The Forkhead Transcription Factor Foxl2 Is Sumoylated in Both Human and Mouse: Sumoylation Affects Its Stability, Localization, and Activity

The FOXL2 forkhead transcription factor is expressed in ovarian granulosa cells, and mutated FOXL2 causes the blepharophimosis, ptosis and epicanthus inversus syndrome (BPES) and predisposes to premature ovarian failure. Inactivation of Foxl2 in mice demonstrated its indispensability for female gonadal sex determination and ovary development and revealed its antagonism of Sox9, the effector of male testis development. To help to define the regulatory activities of FOXL2, we looked for interacting proteins. Based on yeast two-hybrid screening, we found that FOXL2 interacts with PIAS1 and UBC9, both parts of the sumoylation machinery. We showed that human FOXL2 is sumoylated in transfected cell lines, and that endogenous mouse Foxl2 is comparably sumoylated. This modification changes its cellular localization, stability and transcriptional activity. It is intriguing that similar sumoylation and regulatory consequences have also been reported for SOX9, the male counterpart of FOXL2 in somatic gonadal tissues

Heritability of Cardiovascular and Personality Traits in 6,148 Sardinians

In family studies, phenotypic similarities between relatives yield information on the overall contribution of genes to trait variation. Large samples are important for these family studies, especially when comparing heritability between subgroups such as young and old, or males and females. We recruited a cohort of 6,148 participants, aged 14–102 y, from four clustered towns in Sardinia. The cohort includes 34,469 relative pairs. To extract genetic information, we implemented software for variance components heritability analysis, designed to handle large pedigrees, analyze multiple traits simultaneously, and model heterogeneity. Here, we report heritability analyses for 98 quantitative traits, focusing on facets of personality and cardiovascular function. We also summarize results of bivariate analyses for all pairs of traits and of heterogeneity analyses for each trait. We found a significant genetic component for every trait. On average, genetic effects explained 40% of the variance for 38 blood tests, 51% for five anthropometric measures, 25% for 20 measures of cardiovascular function, and 19% for 35 personality traits. Four traits showed significant evidence for an X-linked component. Bivariate analyses suggested overlapping genetic determinants for many traits, including multiple personality facets and several traits related to the metabolic syndrome; but we found no evidence for shared genetic determinants that might underlie the reported association of some personality traits and cardiovascular risk factors. Models allowing for heterogeneity suggested that, in this cohort, the genetic variance was typically larger in females and in younger individuals, but interesting exceptions were observed. For example, narrow heritability of blood pressure was approximately 26% in individuals more than 42 y old, but only approximately 8% in younger individuals. Despite the heterogeneity in effect sizes, the same loci appear to contribute to variance in young and old, and in males and females. In summary, we find significant evidence for heritability of many medically important traits, including cardiovascular function and personality. Evidence for heterogeneity by age and sex suggests that models allowing for these differences will be important in mapping quantitative traits

FOXL2 stability is increased by SUMO-1 and is not dependent on proteasome-mediated degradation.

<p><i>A.</i> Sumoylation regulates FOXL2 stability: COS-7 cells were co-transfected with pCRUZ-myc-FOXL2 (0.5 µg) and increasing amounts of pCRUZ-HA-UBC9 or pCRUZ-HA-PIAS1(3.5–4.5 µg) or pCRUZ-HA-SUMO-1 (4.5 µg). Lysates treated with and without NEM were analysed by immunoblotting with anti-FOXL2 antibody. The bracket shows slower migrating bands of about 105–160 kDa. Densitometric analysis was performed on sumoylated band using Image J software and is reported compared to that in lane 1. <i>B.</i> FOXL2 stabilization is mediated by SUMO-1 in a dose-dependent manner: COS7 were transfected with 500 ng of pCRUZ-myc-FOXL2 and increasing amounts of pCRUZ-HA-SUMO-1 (0, 1, 2, 3, 4 µg). Immunoblotting against myc shows that FOXL2 increases with the augmentation of SUMO-1. In the lower panel anti-actin is used as loading control. <i>C.</i> FOXL2 stability is not dependent on ubiquitination and proteasome-mediated degradation: COS7 cells were transfected with pCRUZ-HA-FOXL2 alone and with pCRUZ-HA-SUMO-1 in quadruplicate. After 12 h of transfection, cells were lysed (t0) or treated with cycloheximide (inhibitor of protein biosynthesis), cycloheximide and MG132 (proteasome-inhibitor) or neither. After 36 h cells were lysed and 50 ug of protein were blotted and hybridization performed with anti-myc antibody.</p

FOXL2 interacts with sumoylation machinery, is sumoylated and co-localize with SUMO-1 and PIAS1.

<p><i>A., B.</i> FOXL2 interacts with PIAS1 and UBC9: COS-7 cells were co-transfected with pCRUZ-HA-FOXL2 and pCRUZ-myc-PIAS1 (A), or with pCRUZ-myc-FOXL2 and pCRUZ-HA-UBC9 (B) or with HA and myc empty vectors (−). Lysates were immunoprecipitated with anti-myc agarose conjugated antibody and analysed by western blotting with anti-FOXL2, anti-PIAS1 and anti-UBC9 antibodies. Mock IP consisted in an immunoprecipitation with only protein-A beads, without antibody. Expression of all proteins was also analysed in total lysates (input). <i>C.</i> FOXL2 is sumoylated in transfected COS-7 cells: COS-7 cells were transfected with pCRUZ-myc-FOXL2 (0.5 µg) alone or with pCRUZ-HA-SUMO-1 (4.5 µg), or with HA and myc empty vectors (−). Lysates were immunoprecipitated with anti-myc agarose conjugated antibody, and analysed by western blotting using anti-FOXL2 antibody (upper panel) or anti-SUMO-1 antibody (lower panel). Both antibodies recognized band(s) of about 105-160-kDa indicated by brackets (<b>[</b>), not present in the transfection of FOXL2 alone. <i>D.,E.</i> FOXL2 is sumoylated <i>in vivo</i> in physiological conditions: (D) Immunoprecipitation was done using anti-FOXL2 antibody on α-T31 cell lysate and the eluate analyzed by western blotting using anti-FOXL2 antibody (left panel) or anti-SUMO-1 antibody (right panel). Both antibodies recognized band(s) of about 105–160-kDa indicated by brackets (<b>[</b>). The asterisk indicates the FOXL2 signal from the previous hybridization. (E) 4-week old mouse ovaries were lysed with or without NEM, 60 µg of protein was loaded on SDS PAGE, electrophoresed, and then immunoblotted. Western blotting with an anti-Foxl2 antibody showed a 45 kDa band corresponding to native Foxl2 and a slower migrating band of about 105–160 kDa, also recognised by anti-Sumo-1 antibody (not shown). <i>F.</i> FOXL2 and SUMO-1 co-localize in the nucleus: pCRUZ-myc-FOXL2 and pCRUZ-HA-SUMO-1 were co-transfected into COS7 cells, and immunofluorescence was performed using anti-myc and anti-HA antibody. In red (Alexa 633) is shown FOXL2, in green (Alexa 488) SUMO-1. The yellow colour indicates co-localization, and is particularly seen in spots resembling PML bodies. <i>G.</i> Wild type FOXL2 co-localizes with PIAS1 in the nucleus: COS-7 cells were co-transfected with pCRUZ-myc-FOXL2 and pCRUZ-HA-PIAS1. The intracellular distribution of FOXL2 (red) and PIAS1 (green) was detected by indirect immunofluorescence with mouse anti-myc and rabbit anti-HA primary antibodies and Alexa Fluor 633 anti-mouse and Alexa Fluor 488 anti-rabbit secondary antibodies. <i>H.</i> PIAS1 enhances FOXL2 sumoylation: COS-7 cells were co-transfected with pCRUZ-myc-FOXL2 and pCRUZ-HA-PIAS1. And lysed with or without NEM. Lysates were immunoblotted with anti-FOXL2 antibody and densitometric analysis was performed on sumoylated band using Image J software. Densitometric analysis is reported compared to that in lane 1.</p

Foxl2, Sumo-1 and Ubc9 are expressed in mouse ovary.

<p><i>A.</i> Pias1, Ubc9 and Sumo-1 are expressed in 4 week mouse ovaries: 4-week old mouse ovaries were lysed and analysed by immunoblotting with anti- Pias1, Ubc9, and Sumo-1 specific antibodies. 50 ug of protein were loaded in each lane. <i>B.</i> Co-Localization of Foxl2, Sumo-1 and Ubc9 in 4 week-old mouse ovary: Hybridization with anti-Foxl2 (red) and anti-Ubc9 (green) or anti-Sumo-1 (green) antibodies. Yellow spots in the 40× magnification show the co-localization of Foxl2 with Ubc9 and Sumo-1, especially in primordial and primary follicle granulosa cells. Immunofluorescence analysis was performed using a Leica DMIRE2-TCS-SL Confocal Laser Scanning microscope (from 488 to 633 excitation wavelength).</p

PIAS1 and UBC9 in contrast to FOXL2, activate <i>StAR</i> promoter.

<p>COS-7 cells were co-transfected with pGL3-StAR luciferase reporter vector, and increasing amounts (1.5, 3 µg) of PIAS1 or UBC9, with or without FOXL2 (1 µg). Relative luciferase activity is compared to that of promoter activity alone. Luciferase activity is reported as relative activity, as the mean of at least 4 independent experiments, normalized to the reporter gene <i>Renilla</i>, encoded by pTRLK vector, used as an internal control, and compared to promoter activity alone. Statistical significance was estimated with Student's t-Test, p-values <0.05 are significative, (*) p<0.01, (**) p<0.001.</p

Sumoylation sites prediction.

<p><i>A.</i> The SUMOplot™ Analysis Program (<a href="http://www.abgent.com/sumoplot.html" target="_blank">http://www.abgent.com/sumoplot.html</a>) was used to predict and score sumoylation sites within the FOXL2 protein (upper panel). <i>B.</i> Sequence alignment of the FOXL2 region surrounding the putative sumoylation sites using the ClustalW2 Web based tool (right panel). In grey is highlighted the forkhead domain. In pink the SUMO consensus sequences K25, K87, K114, K150, with higher score, in blue those with lower scores (K36, K48, K54).</p