Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY, USA.
Transcription. 2021 Apr-Jun;12(2-3):72-88. doi: 10.1080/21541264.2021.1963627. Epub 2021 Aug 17.
The N-terminal methyltransferase NRMT1 is an important regulator of protein/DNA interactions and plays a role in many cellular processes, including mitosis, cell cycle progression, chromatin organization, DNA damage repair, and transcriptional regulation. Accordingly, loss of NRMT1 results in both developmental pathologies and oncogenic phenotypes. Though NRMT1 plays such important and diverse roles in the cell, little is known about its own regulation. To better understand the mechanisms governing NRMT1 expression, we first identified its predominant transcriptional start site and minimal promoter region with predicted transcription factor motifs. We then used a combination of luciferase and binding assays to confirm CREB1 as the major regulator of NRMT1 transcription. We tested which conditions known to activate CREB1 also activated NRMT1 transcription, and found CREB1-mediated NRMT1 expression was increased during recovery from serum starvation and muscle cell differentiation. To determine how NRMT1 expression affects myoblast differentiation, we used CRISPR/Cas9 technology to knock out NRMT1 expression in immortalized C2C12 mouse myoblasts. C2C12 cells depleted of NRMT1 lacked expression and were unable to proceed down the muscle differentiation pathway. Instead, they took on characteristics of C2C12 cells that have transdifferentiated into osteoblasts, including increased alkaline phosphatase and type I collagen expression and decreased proliferation. These data implicate NRMT1 as an important downstream target of CREB1 during muscle cell differentiation.
N 端甲基转移酶 NRMT1 是蛋白质/DNA 相互作用的重要调节剂，在许多细胞过程中发挥作用，包括有丝分裂、细胞周期进展、染色质组织、DNA 损伤修复和转录调控。因此，NRMT1 的缺失既导致发育病理学又导致致癌表型。尽管 NRMT1 在细胞中发挥如此重要和多样化的作用，但对其自身的调节知之甚少。为了更好地理解调控 NRMT1 表达的机制，我们首先确定了其主要转录起始位点和最小启动子区域，预测了转录因子基序。然后，我们使用荧光素酶和结合测定的组合来确认 CREB1 是 NRMT1 转录的主要调节剂。我们测试了已知激活 CREB1 的条件是否也激活 NRMT1 转录，发现 CREB1 介导的 NRMT1 表达在血清饥饿和肌肉细胞分化的恢复过程中增加。为了确定 NRMT1 表达如何影响成肌细胞分化，我们使用 CRISPR/Cas9 技术敲除永生化 C2C12 小鼠成肌细胞中的 NRMT1 表达。NRMT1 缺失的 C2C12 细胞缺乏 表达，无法沿着肌肉分化途径进行。相反，它们表现出已经转分化为成骨细胞的 C2C12 细胞的特征，包括碱性磷酸酶和 I 型胶原表达增加，增殖减少。这些数据表明 NRMT1 是肌肉细胞分化过程中 CREB1 的重要下游靶标。
