Downregulation of MLF1 safeguards cardiomyocytes against senescence-associated chromatin opening

Previous studies found that MLF1 mRNA levels tended to decline in aging hearts. Functional studies found that knockdown of MLF1 ameliorated the drug-induced cellular senescence phenotype. Here, we apply CUT&Tag to explore the underlying molecular mechanisms. Cardiomyocyte AC16 cells were cultured in DMEM/F12 . DMEM/F12 supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin, and 100 μg/ml streptomycin. Medium without FBS was replaced before transfection. Negative control siRNA and Mlf1 siRNA or SUZ12 siRNA were transfected into AC16 cells at ~70% confluence using jetPRIME® (Polyplus-transfection, NY, USA). After 12 h of transfection, fresh complete medium was replaced. Utilizing the Hyperactive® Universal CUT&Tag Assay Kit for Illumina (TD903), we performed CUT&Tag experiments as follows: AC16 cells were harvested at room temperature and incubated with ConA Beads for 10 minutes. They were then incubated with anti-MLF1 (ABclonal, A13329), anti-SUZ12 antibody (CST, 3737S, 1:50), anti-EP300 (CST, #57625), anti-H3K27ac (CST, #4353S), or anti-H3K27me3 (CST, #9733) antibodies overnight at 4°C. On the next day, the samples were incubated with a secondary antibody at room temperature for 1h, followed by incubation with pA/G-Tnp for 1h. After DNA purification with the MinElute kit, and the PCR was performed to amplify the library for 12 cycles and libraries were purified with the MinElute kit. Amplified libraries were sequenced on the Illumina Novaseq 6000 platform.

既往研究显示，衰老心脏中髓系白血病因子1（MLF1）的mRNA水平呈下降趋势。功能研究证实，敲低MLF1可改善药物诱导的细胞衰老表型。本研究采用CUT&Tag技术探索其潜在分子机制。实验以AC16心肌细胞为研究对象，采用DMEM/F12培养基进行培养，该培养基添加有10%胎牛血清（FBS, Gibco, 美国）、100 U/mL青霉素及100 μg/mL链霉素。转染前更换无FBS培养基。当细胞汇合度达到约70%时，使用jetPRIME®（Polyplus-transfection, 美国纽约）将阴性对照siRNA、MLF1 siRNA或SUZ12 siRNA转染至AC16细胞中。转染12小时后，更换新鲜完全培养基。本研究使用适配Illumina平台的Hyperactive®通用CUT&Tag检测试剂盒（货号TD903）开展CUT&Tag实验，具体步骤如下：室温下收集AC16细胞，与刀豆蛋白A（ConA）磁珠孵育10分钟；随后分别与抗MLF1抗体（ABclonal, A13329）、抗SUZ12抗体（CST, 3737S, 稀释比例1:50）、抗EP300抗体（CST, #57625）、抗组蛋白H3K27乙酰化（H3K27ac）抗体（CST, #4353S）或抗组蛋白H3K27三甲基化（H3K27me3）抗体（CST, #9733）在4℃下孵育过夜。次日，样本于室温下与二抗孵育1小时，随后与蛋白A/G融合转座酶（pA/G-Tnp）孵育1小时。使用MinElute试剂盒完成DNA纯化后，通过PCR扩增文库12个循环，再使用MinElute试剂盒纯化文库。最终将扩增得到的文库在Illumina NovaSeq 6000平台上进行测序。