The Key Regulator for Language and Speech Development, FOXP2, is a Novel Substrate for SUMOylation

Abstract

Transcription factor forkhead box protein P2 (FOXP2) plays an essential role in the development of language and speech. However, the transcriptional activity of FOXP2 regulated by the post-translational modifications remains unknown. Here we demonstrated that FOXP2 is clearly defined as a SUMO target protein at the cellular levels as FOXP2 is covalently modified by both SUMO1 and SUMO3. Furthermore, SUMOylation of FOXP2 was significantly decreased by SENP2 (a specific SUMOylation protease). We further showed that FOXP2 is selectively SUMOylated in vivo on a phylogenetically conserved lysine 674 but the SUMOylation does not alter subcellular localization and stability of FOXP2. Interestingly, we observed that human etiological FOXP2 R553H mutation robustly reduces its SUMOylation potential as compared to wild-type FOXP2. In addition, the acidic residues downstream the core SUMO motif on FOXP2 are required for its full SUMOylation capacity. Finally, our functional analysis using reporter gene assays showed that SUMOylation may modulate transcriptional activity of FOXP2 in regulating downstream target genes (DISC1, SRPX2 and MiR200c). Altogether, we provide the first evidence that FOXP2 is a substrate for SUMOylation and SUMOylation of FOXP2 plays a functional role in regulating its transcriptional activity.

Sumoylation of FOXP2 Regulates Motor Function and Vocal Communication Through Purkinje Cell Development

“…Moreover, loss of the lysine residue of FOXP2 at K647 could have additional detrimental effects in addition to loss of sumoylation, such as conformational changes of FOXP2 leading to altered protein-protein interaction or DNA binding, alterations of other post-transcriptional modifications like ubiquitination, or protein stability. We investigated a number of these possibilities, but there were no differences between FOXP2 WT and KR in our experiments (Figures S6 and S9 in Supplement 1, data not shown), consistent with a recent study showing that FOXP2 sumoylation does not affect protein stability (26). …”

“…When we expressed wild type FOXP2 (FOXP2 WT) in 293T cells, we also observed a higher molecular weight band recognized by a FOXP2 antibody that is decreased by H 2 O 2 treatment, a mechanism for reversible inhibition of SUMO conjugating enzymes (31) (Figure 1C) and ginkgolic acid (26, 32), a sumoylation-specific inhibitor (Figure 1D). Furthermore, in both 293T cells and mouse cerebellum, this high molecular weight band is recognized by either FOXP2 or SUMO-1 antibody in lysates that have undergone immunoprecipitation with an antibody recognizing SUMO-1 or FLAG (to capture FLAG-tagged FOXP2) respectively (Figure 1B–D).…”

“…Furthermore, in both 293T cells and mouse cerebellum, this high molecular weight band is recognized by either FOXP2 or SUMO-1 antibody in lysates that have undergone immunoprecipitation with an antibody recognizing SUMO-1 or FLAG (to capture FLAG-tagged FOXP2) respectively (Figure 1B–D). Based on these observations, we identified a conserved consensus sumoylation site (ψKXE) at K674 (K673 in mouse) that is outside of the annotated functional domains of FOXP2 (26) (Figure 1E, F). Upon mutation of this lysine to arginine (the non-sumoylated form of FOXP2 K674R , FOXP2 KR), the high molecular weight band was not observed (Figure 1G), however this point mutation does not affect the protein expression of FOXP2 in 293T cells, nor the amount immunoprecipitated by an antibody (Figure S1 in Supplement 1).…”

“…50 mM N-ethylmaleimide (NEM) (#E3876, Sigma-Aldrich, St. Louis, MO) was used as a SUMO protease inhibitor. 1 mM hydrogen peroxide (H 2 O 2 ) (31) and 100 μM ginkgolic acid (#75741, Sigma-Aldrich, St. Louis, MO) (26, 32) were used as sumoylation inhibitors.…”

“…Disruption of sumoylation can affect pathology in brain disorders such as Huntington’s disease (Htt), spinal and bulbar muscular atrophy (SUMO-1 positive intranuclear inclusions), spinocerebellar ataxias (Ataxin-1, 3, 7), Alzheimer’s disease (APP, Tau), Parkinson’s disease (α-Synuclein, Parkin, DJ-1) and ischemia (increase of SUMO-2/3, Drp1) (22, 24). A recent report has shown that FOXP2 is a substrate for sumoylation in transformed cell lines (26), however the role of sumoylation and potentially other post-translational modifications of FOXP2 in the CNS is completely unknown.…”