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. 2009 Dec;21(12):1935-44.
doi: 10.1016/j.cellsig.2009.09.001. Epub 2009 Sep 8.

Sumoylation of forkhead L2 by Ubc9 is required for its activity as a transcriptional repressor of the Steroidogenic Acute Regulatory gene

Fang-Ting Kuo  1 , Ikuko K Bentsi-Barnes, Gillian M Barlow, Jeehyeon Bae, Margareta D Pisarska
Affiliations

Sumoylation of forkhead L2 by Ubc9 is required for its activity as a transcriptional repressor of the Steroidogenic Acute Regulatory gene

Fang-Ting Kuo et al. Cell Signal. 2009 Dec.

Abstract

Forkhead L2 (FOXL2) is a member of the forkhead/hepatocyte nuclear factor 3 (FKH/HNF3) gene family of transcription factors and acts as a transcriptional repressor of the Steroidogenic Acute Regulatory (StAR) gene, a marker of granulosa cell differentiation. FOXL2 may play a role in ovarian follicle maturation and prevent premature follicle depletion leading to premature ovarian failure. In this study, we found that FOXL2 interacts with Ubc9, an E2-conjugating enzyme that mediates sumoylation, a key mechanism in transcriptional regulation. FOXL2 and Ubc9 are co-expressed in granulosa cells of small and medium ovarian follicles. FOXL2 is sumoylated by Ubc9, and this Ubc9-mediated sumoylation is essential to the transcriptional activity of FOXL2 on the StAR promoter. As FOXL2 is endogenous to granulosa cells, we generated a stable cell line expressing FOXL2 and found that activity of the StAR promoter in this cell line is greatly decreased in the presence of Ubc9. The sumoylation site was identified at lysine 25 of FOXL2. Mutation of lysine 25 to arginine leads to loss of transcriptional repressor activity of FOXL2. Taken together, we propose that Ubc9-mediated sumoylation at lysine 25 of FOXL2 is required for transcriptional repression of the StAR gene and may be responsible for controlling the development of ovarian follicles.

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Conflict of interest statement

Disclosure Statement: F-TK, I B-B, JB and GB have nothing to disclose. MP is supported by grants from the NICHD/ORWH (R01HD047603) and the Helping Hands of Los Angeles, Inc.

Figures

Fig. 1
Fig. 1
FOXL2 interacts with Ubc9 in CHO cells. CHO cells were transfected with different combinations of FLAG or HA tagged FOXL2 and Ubc9. The cell lysates were immunoprecipitated with an anti-FLAG antibody, and proteins in the cell lysates and immunoprecipitates (IP) were resolved by SDS-PAGE and immunoblotted with antibodies to FLAG and HA tags. A. HA-tagged Ubc9 (HA-Ubc9) was co-transfected with or without FLAG-tagged FOXL2 (FLAG-FOXL2) into CHO cells. When cells were transfected with HA-Ubc9 and FLAG-FOXL2, a 60 kDa FLAG-FOXL2 band was present in the cell lysates and immunoprecipitates. When cells transfected with HA-tagged Ubc9 alone or combined with FLAG-FOXL2, a 20 kDa HA-Ubc9 was expressed in the cell lysates. Moreover, HA-Ubc9 was observed co-immunoprecipitating with FLAG-tagged FOXL2 (IP, HA-Ubc9), when cells co-expressed FLAG-FOXL2 and HA-Ubc9. B. HA-tagged FOXL2 (HA-FOXL2) was co-transfected with or without FLAG-tagged Ubc9 in CHO cells. When cells were transfected with FLAG-Ubc9, a 20 kDa FLAG-Ubc9 band was present in the cell lysates and immunoprecipitates. When cells were transfected with HA-FOXL2 alone or combined with FLAG-Ubc9, cells expressed a 60 kDa HA-FOXL2 in the cell lysates. Similar to the previous result (A), HA-FOXL2 co-immunoprecipitated with FLAG-Ubc9 (IP, HA-FOXL2) when cells co-expressed FLAG-Ubc9 and HA-FOXL2. The experiment was repeated three times with similar results.
Fig. 2
Fig. 2
FOXL2 and Ubc9 are co-expressed in mouse granulosa cells. Immunohistochemical staining was performed on serial sections of ovaries from 21 day-old immature mice using antibodies to FOXL2 or Ubc9, followed by counterstaining with hematoxylin. Both FOXL2 (left) and Ubc9 (right) are expressed in granulosa cells of immature ovarian follicles, small (arrow) and medium (arrowhead) follicles. The experiment was performed three times with identical results.
Fig. 3
Fig. 3
FOXL2 is sumoylated in vivo and in vitro. A. CHO cells were transfected with FLAG-tagged FOXL2. The cell lysates were immunoprecipitated with an anti-FLAG antibody, and proteins in the cell lysates and immunoprecipitates (IP) were resolved by SDS-PAGE and immunoblotted with antibodies to FOXL2 and SUMO1. Endogenous FOXL2 was not detected in CHO cells, but cells transfected with FLAG-FOXL2 expressed a 60 kDa FLAG-FOXL2 band (arrowhead, cell lysates). This 60 kDa FLAG-FOXL2 band and a 120 kDa band (asterisk) were seen in the IP products when immunoblotted with an antibody to FOXL2. The 120 kDa band was then confirmed to be SUMO1 modified FLAG-tagged FOXL2 (asterisk, SUMO-FLAG-FOXL2) by immunoblotting with a SUMO1 antibody. The blots of the cell lysates were then re-probed with a tubulin antibody to control for equal loading. The experiment is representative of three similar experiments. B. A 45 kDa FOXL2 protein (arrowhead) was synthesized from the FOXL2/pcDNA3 construct using TNT Quick Coupled Transcription/Translation Systems, and was then used as a substrate for in vitro SUMO1-sumoylation reactions. When Ubc9 was present in the reaction, a 110 kDa band (asterisk) was observed when the assay products were immunoblotted with an antibody to FOXL2. When Ubc9 was absent from the reaction, this 110 kDa band was not observed. This 110 kDa band was then confirmed to be SUMO1-modified FOXL2 (asterisk, SUMO-FOXL2) by immunoblotting the assay products with a SUMO1 antibody. The experiment is representative of three similar experiments.
Fig. 4
Fig. 4
Overexpression of Ubc9 enhances the sumoylation of FOXL2. CHO cells were transfected with 0.4μg FLAG-FOXL2 plasmid alone, 0.4μg FLAG-FOXL2 and 2μg Ubc9 plasmids, or 0.4μg FLAG-FOXL2 and 2μg dominant negative mutant Ubc9 C93S plasmids. The cell lysates were immunoprecipitated. Proteins in the cell lysates and immunoprecipitates were resolved by SDS-PAGE and immunoblotted with an antibody to FOXL2 or SUMO1 (A) or Ubc9 (B). A. Cells transfected with FOXL2 along or with wild type or mutant Ubc9 exhibit a 60 kDa FLAG-FOXL2 product (cell lysate, lane 2, 3 & 4). After immunoprecipitation of FLAG-tagged proteins, cells transfected with FLAG-FOXL2 alone exhibit both a 60 kDa FLAG-FOXL2 product and a 120 kDa sumoylated FLAG-FOXL2 product (SUMO-FLAG-FOXL2) (lane 6). Co-expression of FLAG-FOXL2 with Ubc9 increased the amount of the 120 kDa product (lane 7), but co-expression with dominant negative mutant Ubc9 C93S did not (lane 8). B. Transfection with wild type Ubc9 or dominant negative mutant Ubc9 C93S increased the amount of Ubc9 in these cells. The blots of the cell lysates were then re-probed with a tubulin antibody to control for equal loading. The experiment was repeated three times with similar results.
Fig. 5
Fig. 5
Ubc9 is required for the sumoylation of FOXL2. A. CHO cells were transfected with 5nM non-silencing siRNA or 5nM Ubc9 siRNA. After 24h, the cells were transfected with FLAG-FOXL2, cultured for another 24 h, lysed and immunoblotted with an antibody to Ubc9. Transfection with Ubc9 siRNA depleted the amounts of Ubc9 (18 kDa) in CHO cells. B. The cell lysates were immunoprecipitated, and probed with an antibody to FOXL2 or SUMO1. The amount of sumoylated FLAG-FOXL2 (SUMO-FLAG-FOXL2, indicated by the bracket) decreased following transfection with Ubc9 siRNA (lane 4), but not after transfection with non-silencing siRNA (lane 2). The experiment was repeated three times with similar results.
Fig. 6
Fig. 6
Ubc9 is essential for the repression of the StAR gene promoter by FOXL2. A. CHO cells were co-transfected with −95-bp StAR promoter-luciferase and either 20ng FLAG-FOXL2 alone, 20ng FLAG-FOXL2 and 200ng Ubc9, or 20ng FLAG-FOXL2 and 200ng Ubc9 C93S, and the resulting luciferase activities were determined. Luciferase activity values were normalized against activity levels in cells transfected with the StAR promoter plasmid and pcDNA3 backbone vector (Control, value = 1). StAR promoter activity is repressed following transfection with FOXL2, and is further repressed following transfection with FOXL2 and Ubc9. However, in the presence of the mutant Ubc9 C93S, FOXL2 repressor activity is lost. B. CHO cells were transfected with 5nM non-silencing siRNA or 5nM Ubc9 siRNA. After 24 h, the cells were transfected with the −95-bp StAR promoter-luciferase alone or with 50ng FLAG-FOXL2, incubated for 24 h and then lysed for determination of luciferase activity. Luciferase activity values were normalized as described above. FOXL2 maintains its ability to repress StAR promoter activity when co-transfected with non-silencing siRNA, but loses this repressor activity when co-transfected with Ubc9 siRNA. The results were similar for at least three separate experiments. The standard error (±SE) and statistically significant differences are shown (*, p<0.001).
Fig. 7
Fig. 7
FOXL2 and Ubc9 are localized to the nucleus. CHO cells stably expressing FOXL2 were grown on poly-L-lysine coverslips for 24 h. Cells were then immunostained with FOXL2 and Ubc9 antibodies. The cell nucleus was identified by DAPI staining (blue). Both FOXL2 (green) and Ubc9 (red) are localized in the cell nucleus. The experiment is representative of three separate experiments.
Fig. 8
Fig. 8
Co-expression of FOXL2 and Ubc9 is required for repression of the StAR gene promoter. A. Native CHO cells and CHO cells stably expressing FOXL2 were transfected with 5nM non-silencing siRNA or 5nM Ubc9 siRNA. After 24 h, the cells were transfected with −95-bp StAR promoter-luciferase, incubated for 24 h and then lysed for determination of luciferase activity. Luciferase activity values were normalized against activity levels in cells transfected with StAR promoter plasmid and pcDNA3 backbone vector (Control, value = 1). StAR promoter activity is repressed in cells stably expressing FOXL2, compared to that in native CHO cells which lack FOXL2 expression. This repression is unaffected by non-silencing siRNA, but is reduced following transfection with Ubc9 siRNA. B. and C. CHO cells stably expressing FOXL2 were transfected with 5nM non-silencing siRNA or 5nM Ubc9 siRNA. Forty-eight hours after siRNA transfection, cells were lysed and Ubc9 (B) and FOXL2 protein expression (C) in cell lysates were detected by immunoblotting. Transfection with Ubc9 siRNA reduces the amounts of Ubc9 in these cells (B) and also reduces the amount of SUMO-modified FOXL2 (SUMO-FOXL2, indicated by the bracket) (C). The results were similar for at least three separate experiments. The standard error (±SE) and statistically significant differences are shown (*, p<0.001).
Fig. 9
Fig. 9
Sumoylation consensus site aligned in sequences of FOXL2 from different species. The sumoylation consensus site is ψKXE/D with a hydrophobic residue, such as Valine (V), Isoleucine (I), or Leucine (L). Sequences of FOXL2 of nile tilapia, chicken, goat, mouse, chimpanzee, and human, and FOXL of yellow fever mosquito were aligned. A consensus sumoylation site is localized in FOXL of yellow fever mosquito, 91IKTD94 (underlined), and is highly homologous to sequences of other FOXL2 protein sequences (red box).
Fig. 10
Fig. 10
Sumoylation mutant of FOXL2 loses transcriptional repression activity. A. CHO cells were transfected with wild type (Wt) or sumoylation mutant (K25R) FLAG-tagged FOXL2. The cell lysates were immunoprecipitated with an anti-FLAG antibody, and proteins in immunoprecipitates were resolved by SDS-PAGE and immunoblotted with M2 FLAG or SUMO1 antibodies. Cells transfected with wild type FOXL2 (Wt) exhibit both a 60 kDa FLAG-FOXL2 product and a 120 kDa sumoylated FLAG-FOXL2 product (SUMO-FLAG-FOXL2). A 60 kDa FLAG-FOXL2 product with similar intensity was also present in cells transfected with mutant FOXL2 K25R. However, the 120 kDa sumoylated FLAG-FOXL2 product (SUMO-FLAG-FOXL2) was markedly diminished. B. CHO cells were co-transfected with −95-bp StAR promoter-luciferase and either 10ng wild type FOXL2 or 10ng sumoylation mutant FOXL2 (K25R), and the resulting luciferase activities were determined. Luciferase activity values were normalized against activity levels in cells transfected with StAR promoter plasmid and FLAG-CMV2 backbone vector (Control, value = 1). StAR promoter activity is repressed following transfection with wild type FOXL2, but repression is diminished following transfection with the sumoylation mutant FOXL2 K25R. The results were similar for at least three separate experiments. The standard error (±SE) and statistical significances were shown (*, p<0.001).
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