In this report we provide evidence that Gli proteins are targets of SUMOylation and that Pias1, in a manner dependant on its ability to conjugate SUMO, affects the transcriptional activity of Gli proteins in both cultured cells and in embryos. Each of the Gli proteins has several phylogenetically conserved consensus motifs for SUMOylation and we provide evidence that SUMO1 can be conjugated to the lysines in several of these motifs. The addition of SUMO to Gli proteins appears to boost their transcriptional activating function and this enhancement appears to be necessary for the induction of at least one known target of Gli proteins, Nkx2.2, in the developing neural tube 
. Consistent with these data, PKA activity, which negatively regulates Gli2 and Gli3, appears to block the SUMOylation of Gli2 and Gli3. Together these data identify SUMOylation as a post-translational modification of Gli proteins that influences the activity of this important family of transcription factors.
SUMOylation appears to correlate with, and to increase, the transcriptional activating function of Gli proteins. SUMOylation of transcription factors is most frequently associated with transcriptional repression 
, however SUMOylation has previously been shown to increase the activity of several transcription factors including p53 and Tcf4 (reviewed in 
). Our data therefore add to the evidence that SUMOylation can have positive as well as inhibitory effects of the activity of transcription factors. Although we have been unable to detect SUMOylation of endogenous Gli proteins, due to the lack of appropriate reagents, the evidence supports a model in which SUMOylation can regulate endogenous Gli activity in a physiological context. Importantly, SUMO E3 ligase, Pias1, enhances Gli activity in cultured cells and in embryos and this appears to be a consequence of its ability to catalyze the covalent addition of SUMO to Gli proteins (). First, a point mutant of Pias1 that lacks ligase activity does not affect Gli1 activity. Second, Pias1 does not affect the activity of a form of Gli1 in which the SUMOylation sites have been mutated to arginine, a residue incapable of SUMO conjugation. Third, over-expression of SUMO1 in cells also increases the activity of Gli1. Together these data favour the idea that direct SUMOylation of a Gli protein alters its transcriptional activity and argue against models in which the effect of SUMOylation is indirect, mediated through the regulated SUMOylation of other proteins. These data also rule out the possibility that the effects of Pias1 on Gli proteins are due to an E3-ligase independent activity of Pias1. Nevertheless, we have not consistently found an increase in SUMOylation levels of Gli proteins in cells transfected with Pias1. However, our experiments relied on the overexpression of tagged SUMO1 and this might make it difficult to detect any effect of increased Pias1 expression on the SUMOylation. How SUMO conjugation contributes to an increase of Gli activity remains unresolved. Although we have not obtained consistent evidence of an increase in the nuclear localization or stability of Gli proteins in the presence of Pias1 (data not shown), we cannot rule out the possibility that changes in the location or stability of a small yet crucial pool of Gli proteins is responsible for the observed effects. Moreover, we do not exclude the possibility that other components of the Hh signaling pathway might also be susceptible to SUMO regulation. In this regard it is notable that Pias1 has been shown to interact with Supressor of fused (Sufu) 
, a negative regulator of the Hh pathway that interacts with Gli proteins 
. It is possible therefore that sumoylation regulates the Hh pathway at multiple levels.
The mutational analysis of Gli proteins suggest suggests several lysines are potentially conjugated to SUMO. Whether there is a hierarchical relationship between these sites and whether an individual Gli protein harbours multiple SUMOylations is not known. Moreover, the presence of multiple distinct SUMO proteins within cells and the potential for some of these forms to generate polySUMO conjugates 
raises the possibility that a “SUMO” code exists. This code might result in distinct combinations SUMO modifications at different sets of lysine residues of Gli proteins with each combination resulting in a different effect on the activity of the Gli protein. Further analysis will be required to determine if different patterns of SUMOylation are found on different Gli proteins and to resolve whether these elicit different effects on Gli activity.
PKA is a well established negative regulator of Hh signaling that directly phosphorylates Gli2 and Gli3 to promote their processing by the proteasome 
. Our data indicate that PKA activity also antagonizes SUMOylation of Gli2 and Gli3. We provide evidence that this antagonism is a consequence of PKA phosphorylation of Gli proteins () and not an indirect effect of PKA on the SUMOylation or proteasome machinery. It was also notable that little, if any, SUMO conjugated partially processed Gli3 protein was observed. Together these data suggest that PKA dependent processing of Gli proteins and SUMOylation of Gli proteins are mutually exclusive. In contrast to Gli2 and Gli3, the SUMOylation of Gli1 was less affected by PKA activity. Differences in the distance between the PKA phosphorylation sites and the SUMOylatable lysines in Gli1 and Gli2/3, might account for this observation. Irrespective of the explanation, the data further support the contention that the reduced SUMOylation of Gli2 and Gli3 in the presence of activated PKA is a specific and direct effect.
As Pias1, like other SUMO pathway components, is expressed in the developing neural tube (), it is conceivable that it is capable of regulating the activity of Gli proteins within the neural tube. Here we provide evidence that Pias1 can increase Gli transcriptional activity within the neural tube of chick embryos (). Strikingly, Pias1 can induce ectopic expression of Nkx2.2, a homeodomain transcription factor (), the activation of which has been shown to require high levels of Gli activity 
. Conversely, inhibition of Pias1 activity blocks Nkx2.2 expression. Taken together, our results suggest a scenario in which Gli proteins require SUMOylation in order to achieve the highest levels of activity necessary to induce the most ventral cell identities within the neural tube.
The finding that Pias1, through SUMOylation, regulates Gli activity within the neural tube raises the possibility that patterning throughout the neural tube may be affected by the SUMOylation machinery. As Pias1 and SUMOylation have been shown to be involved in the regulation of members of Wnt and BMP pathways in other systems 
and Wnt and BMP signaling, together with Shh, play fundamental roles in the control of cell fate specification and proliferation within the neural tube 
, it is possible that Pias/SUMO regulates the intrinsic activity of proteins that define cell identity, namely members of the homeodomain and basic helix-loop-helix protein families. The extent of the role of SUMO conjugation within the neural tube system remains unknown and will require further investigation.