Calmodulin-dependent nuclear import of HMG-box family nuclear factors: Importance of the role of SRY in sex reversal

The high mobility group (HMG)-box containing chromatin remodeling factor, SRY (sex determining region on the Y chromosome) plays a key role in sex determination. Its role in the nucleus being critically dependent on two nuclear localisation signals (NLSs) that flank its HMG domain: the C-terminally located beta-NLS that mediates nuclear transport through Importin beta1 (Imp beta1) and the N-terminally located CaM-NLS which is known to recognize the calcium-binding protein calmodulin (CaM). In this study we examine a number of missense mutations in the SRY CaM-NLS from human XY sex-reversed females for the first time, showing that they result in significantly reduced nuclear localisation of GFP-SRY fusion proteins in transfected cells compared to wild type. The CaM antagonist, calmidazolium chloride (CDZ), was found to significantly reduce wild type SRY nuclear accumulation, indicating dependence of SRY nuclear import on CaM. Intriguingly, the CaM-NLS mutants were all resistant to CDZ s effects, implying a loss of interaction with CaM, which was confirmed by direct binding experiments. CaM binding/resultant nuclear accumulation was the only property of SRY found to be impaired by two of the CaM-NLS mutations, implying that inhibition of CaM-dependent nuclear import is the basis of sex reversal in these cases. Importantly, the CaM-NLS is conserved in other HMG box domain-containing proteins such as SOX-2, -9, -10 and HMGN1, all of which were found here for the first time to rely on CaM for optimal nuclear localisation. CaM-dependent nuclear translocation is thus a common mechanism for this family of important transcription factors.