BRCA1 Regulates Carbohydrate Metabolism Through its RING Domain and Transcription Factor Oct1

The tumor suppressor BRCA1 regulates DNA damage responses and multiple other processes. Among these, BRCA1 heterodimerizes with BARD1 to ubiquitylate targets via its N-terminal RING domain. Here we show that BRCA1 promotes oxidative metabolism via degradation of Oct1, a transcription factor with pro-glycolytic/tumorigenic effects. BRCA1 E3 ubiquitin ligase mutation skews cells towards a glycolytic metabolic profile while elevating Oct1 protein. CRISPR-mediated Oct1 deletion reverts the glycolytic phenotype. RNAseq confirms the deregulation of metabolic genes. BRCA1 mediates direct Oct1 ubiquitylation and degradation, and mutation of two ubiquitylated Oct1 lysines insulates the protein against BRCA1-mediated destabilization. Oct1 deletion in MCF-7 breast cancer cells does not perturb growth in standard culture, but inhibits growth in soft agar and xenografts. Oct1 protein levels correlate positively with tumor aggressiveness, and inversely with BRCA1, in primary breast cancer samples. These results identify BRCA1 as an Oct1 ubiquitin ligase that catalyzes Oct1 degradation to promote oxidative metabolism. Overall design: mRNA profiles of BRCA1-I26A mutant MEFs treated with control CRISPR lentiviral vector, or an Oct1-specific CRISPR construct

肿瘤抑制因子BRCA1可调控DNA损伤应答及多种其他生物学过程。其中，BRCA1与BARD1形成异二聚体，通过其N端RING结构域对靶标蛋白进行泛素化修饰。本研究发现，BRCA1可通过降解转录因子Oct1（八聚体结合转录因子1）促进氧化代谢，而Oct1兼具促糖酵解与促肿瘤发生的功能。BRCA1的E3泛素连接酶（E3 ubiquitin ligase）发生突变后，会使细胞的代谢模式转向糖酵解型，同时升高Oct1蛋白水平。经CRISPR（成簇规律间隔短回文重复序列）介导的Oct1敲除可逆转该糖酵解表型。RNA测序（RNA-seq）结果证实了代谢相关基因的表达失调。BRCA1可直接介导Oct1的泛素化与降解，而Oct1上两个泛素化赖氨酸位点的突变可使该蛋白免受BRCA1介导的降解作用。在MCF-7乳腺癌细胞中敲除Oct1，不会影响常规培养条件下的细胞增殖，但可抑制其在软琼脂集落形成实验与异种移植瘤模型中的生长。在原发性乳腺癌临床样本中，Oct1蛋白水平与肿瘤侵袭性呈正相关，而与BRCA1表达呈负相关。上述研究结果证实，BRCA1是一种可催化Oct1降解以促进氧化代谢的Oct1泛素连接酶。实验整体设计：经对照CRISPR慢病毒载体或Oct1特异性CRISPR构建体处理的BRCA1-I26A突变小鼠胚胎成纤维细胞（MEFs，Mouse Embryonic Fibroblasts）的mRNA表达谱。