Epigenetic regulation of hormone action in Red flour beetle, Tribolium castaneum

N-terminal acetylation is a ubiquitous protein modification conserved from yeast to human, which has a widespread biological role. Extensive research about histone and other proteins revealed the role of co-translational and post-transcriptional modifications and their impact on chromatin folding and activity of relevant genes. We used Tribolium castaneum, a model insect, to identify the role of individual histone acetyltransferase in hormone action. We performed RNAi mediated knockdown studies of various HAT genes. In this study, we show that, N-alpha-acetyltransferase, Tc015921 (NAA40) plays a significant role in metamorphosis and development. Developmental profiles of TC015921 mRNA showed increases at the later stages of last instar larvae and early pupal stage. The knockdown of the TC015921 gene showed a larval phenotype with dorsal split. Ecdysone analog, 20E induced Tc015921 expression in TcA Cells. Knockdown of TC015921 in the final instar larvae suppressed the expression of genes coding for proteins involved in ecdysone action. RNA-seq analysis of dsTc015921 identified 248 DEG at 24 h and 752 genes at 72 h and 580 genes at 120 h after dsRNA injection. We found cuticular protein significantly downregulated in all the time points. Furthermore, western blot identified changes in acetylation of Histone H4 status in Tc015921 knockdown larvae. Together, our result suggests that TC015921 affects the acetylation of histone, resulting in a change in chromatin status and expression of genes involved in development and metamorphosis.

N端乙酰化（N-terminal acetylation）是一种普遍存在的蛋白质修饰方式，从酵母到人类均保守，具有广泛的生物学功能。针对组蛋白（histone）及其他蛋白质的大量研究揭示了共翻译修饰与转录后修饰的作用，以及它们对染色质折叠及相关基因活性的影响。本研究以模式昆虫赤拟谷盗（Tribolium castaneum）为实验材料，探究单一组蛋白乙酰转移酶（histone acetyltransferase, HAT）在激素信号通路中的作用，并对多种HAT基因开展了RNA干扰（RNA interference, RNAi）介导的基因敲低实验。本研究证实，Nα-乙酰转移酶（N-alpha-acetyltransferase）Tc015921（NAA40）在昆虫变态发育与生长过程中发挥重要作用。Tc015921 mRNA的发育表达谱显示，其在末龄幼虫后期及蛹早期的转录水平显著上调。敲低Tc015921基因可导致幼虫出现背部开裂的表型。蜕皮激素类似物20-羟基蜕皮酮（20E）可诱导TcA细胞中Tc015921的表达。在末龄幼虫中敲低Tc015921基因，可抑制参与蜕皮激素信号通路的蛋白质编码基因的表达。对注射靶向Tc015921的双链RNA（dsTc015921）后的样本进行RNA测序（RNA-seq）分析发现，注射后24小时可检测到248个差异表达基因（differentially expressed genes, DEG），72小时为752个，120小时为580个。我们发现，在所有检测时间点，表皮蛋白的转录水平均显著下调。此外，蛋白质免疫印迹（Western blot）实验显示，Tc015921基因敲低的幼虫体内组蛋白H4（histone H4）的乙酰化水平发生了改变。综上，本研究结果表明，Tc015921可通过影响组蛋白乙酰化水平，改变染色质状态，进而调控发育与变态相关基因的表达。