MOF haploinsufficiency triggers diet-induced obesity resistance (ChIP-seq). MOF haploinsufficiency triggers diet-induced obesity resistance (ChIP-seq)

Constant crosstalk between epigenetic regulators and metabolism homeostasis ensures that several tissues can respond and adapt to environmental cues. Decreased levels of the lysine acetyltransferase (KAT), MOF, was recently associated with cerebral development and syndromic intellectual disability. However, the consequences of having a chronic reduction of MOF levels are still unveiled. Here we characterized by FIA-MS/MS the metabolic profile of Mof heterozygous animals. We generated the profile of 7 different organs that have distinct metabolic demands. Overall our analysis reveled that Mof heterozygous mice have impaired glucose homeostasis, fatty acids metabolism, and amino acid accumulation. Furthermore, when exposed ad libitum to high-fat diet those animals failed to gain fat mass, while the lean mass remains unalterable. Accordingly, at the molecular level, using the adipocyte model we found that Mof regulates the expression of PPARs and Slc2a4. In summary, we identified the first KAT that shows high-fat diet resistance and we propose that the chronic reduction of Mof has a strong impact on metabolic disorders.​ Overall design: Experimental design: Visceral white adipocyte (WAT) from 2 control and 3 Mof +/- was extracted. WAT of 8 week old SD treated animals was minced and transferred into a Dounce homogenizer (loose pestle), covered with 1% formaldehyde in PBS, and dissociated, followed by 15 min incubation at room temperature (RT). During incubation, the tissue suspension was filtered using a nylon 70 µM cell strainer (Falcon, 352350). 125 mM glycine final was added to the sample, incubated at RT for 5 min and the suspension was pelleted for 5 min at 500 g. Cell pellets were washed twice in ice cold PBS and pelleted for 5 min at 500 g. For NEXON based nuclei isolation cell pellets were resuspended in 1ml of lysis buffer (10mM Tris-HCl pH 8, 10mM NaCl, 0.2% Igepal, protease inhibitor cocktail. Cell suspension was transferred into Covaris MilliTube (cat. No. 520130) and sonicated in Covaris instrument (E220) for 30 sec at peak power 75W, duty factor 10% and 200 cycles/burst. Nuclei were pelleted at 1000g at 20°C for 5min. Pellets were resuspended in 0.5% SDS and incubated at RT for 10min. Sample was diluted 1:5 in the digestion buffer (1.1% Triton, 1x CutSmart buffer , H2Odest). Up to 1*10^6 nuclei were digested with Cvik1 (5U/100.000 cells) at 25°C for 17hrs, shaking at 800rpm. Nuclei were pelleted for 5min at 1000g and washed in a nuclei wash solution (10mM Tris-HCl pH 8, 0.25% Triton, 0.2mg/ml BSA ) until residual fat was removed. Nuclei were pelleted at 5000g for 10min, resuspend in the shearing buffer (10mM Tris-HCl pH 8, 0.1% SDS, 1mM EDTA), transferred into Covaris MicroTube (cat. No. 520052) and sonicated for 6min at peak power 105W, duty factor 2% and 200 cycles/burst. Sample was diluted 1:6 in 1x iC1 buffer (iDeal ChIP-seq, Diagenode) and incubated with 2ug of antibody o/n at 4°C. The next day protein G Dynabeads were added and incubated for 3 hrs at 4°C. Beads were washed and DNA was eluted according to manufacturer's instructions (iDeal ChIP-seq, Diagenode). DNA was purified using MinElute PCR purification kit (Qiagen) for further analysis.

表观遗传调控因子与代谢稳态之间持续的串扰，可确保多种组织响应并适应环境信号。近期研究发现，赖氨酸乙酰转移酶（lysine acetyltransferase, KAT）MOF的水平降低与大脑发育及综合征性智力障碍密切相关。然而，MOF水平长期降低所产生的具体后果尚未被阐明。本研究通过FIA-MS/MS（流动注射-串联质谱）对Mof杂合小鼠的代谢谱进行了系统表征，成功获取了7种具有差异化代谢需求的器官的代谢特征。整体分析结果显示，Mof杂合小鼠存在葡萄糖稳态受损、脂肪酸代谢异常以及氨基酸蓄积的表型。此外，当这些小鼠自由进食高脂饲料时，无法正常增加脂肪质量，而瘦体重则保持稳定。在分子层面，本研究通过脂肪细胞模型进一步证实，MOF可调控过氧化物酶体增殖物激活受体（Peroxisome Proliferator-Activated Receptors, PPARs）及溶质载体家族2成员4（Solute Carrier Family 2 Member 4, Slc2a4）的表达。综上，本研究鉴定出首个具有高脂饮食抵抗性的KAT，并提出MOF的长期缺失会对代谢紊乱产生显著影响。

整体实验设计：提取2只野生型对照小鼠与3只Mof+/-杂合小鼠的内脏白色脂肪组织（white adipose tissue, WAT）。将8周龄SD（Sprague-Dawley）品系处理小鼠的WAT剪碎后转移至玻璃匀浆器（粗研棒），加入PBS配制的1%甲醛溶液进行固定，随后解离组织，室温孵育15分钟。孵育过程中，使用70 μm尼龙细胞滤器（Falcon, 352350）过滤组织悬液。向样品中加入终浓度为125 mM的甘氨酸以终止固定，室温孵育5分钟后，以500 g离心5分钟收集细胞沉淀。使用预冷PBS洗涤细胞沉淀两次，再以500 g离心5分钟收集沉淀。
采用NEXON法分离细胞核：将细胞沉淀重悬于1 ml裂解缓冲液（10 mM Tris-HCl pH 8、10 mM NaCl、0.2% Igepal、蛋白酶抑制剂混合物），转移至Covaris MilliTube（货号520130），使用Covaris E220超声仪进行超声破碎：峰值功率75W，占空比10%，循环次数200次/爆发，超声时长30秒。以1000 g、20℃离心5分钟收集细胞核。将细胞核沉淀重悬于0.5% SDS溶液，室温孵育10分钟。将样品用消化缓冲液（1.1% Triton、1× CutSmart缓冲液、去离子水）按1:5的比例稀释。取至多1×10^6个细胞核，加入Cvik1酶（5U/100,000个细胞），25℃振荡孵育17小时，振荡速率为800 rpm。以1000 g离心5分钟收集细胞核，使用细胞核洗涤液（10 mM Tris-HCl pH 8、0.25% Triton、0.2 mg/ml BSA）洗涤直至残留脂肪完全去除。以5000 g离心10分钟收集细胞核，重悬于剪切缓冲液（10 mM Tris-HCl pH 8、0.1% SDS、1 mM EDTA），转移至Covaris MicroTube（货号520052），使用超声仪进行第二次超声破碎：峰值功率105W，占空比2%，循环次数200次/爆发，超声时长6分钟。将样品用1× iC1缓冲液（iDeal ChIP-seq试剂盒，Diagenode）按1:6的比例稀释，加入2 μg对应抗体，4℃孵育过夜。次日加入Protein G Dynabeads，4℃孵育3小时。按照试剂盒说明书（iDeal ChIP-seq试剂盒，Diagenode）对磁珠进行洗涤并洗脱DNA。使用MinElute PCR纯化试剂盒（Qiagen）纯化DNA，用于后续实验分析。