Deciphering the roles of subcellular distribution and interactions involving the MEF2 binding region, the ankyrin repeat binding motif and the catalytic site of HDAC4 in Drosophila neuronal morphogenesis

Abstract Background Dysregulation of nucleocytoplasmic shuttling of histone deacetylase 4 (HDAC4) is associated with several neurodevelopmental and neurodegenerative disorders. Consequently, understanding the roles of nuclear and cytoplasmic HDAC4 along with the mechanisms that regulate nuclear entry and exit is an area of concerted effort. Efficient nuclear entry is dependent on binding of the transcription factor MEF2, as mutations in the MEF2 binding region result in cytoplasmic accumulation of HDAC4. It is well established that nuclear exit and cytoplasmic retention are dependent on 14–3-3-binding, and mutations that affect binding are widely used to induce nuclear accumulation of HDAC4. While regulation of HDAC4 shuttling is clearly important, there is a gap in understanding of how the nuclear and cytoplasmic distribution of HDAC4 impacts its function. Furthermore, it is unclear whether other features of the protein including the catalytic site, the MEF2-binding region and/or the ankyrin repeat binding motif influence the distribution and/or activity of HDAC4 in neurons. Since HDAC4 functions are conserved in Drosophila, and increased nuclear accumulation of HDAC4 also results in impaired neurodevelopment, we used Drosophila as a genetic model for investigation of HDAC4 function. Results Here we have generated a series of mutants for functional dissection of HDAC4 via in-depth examination of the resulting subcellular distribution and nuclear aggregation, and correlate these with developmental phenotypes resulting from their expression in well-established models of neuronal morphogenesis of the Drosophila mushroom body and eye. We found that in the mushroom body, forced sequestration of HDAC4 in the nucleus or the cytoplasm resulted in defects in axon morphogenesis. The actions of HDAC4 that resulted in impaired development were dependent on the MEF2 binding region, modulated by the ankyrin repeat binding motif, and largely independent of an intact catalytic site. In contrast, disruption to eye development was largely independent of MEF2 binding but mutation of the catalytic site significantly reduced the phenotype, indicating that HDAC4 acts in a neuronal-subtype-specific manner. Conclusions We found that the impairments to mushroom body and eye development resulting from nuclear accumulation of HDAC4 were exacerbated by mutation of the ankyrin repeat binding motif, whereas there was a differing requirement for the MEF2 binding site and an intact catalytic site. It will be of importance to determine the binding partners of HDAC4 in nuclear aggregates and in the cytoplasm of these tissues to further understand its mechanisms of action.