ABSTRACT

Macroautophagy/autophagy is a conserved catabolic pathway that targets cytoplasmic components for their degradation and recycling in an autophagosome-dependent lysosomal manner. Under physiological conditions, this process maintains cellular homeostasis. However, autophagy can be stimulated upon different forms of cellular stress, ranging from nutrient starvation to exposure to drugs. Thus, this pathway can be seen as a central component of the integrated and adaptive stress response. Here, we report that even brief induction of autophagy is coupled in vitro to a persistent downregulation of the expression of MAP1LC3 isoforms, which are key components of the autophagy core machinery. In fact, DNA-methylation mediated by de novo DNA methyltransferase DNMT3A of MAP1LC3 loci upon autophagy stimulation leads to the observed long-term decrease of MAP1LC3 isoforms at transcriptional level. Finally, we report that the downregulation of MAP1LC3 expression can be observed in vivo in zebrafish larvae and mice exposed to a transient autophagy stimulus. This epigenetic memory of autophagy provides some understanding of the long-term effect of autophagy induction and offers a possible mechanism for its decline upon aging, pathological conditions, or in response to treatment interventions.

Abbreviations: ACTB: actin beta; ATG: autophagy-related; 5-Aza: 5-aza-2’-deoxycytidine; BafA1: bafilomycin A₁; CBZ: carbamazepine; CDKN2A: cyclin dependent kinase inhibitor 2A; ChIP: chromatin immunoprecipitation; Clon.: clonidine; CpG: cytosine-guanine dinucleotide: DMSO: dimethyl sulfoxide; DNA: deoxyribonucleic acid; DNMT: DNA methyltransferase; DNMT1: DNA methyltransferase 1; DNMT3A: DNA methyltransferase alpha; DNMT3B: DNA methyltransferase beta; dpf: days post-fertilization; EBSS: Earle’s balanced salt solution; EM: Zebrafish embryo medium; GABARAP: GABA type A receptor associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GRO-Seq: Global Run-On sequencing; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MAP1LC3B2: microtubule-associated protein 1 light chain 3 beta 2; MEM: minimum essential medium; MEF: mouse embryonic fibroblasts; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin kinase; PBS: phosphate-buffered saline; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RT-qPCR: quantitative reverse transcription polymerase chain reaction; SQSTM1/p62: sequestosome 1; Starv.: starvation; Treh.: trehalose; ULK1: unc-51 like autophagy activating kinase 1.

 抽象的

巨自噬/自噬是一种保守的分解代谢途径，其目标是细胞质成分以自噬体依赖性溶酶体方式降解和回收。在生理条件下，这个过程维持细胞稳态。然而，不同形式的细胞应激（从营养匮乏到接触药物）都会刺激自噬。因此，该途径可以被视为综合和适应性应激反应的核心组成部分。在这里，我们报告即使是短暂的自噬诱导在体外也会与 MAP1LC3 亚型表达的持续下调相结合，而 MAP1LC3 亚型是自噬核心机制的关键组成部分。事实上，在自噬刺激下， MAP1LC3位点的从头DNA 甲基转移酶 DNMT3A 介导的 DNA 甲基化导致观察到的MAP1LC3亚型在转录水平的长期减少。最后，我们报告在暴露于瞬时自噬刺激的斑马鱼幼虫和小鼠体内可以观察到 MAP1LC3 表达的下调。这种自噬的表观遗传记忆提供了对自噬诱导的长期影响的一些理解，并提供了自噬在衰老、病理条件或治疗干预反应中下降的可能机制。

缩写： ACTB：肌动蛋白β； ATG：自噬相关； 5-氮杂：5-氮杂-2'-脱氧胞苷； BafA1：巴弗洛霉素A ₁ ； CBZ：卡马西平； CDKN2A：细胞周期蛋白依赖性激酶抑制剂2A； ChIP：染色质免疫沉淀；克隆：可乐定； CpG：胞嘧啶-鸟嘌呤二核苷酸：DMSO：二甲亚砜； DNA：脱氧核糖核酸； DNMT：DNA甲基转移酶； DNMT1：DNA甲基转移酶1； DNMT3A：DNA甲基转移酶α； DNMT3B：DNA 甲基转移酶 β； dpf：受精后天数； EBSS：厄尔平衡盐溶液； EM：斑马鱼胚胎培养基； GABARAP：GABA A 型受体相关蛋白； GABARAPL1：GABA A 型受体相关蛋白样 1； GABARAPL2：GABA A 型受体相关蛋白样 2； GAPDH：3-磷酸甘油醛脱氢酶； GRO-Seq：全局连续测序； MAP1LC3/LC3：微管相关蛋白1轻链3； MAP1LC3A：微管相关蛋白1轻链3α； MAP1LC3B：微管相关蛋白1轻链3β； MAP1LC3B2：微管相关蛋白1轻链3β2； MEM：最低必需培养基； MEF：小鼠胚胎成纤维细胞； mRNA：信使RNA； MTOR：雷帕霉素激酶的机制靶点； PBS：磷酸盐缓冲盐水； PIK3C3：磷脂酰肌醇3-激酶催化亚基3型； RB1CC1/FIP200：RB1感应线圈1； RT-qPCR：定量逆转录聚合酶链式反应； SQSTM1/p62: 隔离体 1; Starv. 饥饿的英文Treh.：海藻糖； ULK1：unc-51 样自噬激活激酶 1。