Mutational probing of the forkhead domain of the transcription factor FOXL2 provides insights into the pathogenicity of naturally occurring mutations

Abstract

Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development, lead to the Blepharophimosis Syndrome. Here, we have systematically replaced the amino acids of the helices of the forkhead domain (FHD) of FOXL2 by glycine residues to assess the impact of such substitutions. A number of mutations lead to protein mislocalization, aggregation and to partial or complete loss of transactivation ability on a series of luciferase reporter systems. To rationalize the results of this glycine mutation scan, we have modeled the structure of the FHD by comparison with crystallographic data available for other FHDs. We failed to detect a clear-cut correlation between protein mislocalization or aggregation and the position of the mutation. However, we found that the localization of the side chain of each amino acid strongly correlated with the impact of its mutation on FOXL2 transactivation capacity. Indeed, when the side chains of the amino acids involved in the helices of the forkhead are supposed to point towards the hydrophobic core formed by the three main helices, a loss of function was observed. On the contrary, if the side chains point outward the hydrophobic core, protein function was preserved. The extension of this analysis to natural mutants shows that a similar correlation can be found for BPES mutations associated or not with ovarian dysfunction. Our findings reveal new insights into the molecular effects of FOXL2 mutations affecting the FHD, which represent two-thirds of intragenic mutations, and provide the first predictive tool of their effects.

Characterization of endocrine features and genotype–phenotypes correlations in blepharophimosis–ptosis–epicanthus inversus syndrome type 1

“…Beysen et al [13] described a diffuse nuclear distribution of the 104R mutant FOXL2 protein (as for the wild-type) and a normal transcriptional activation. Similar findings were reported for the 104 N and 104G mutants [14,15], involving the same amino acid position. These authors proposed a functional classification of FOXL2 missense mutations, correlating the subcellular localization and aggregation of FOXL2 mutants, transactivation activities and the type of BPES.…”

<i>FOXL2 </i>Impairment in Human Disease

“…A clear correlation was found between the transcriptional activity of FOXL2 mutations on two different reporter promoters and the BPES type [25]. In a very recent study by Todeschini et al [26], the amino acids of the helices of the forkhead domain of FOXL2 were systematically replaced by glycine residues to assess the impact of these artificial mutations. A number of mutations led to protein mislocalization, aggregation and to partial or complete loss of transactivation ability on a dozen of luciferase reporter systems.…”

“…Extrapolation of this analysis to natural mutations was in agreement with the findings obtained for the artificial mutations. This study brought important insights into the molecular effects of FOXL2 missense mutations located in the forkhead domain, and provided an apparently reliable in silico predictive tool for their phenotypic effects [26]. …”

The transcription factor FOXL2 mobilizes estrogen signaling to maintain the identity of ovarian granulosa cells

“…In humans, FOXL2 heterozygous mutations are responsible for the blepharophimosis-ptosis-epicanthus inversus syndrome (BPES), characterized by facial malformations often associated with primary ovarian insufficiency (POI) (Crisponi et al, 2001). Recent works have shown that FOXL2 mutations impairing its DNA binding and/or transcriptional activity are responsible for POI occurrence in BPES (Dipietromaria et al, 2009; Todeschini et al, 2011). Interestingly, a specific somatic mutation of FOXL2 has been identified in more than 95% of adult-type granulosa cell tumors (GCTs) confirming the strong association of FOXL2 with granulosa cell fate and function (Shah et al, 2009; Jamieson and Fuller, 2012).…”