背景:组蛋白脱乙酰酶4(HDAC4)的核质穿梭失调与几种神经发育和神经退行性疾病有关。因此,了解细胞核和细胞质HDAC4的作用以及调节核进出的机制是一个共同努力的领域。有效的核进入依赖于转录因子MEF2的结合,因为MEF2结合区中的突变导致HDAC4的细胞质积累。众所周知,细胞核退出和细胞质保留依赖于14-3-3结合,和影响结合的突变被广泛用于诱导HDAC4的核积累。虽然HDAC4穿梭的调节显然很重要,在理解HDAC4的核和细胞质分布如何影响其功能方面存在差距。此外,目前还不清楚蛋白质的其他特征,包括催化位点,MEF2结合区和/或锚蛋白重复结合基序影响HDAC4在神经元中的分布和/或活性。由于HDAC4功能在果蝇中是保守的,HDAC4的核积累增加也导致神经发育受损,我们使用果蝇作为研究HDAC4功能的遗传模型。
结果:在这里,我们通过对由此产生的亚细胞分布和核聚集的深入检查,产生了一系列用于HDAC4功能解剖的突变体,并将它们与在果蝇蘑菇体和眼的神经元形态发生模型中表达的发育表型相关联。我们发现在蘑菇体内,HDAC4在细胞核或细胞质中的强制隔离导致轴突形态发生缺陷。HDAC4导致发育受损的作用依赖于MEF2结合区,由锚蛋白重复结合基序调制,并且很大程度上独立于完整的催化位点。相比之下,眼发育的中断在很大程度上与MEF2结合无关,但催化位点的突变显着降低了表型,表明HDAC4以神经元亚型特异性方式起作用。
结论:我们发现,锚蛋白重复结合基序的突变加剧了HDAC4核积累对蘑菇体和眼睛发育的损害,而对MEF2结合位点和完整催化位点有不同的要求。确定HDAC4在核聚集体和这些组织的细胞质中的结合配偶体以进一步理解其作用机制将是重要的。
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.
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.
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.