpression of SUMO-1, may be due to the inhibition of SUMO-2 or SUMO-3 conjugation.
Repression of transcription factor function by SUMO modification has been observed for a number of transcription factors. There are several possible mechanisms by which SUMO represses the activity of target proteins (reviewed in Gill, 2003, and Verger et al., 2003).
SUMO-1 fused to the DNA binding domain of GAL4 has been shown to repress transcription from a GAL4-responsive promoter (Ross et al., 2002; Yang et al., 2003).
Therefore, SUMO-1 conjugation may be directly repressing the transactivating properties of CRTR-1, as has been reported for Elk-1 (Yang et al., 2003), possibly through recruitment of corepressors such as HDACs (Yang and Sharrocks, 2004). The critical
SUMO acceptor site, lysine 30, is located within the sumoylation consensus sequence,
FKQE, which is conserved in mouse CP2, NF2d9 and altNF2d9 proteins and lies within their activation domains. This suggests that transactivation by other CP2 family members may also be regulated by SUMO modification. Ubc9 and SUMO-1 have also been reported to be putative CP2-interacting proteins from a yeast-2-hybrid screen (Kang et al.,
2005a). However, sumoylation of these proteins has not been demonstrated. CRTR-1 contains a putative phosphorylation-dependent sumoylation motif (PDSM) (reviewed in
(Anckar and Sistonen, 2007) adjacent to the K30 residue which is absent in the other proteins. Whether the presence or absence of this motif influences sumoylation of CRTR-
1 may provide insight into both the role of CRTR-1 sumoylation and its regulation.
Sumoylation and ubiquitination can occur on the same lysine residue. Therefore, competition by SUMO for ubiquitination sites in a protein can protect them from degradation, leading to increased protein stability, which has been observed on SUMO modification of a number of proteins including IκBα, Mdm2, CREB and Smad4
(Buschmann et al., 2000; Comerford et al., 2003; Desterro et al., 1998; Lin et al., 2003).
The CRTR-1 sumoylation defective mutants appeared to be expressed at relatively similar
89 levels to that of wild type CRTR-1 (Figure 7.2 C), suggesting that SUMO modification of
CRTR-1 may not affect its stability. This could be confirmed by comparing the protein turnover rate of wild type CRTR-1 with that of the sumoylation defective mutant following treatment of cells overexpressing these proteins with cycloheximide to prevent new protein synthesis. The half-life of the proteins can be assessed by harvesting these cells at various time points and measuring the level of CRTR-1 protein by western blot analysis.
As only a small percentage of CRTR-1 is sumoylated in the cell at any one time, there are likely to be detection sensitivity problems. Therefore, cotransfection of CRTR-1 and
FLAG-SUMO-1 constructs may be required. Cells can also be treated with the proteasome inhibitor MG132 to determine whether there is an association between suppression of CRTR-1 transactivation by sumoylation and protein degradation.
Sumoylation can alter the localization of several target proteins (Hamard et al., 2007; Lin et al., 2003; Ross et al., 2002; Sachdev et al., 2001; Salinas et al., 2004). It has been shown that a number of proteins localize to PML bodies on sumoylation leading to inhibition of transactivation (Dahle et al., 2003; Ross et al., 2002; Sachdev et al., 2001;
Schmidt and Muller, 2002). However, no obvious change in the localization of CRTR-1 was observed on overexpression of SUMO-1 and SUMO-1 was not detected in nuclear speckles in COS-1 cells, suggesting that sumoylated CRTR-1 does not accumulate in these speckles. Obvious changes in localization between wild type CRTR-1 and sumoylation defective mutants were not observed, suggesting that the mechanism by which SUMO-1 regulates CRTR-1 activity is not likely to be due to altered protein localization. However, since only a small fraction of CRTR-1 is sumoylated, the possibility that sumoylation can alter CRTR-1 localization cannot be excluded.
Inhibition of transcription factor transactivation by sumoylation can also be mediated through decreased DNA binding (Anckar et al., 2006; Baba et al., 2005; Tsuruzoe et al.,
2006). To determine whether SUMO-1 conjugation to CRTR-1 affects its DNA binding
90 activity, EMSA experiments can be performed using in vitro modified CRTR-1 protein.
Recombinant proteins have been shown to be efficiently SUMO modified in E. coli when coexpressed with SUMO-1 and the sumoylation enzymes Aos1/Uba2 (E1) and Ubc9 (E2)
(Tsuruzoe et al., 2006; Uchimura et al., 2004).
SUMO has also been shown to mediate transcriptional repression of Sp100, Daax, and
Lef1 by modulating their interaction with other proteins (Jang et al., 2002; Sachdev et al.,
2001; Sternsdorf et al., 1999). At high concentrations, the CRTR-1 sumoylation site mutants appear to differ from that of wildtype CRTR-1 in their ability to modulate the activities of CP2 family members. A possible explanation could be that CP2 family proteins form a more stable heteromeric complex with CRTR-1 sumoylation site mutants than with wild type CRTR-1. Sumoylation may therefore be suppressing the transactivating function of CRTR-1 by promoting its ability to form homomeric complexes.
Co-immunoprecipitation experiments are required to confirm the interaction between the sumoylation defective CRTR-1 mutant and CP2 family proteins.
7.3.3. Enhancement of CRTR-1 sumoylation by Ubc9 and PIAS1
Although a previous study identified Ubc9 and PIAS1 as CRTR-1-interacting partners
(Rodda, 2003), a mutant CRTR-1 expression construct was used. Standard co- immunoprecipitation conditions used in this thesis failed to confirm the interaction with wild type CRTR-1. The interaction between Ubc9 or PIAS1 with mutant CRTR-1 was previously confirmed by affinity co-purification using histidine-tagged Ubc9 or PIAS1
(Rodda, 2003). Therefore, it is possible that an interaction between these proteins and wild type CRTR-1 could be detected using this method. Alternatively, it is possible that the failure to demonstrate an interaction by co-immunoprecipitation may be due to the transient nature of this interaction.
91 Ubc9 and PIAS1 have been shown to enhance sumoylation of several proteins including
Sp3, Net, Ad4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1), and EKLF (Komatsu et al., 2004; Sapetschnig et al., 2002; Siatecka et al., 2007; Wasylyk et al., 2005). The increase in the intensity of the ~80 kD sumoylated form of CRTR-1 on coexpression with
Ubc9 or PIAS1 together with SUMO-1 suggests that Ubc9 and PIAS1 can act as SUMO-1
E2 conjugating enzyme and SUMO-1 E3 ligase, respectively, for CRTR-1 (Figure 7. 9).
Knocking down endogenous Ubc9 or PIAS1 with small interference RNA (siRNA) can be performed to confirm that these enzymes mediate SUMO conjugation to CRTR-1.
7.3.4. Enhancement of CRTR-1 activity by Ubc9 and PIAS1
Co-expression of CRTR-1 with Ubc9 or PIAS1 resulted in a marked increase in CRTR-1 transcriptional activity. Coexpression of the sumoylation defective K30A CRTR-1 mutant with Ubc9 also resulted in increased transactivation, suggesting that Ubc9 may be functioning as a CRTR-1 coactivator in addition to its role as a SUMO conjugating enzyme
(PIAS1 coexpression with K30A has not been tested). Ubc9 has been shown to mediate the sumoylation of the mineralcorticoid receptor (MR) as well as functioning as an MR coactivator using the sumoylation defective mutant Ubc9 (C93S) (Yokota et al., 2007).
PIAS1 has also been shown to function as a coactivor independent of sumoylation using the sumoylation defective mutant PIAS1 (C351S) (Lee et al., 2003a). The ability of these sumoylation defective mutants Ubc9 (C93S) or PIAS1 (C351S) to enhance CRTR-1 activity would confirm that Ubc9 and PIAS1 enhance CRTR-1 activity in a sumoylation- independent manner.
Ubc9 and PIAS1 could also enhance CRTR-1 activity indirectly. They could act, for example, to increase SUMO conjugation to endogenous CRTR-1-interacting coactivators whose activation capabilities are enhanced on SUMO conjugation. SRC-1 and GRIP1 are examples of coactivators whose activities are enhanced by sumoylation (Chauchereau et
92 al., 2003; Kotaja et al., 2002). It is also possible that CP2 family proteins are sumoylated and this modification may enhance their transactivating capacity.
93 CHAPTER 8 Final discussion and conclusions 8. FINAL DISCUSSION AND CONCLUSIONS
8.1. Introduction
The transcription factor CRTR-1 is expressed in the pluripotent ICM of the early mammalian blastocyst, ES cells, the developing kidney and salivary glands (Pelton et al.,
2002; Rodda et al., 2001; Yamaguchi et al., 2005; Yamaguchi et al., 2006). Mice lacking
CRTR-1 appeared to have no defect in the early embryo , suggesting that it doesn’t have a vital role in the ICM. However, recent studies have shown a connection between
CRTR-1 (Tcfcp2l1) and the complex transcription factor network responsible for the maintenance of pluripotency in mouse ES cells. The pluripotency-associated factors
Oct4, Nanog and Jmjd1a have been shown to bind and regulate CRTR-1 expression (Loh et al., 2006; Loh et al., 2007). CRTR-1 has also been shown to bind to the regulatory regions of the Oct4 and Nanog genes, as well as other critical components of this network, including the Sox2 and Klf4 genes (Chen et al., 2008). This suggests a putative role for CRTR-1 in the expression of genes required for pluripotency. This thesis reports the characterization of the CRTR-1 protein at the molecular level in order to better understand the function of CRTR-1 in ES cells. The transcriptional activity, DNA binding properties, sumoylation, and subcellular localization of CRTR-1 were investigated, as well as its ability to regulate the activity of other CP2 family members.
8.2. Mechanisms regulating CRTR-1 activity
Data presented in this thesis demonstrate for the first time that CRTR-1 is able to act as both a transcriptional activator and suppressor, depending on the cell type and the complement of other CP2 family members present within the cell. It is possible that the context of the promoter may also play a role in determining CRTR-1 activity, but this has not been investigated here. Although the transactivation domain of CP2, NF2d9, and
94 altNF2d9 have been mapped to the extreme amino terminus (Kang et al., 2005a), GAL4- deletion assays were unable to detect any region of CRTR-1 with transactivation properties but mapped a repression domain to residues 48-200. The fact that CRTR-1 repressed transcription when linked to a heterologous DNA binding domain suggests that
CRTR-1 may function as a classic repressor by recruiting corepressors to the promoter.
Since CP2 has been shown to recruit the corepressors YY1, HDAC1 and 2, RING1, and
Sin3A to mediate transcriptional repression (Coull et al., 2000; Drouin et al., 2002;
Romerio et al., 1997; Tuckfield et al., 2002), these corepressors may also be recruited to promoters by CRTR-1.
CRTR-1 was shown to enhance CP2- and NF2d9-mediated activation in ES, HEK293T, and COS-1 cells. Coimmunoprecipitation experiments demonstrated that CRTR-1 forms heteromeric complexes with these proteins and EMSA showed that these complexes can bind DNA. Since CP2 and NF2d9 are also expressed in ES and HEK293T cells (Kang et al., 2005a), CRTR-1 activity may be mediated through the formation of heteromeric complexes with endogenous CP2 family members. In COS-1 cells, transactivation by
CRTR-1 was rarely greater than two-fold (Figure 4. 4 B), suggesting that COS-1 cells may lack or express low levels of endogenous CP2-like proteins compared to ES and
HEK293T cells. When CRTR-1 was coexpressed with CP2 and NF2d9 in COS-1 cells, enhancement of CP2- and NF2d9-mediated activation was observed (Figure 4. 10). The activity of the CRTR-1 sumoylation defective mutant, K30A, was up to 6-fold greater than wild type CRTR-1 in COS-1 cells (Figure 7. 4 B), suggesting that the limited activity may also be due to higher level sumoylation in COS-1 cells compared to HEK293T or ES cells.
Enhancement of CP2- and NF2d9-mediated activation by CRTR-1 may be as a result of the formation of a more favourable conformation for either increased DNA binding or for the recruitment of coactivators. Maximal enhancement of CP2- and NF2d9-mediated activation was observed with lower amounts of CRTR-1 and decreased with increasing
95 amounts of CRTR-1. CP2 family proteins bind DNA as a tetramer (Sueyoshi et al., 1995;
Yoon et al., 1994). Coexpression of CRTR-1 and CP2 or NF2d9 could result in the formation of heterotetramers containing different numbers of CRTR-1 and CP2 family member subunits: 3:1, 2:2, and 1:3 ratios of the two proteins respectively. Thus, at higher amounts of CRTR-1, heterotetramers containing a 1:3 ratio of activating CP2 subunits to repressive CRTR-1 subunits would be expected to form, resulting in suppression of CP2- or NF2d9-mediated activation. Alternatively, at high CRTR-1 concentrations, CRTR-1 may be sequestering factors required for transcriptional activation at a non-specific site away from the promoter.
CRTR-1 suppressed altNF2d9-mediated activation in ES and HEK293T cells, consistent with the ability of LBP-9 to suppress LBP-1b-mediated activation in human placental JEG-
3 cells (Huang and Miller, 2000). AltNF2d9 alone may already be in its most favourable
DNA binding conformation, possibly due to the extra 36 amino acid insert. Therefore, when altNF2d9 complexes with CRTR-1, the repressive CRTR-1 subunits would be suppressing altNF2d9-mediated activation. In contrast to the phenomenon in ES and
HEK293T cells, CRTR-1 enhanced altNF2d9-mediated activation in COS-1 cells.
Coimmunoprecipitation experiments demonstrated that CRTR-1 forms heteromeric complexes with altNF2d9 and EMSA showed that this complex can bind DNA. Given that
CP2 enhances altNF2d9-mediated activation (Kang et al., 2005a) and the fact that potent transactivation by altNF2d9 was not observed in COS-1 cells (Figure 4.10), CRTR-1 may be compensating for the low level of endogenous CP2 in this cell line. Alternatively, differences in the subcellular localization pattern of altNF2d9 in COS-1, ES and HEK293T cells may also affect its regulation by CRTR-1. Both CRTR-1 and altNF2d9 colocalized in the nucleoplasm when coexpressed in ES cells. However, both accumulated in nuclear speckles when coexpressed in COS-1 cells. The colocalization of these proteins to nuclear speckles may be responsible for the increased activation either due to post- translational modification or interaction with cofactors. In addition to the possibility that
96 these nuclear structures correspond to PML bodies, they could also be other nuclear speckles such as polycomb bodies to which proteins such as CTCF have been shown to localize to be sumoylated (MacPherson et al., 2009).
8.3. Regulation of CRTR-1 transactivation by sumoylation
The E3 sumo ligase PIAS1 has previously been shown to interact with CP2 and affect its transcriptional activity (Kang et al., 2005a). However, this is the first demonstration that
CP2 family members can be sumoylated and that this modification appears to affect activity. Sumoylation of CRTR-1 was shown to block its maximal transactivation capacity as mutation of the critical SUMO acceptor site (lysine 30) resulted in enhancement of
CRTR-1-mediated transactivation in ES and COS-1 cells, consistent with the common repressive role for sumoylation of a number of transcription factors (reviewed in Lyst and
Stancheva, 2007). Ubc9 and PIAS1 enhanced SUMO-1 conjugation to CRTR-1 but, interestingly, it also enhanced CRTR-1-mediated activation, including that of the CRTR-1 sumoylation defective mutant, suggesting that they also act as coactivators.
There are several possible mechanisms by which SUMO-1 conjugation could suppress the transactivating function of CRTR-1. Alterations in the localization of CRTR-1 on
SUMO conjugation is unlikely to be the major mechanism as changes in the localization of
CRTR-1 on SUMO-1 overexpression was not observed. Furthermore, no obvious changes in localization between wild type CRTR-1 and sumoylation defective mutants were observed. However, as only a small fraction of CRTR-1 is sumoylated in vivo, the possibility that sumoylation can alter CRTR-1 localization cannot be excluded.
Sumoylation could also alter the conformation of CRTR-1, thereby modifying its ability to interact with coregulators and/or its DNA binding affinity. The CRTR-1 sumoylation defective mutants did not appear to affect the activities of other CP2 family members to
97 the same extent as wild type CRTR-1, suggesting that modulation of the activities of CP2 family members by CRTR-1 is regulated by sumoylation to some degree. Since only a small proportion of CRTR-1 is sumoylated in vivo, the interaction of sumoylated CRTR-1 with CP2 family proteins and the DNA binding properties of this complex may be examined by coimmunoprecipitation and EMSA respectively using in vitro modified CRTR-
1 proteins. Larger amounts of SUMO-modified proteins have been efficiently generated in
E. coli when coexpressed with SUMO-1 and the sumoylation enzymes Aos1/Uba2 (E1) and Ubc9 (E2) compared to the in vivo sumoylation system (Tsuruzoe et al., 2006;
Uchimura et al., 2004).
Given that fusion of SUMO-1 to the DNA-binding domain of GAL4 is sufficient to repress transcription (Ross et al., 2002; Yang et al., 2003), SUMO-1 may be directly suppressing the transactivation by CRTR-1, possibly through recruitment of corepressors such as
HDACs (Yang and Sharrocks, 2004). Although only a small proportion of CRTR-1 is sumoylated, CRTR-1 significantly activated transcription when the sumoylation site was mutated. Two possible mechanisms have been proposed to explain this phenomenon
(Geiss-Friedlander and Melchior, 2007). HDACS could generate a repressive chromatin state that remains even after sumoylation of CRTR-1 is lost. Alternatively, sumoylation of
CRTR-1 could result in the formation of a repressive complex that remain stable following desumoylation of CRTR-1.
8.4. Nucleocytoplasmic shuttling and regulation of CRTR-1 activity
CRTR-1 subcellular localisation appears to be cell type specific, with an exclusively nuclear localisation pattern in ES cells, a predominantly cytoplasmic localisation pattern in
HEK293T cells, and a cytoplasmic and nuclear speckle localisation pattern in COS-1 cells.
LMB studies revealed for the first time that CRTR-1 and other family members shuttle between the nucleus and cytoplasm via a CRM1-dependent pathway. Therefore, the
98 cytoplasmic localization of CRTR-1 in HEK293T and COS-1 cells may be a result of nucleocytoplasmic shuttling kinetics where the rate of export exceeds the rate of import.
Colocalisation studies showed that CRTR-1 re-localizes to the cytoplasm when coexpressed with CP2 or NF2d9. CRM1-dependent shuttling proteins generally contain both an NLS and an NES. However, CP2 and CRTR-1 both lack a classical NLS but appear to contain a putative leucine rich NES-like motif between amino acids 432 and 440 and amino acids 406 and 414 respectively. Disruption of this sequence within CRTR-1 and CP2 by site-directed mutagenesis will be necessary to confirm the presence of a functional NES and the direct effect of LMB on the localization of these proteins. The absence of an NLS within CRTR-1 suggests that its nucleoplasmic and nuclear speckle localization in ES and COS-1 cells respectively may be mediated through an endogenous
NLS-containing protein. Such a protein may be altNF2d9 which has been shown to contain an extra 36 amino acid insert that functions as an NLS and localizes to nuclear speckles corresponding PML bodies in COS-7 cells (Sato et al., 2005). Given that CP2 and NF2d9 localize to nuclear speckles when co-expressed with altNF2d9 in COS-7 cells
(Sato et al., 2005), it is possible that the nuclear import machinery regulated by altNF2d9 may become saturated when CRTR-1 is coexpressed with CP2. As a result, CRTR-1 would not be transported into the nucleus but would be retained in the cytoplasm.
Transcription factor activity can be regulated by nucleocytoplasmic shuttling (Fischer et al., 1995; Mowen and David, 2000; Neufeld et al., 2000; Rodriguez and Henderson, 2000;
Stommel et al., 1999; Watanabe et al., 2000). The ability of CRTR-1 to shuttle between the nucleus and cytoplasm may be a potential mechanism regulating its transcriptional activity with exclusion from the nucleus acting as an inhibitor of its function. Disruption of the putative NES within CRTR-1 by site-directed mutagenesis and analysis of its activity by luciferase reporter assays could determine whether export from the nucleus inhibits its transactivating capacity.
99 Post-translational modifications can affect the nucleocytoplasmic activity of transcription factors. Data presented in this thesis were unable to show alterations in CRTR-1 subcellular localization by SUMO modification. However, other post-translational modification events such as phosphorylation have been shown to alter the balance between nuclear export and import (Dominguez et al., 2003; Okamura et al., 2000). The presence of two potential phosphorylation sites within CRTR-1 (residues S146 and S272) and the detection of two CRTR-1-specific doublets suggest that CRTR-1 may be a potential target for phosphorylation. Treatment of CRTR-1 immunoprecipitates with phosphatase would confirm whether CRTR-1 is phosphorylated and analysis of the subcellular localization of CRTR-1 on mutation of the phosphorylation site would confirm the affect of phosphorylation on CRTR-1 localization.
8.5. Model of CRTR-1 function
Based on the data presented in this thesis, the following model describing the potential mechanism of action of CRTR-1 in ES cells is proposed (Figure 8. 1). CP2 and NF2d9 appear to be able to regulate the cytoplasmic localization of CRTR-1, and the ability of these proteins to shuttle between the nucleus and cytoplasm adds another possible level of control. CRTR-1 and CP2 family proteins could be forming heterotetramers containing
3:1, 2:2, or 1:3 ratios of CRTR-1 to CP2 family proteins respectively. CRTR-1 could be enhancing CP2- and NF2d9-mediated activation by forming heterotetramers with a more favourable conformation for either increased DNA binding or for the recruitment of coactivators. At higher amounts of CRTR-1, heterotetramers would contain more repressive CRTR-1 subunits resulting in decreased enhancement of CP2- and NF2d9- mediated activation. AltNF2d9 may already be in its most favourable DNA binding conformation. Therefore, CRTR-1 could be suppressing altNF2d9-mediated activation by forming a heteromeric complex with reduced DNA binding affinity or the repressive CRTR-
100 Figure 8. 1. Model of CRTR-1 function.
(1) CRTR-1 and CP2 family proteins could form heterotetramers containing different numbers of CRTR-1 and CP2 family member subunits. A complex containing two family members could have 3:1 (A), 2:2 (B), or 1:3 (C) ratios of the two proteins respectively. (2)
The activity of these complexes could be regulated by shuttling between the nucleus and cytoplasm via a CRM1-dependent pathway. (3) Enhancement of CP2- or NF2d9- mediated activation by CRTR-1 may be mediated through the formation of heteromeric complexes with a more favourable conformation for increased DNA binding or for recruitment of coactivators. At higher amounts of CRTR-1, heterotetramers would contain more repressive CRTR-1 subunits which could result in the recruitment of corepressors leading to decreased enhancement of CP2- and NF2d9-mediated activation. (4)
AltNF2d9 is a more potent activator than other family members. In ES cells, addition of
CRTR-1 results in suppression of activity. This could be due to: (i) the formation of heteromeric complexes with reduced DNA binding affinity; (ii) the recruitment of corepressors by the repression domains of CRTR-1; or (iii) the additional 36 amino acid transactivation domain of altNF2d9 could also be blocked on heteromer formation with
CRTR-1. However, this explanation does not hold true for the phenomenon observed in
COS-1 cells where CRTR-1 enhanced altNF2d9-mediated activation. Here, CRTR-1 may be compensating for the low level of endogenous CP2 (or other factors) in this cell line.
(5) The limited activation potential of CRTR-1 in certain cell lines could be due to a lack of required cofactors, which could include CP2 family members, or the ability of CRTR-1 to be SUMO-modified by Ubc9 and PIAS1. Sumoylation could suppress CRTR-1-mediated activation by recruiting corepressors. (6) In addition to the possible enhancement of
SUMO conjugation to CRTR-1 by Ubc9 and PIAS1, these enzymes could also regulate the transactivating function of CRTR-1 by acting as coactivators for CRTR-1. R R A A R R A A 1
ABC
A R A R A A R R A R A R
2 CRM1 cytoplasm nucleus Ubc9 CoA CoA Ubc9 Ubc9 * S PIAS1 A R CoA A R PIAS1 A R A R S S A R 6 A R 3 A R CoR A R CoR 5 PIAS1 CoR 4
6 3 5 A R CoR A R
4
CP2 RE CP2 RE
NOTE:
A R A R A A = OR OR * R R A R A R
LEGEND
CoA Coactivator R CRTR-1 CoR Corepressor
S SUMO-1 CP2 / NF2d9 / altNF2d9 A R Repression domain
A Activation domain 1 subunits could be recruiting corepressors. The additional 36 amino acid transactivation domain of altNF2d9 may also be blocked on heteromer formation with CRTR-1.
The transactivating function of CRTR-1 can be suppressed by sumoylation. SUMO modification may directly suppress CRTR-1-mediated transactivation by recruiting corepressors. Alternatively, sumoylation of CRTR-1 could result in conformational changes that modify its ability to interact with coregulators and/or its DNA binding affinity.
Ubc9 and PIAS1 catalyse the conjugation of SUMO to CRTR-1 but they may also enhance CRTR-1-mediated transactivation by functioning as coactivators.
8.6. Future directions
Little is known about the structure of CRTR-1. Recent analysis of the DNA binding domain of CP2 using structure prediction programs has revealed the presence of an immunoglobulin-like fold homologous to the core domain of the cell cycle regulator p53
(Kokoszynska et al., 2008). The high degree of sequence homology between CRTR-1 and CP2 suggests that the DNA binding domain of CRTR-1 may also form a similar structure. Elucidation of the structure of CRTR-1 will provide insight into the mechanism by which CRTR-1 regulates gene expression. In particular, it will reveal how CRTR-1 recognizes specific DNA sequences, interacts with other CP2 family members and unrelated proteins, as well as the mechanisms by which post-translational modifications regulate its activity.
Proteins that interact with CRTR-1 can also provide insight into the function of CRTR-1, particularly the mechanism regulating CRTR-1 activity. CRTR-1-interacting proteins could be identified using the tandem affinity purification (TAP) method (Puig et al., 2001), which has the advantage of enabling rapid purification of native protein complexes. This system would involve the stable expression of TAP-tagged CRTR-1 protein and the purification of
101 the unknown proteins from the complex mixtures for identification by mass spectrometry.
Identification of proteins that interact with CRTR-1 would provide insight into the mechanisms by which CRTR-1 activates or represses transcription and the regulatory pathways that control CRTR-1 activity. The affect of sumoylation on the interaction between CRTR-1 and these proteins can subsequently be determined by co- immunoprecipitation using the CRTR-1 sumoylation defective mutant.
Mice lacking CRTR-1 appeared to have no defect in the early embryo (Yamaguchi et al.,
2006), suggesting that it does not have a vital role in the ICM. However, given that CP2,
NF2d9 and altNF2d9 bind similar, if not identical, binding sites, form heteromeric complexes with each other, and are also expressed in ES cells (Kang et al., 2005a), functional redundancy between CRTR-1 and other CP2 family members may mask important roles for CRTR-1, including in ICM and ES cells. Therefore, all members should be examined in ES cells to determine the function of this family in the pluripotent transcription factor network. This could be investigated using RNA interference (RNAi) to knockdown the expression of all CP2 family members in ES cells. The effect of knockdown of CP2 family members on pluripotency can be analysed by examining morphological changes, expression levels of key pluripotency genes (such as Oct4 and
Nanog), cytokine dependence and differentiation potential of the knocked down cells.
The ability of CRTR-1 to bind to the regulatory regions of the pluripotency-associated factors Nanog, Oct4, Sox2, and Klf4 (Chen et al., 2008), suggests that CRTR-1 may be inducing the expression of these genes to maintain ES cells in an undifferentiated state while suppressing other genes that are required for further development. The CP2 family members that constitute the CRTR-1-containing DNA binding complex, as well as the promoter context, will dictate the nature of the transcriptional regulation of these genes and can be examined by analysing the expression level of these genes on knockdown of
CRTR-1 in combination with other CP2 family members in ES cells using siRNA.
102 GSK-3 is inactivated following activation of the Wnt signalling pathway in ES cells, leading to maintenance of pluripotency (Sato et al., 2004). Given that GSK-3 contains putative
CP2 binding sites and is upregulated by CP2 (Kosuga et al., 2005), GSK-3 may also be a potential candidate CRTR-1 target gene in ES cells. CRTR-1 may be suppressing the expression of GSK-3, thereby maintaining ES cells in an undifferentiated state. Loss of
CRTR-1 expression would result in induction of GSK-3 expression by other CP2 family members and loss of pluripotency. The ability of CRTR-1 to regulate GSK-3 expression could be examined by cloning the promoter upstream of a luciferase reporter gene and assessing reporter activity on transfection of CRTR-1. Alternatively, ChIP can be performed to show direct binding of CRTR-1 to the GSK-3 promoter. Analysis of the
GSK-3 expression level on knockdown of CRTR-1 would confirm regulation of GSK-3 expression by CRTR-1 in ES cells.
In summary, this thesis has shown that CRTR-1 is able to act both as an activator and repressor of transcription, form DNA-binding heteromers with other CP2 family members and modulate their activity. CRTR-1 function appears to be dependent on the complement of other CP2 family members expressed in a given cell type or tissue of interest, suggesting that CRTR-1 should be studied as a family rather than as an individual member. Given that CP2, NF2d9, and altNF2d9 are also expressed in ES cells
(Kang et al., 2005a), all these proteins should be examined in ES cells to determine the function of this family in the pluripotent transcription factor network. It is likely that this would identify new biological roles for these proteins.
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