Kurasawa Takeshi, Muto Akihiko, Matsumoto Mitsuyo, Ochiai Kyoko, Murayama Kazutaka, Igarashi Kazuhiko
Department of Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan.
J Biochem. 2024 Dec 2;176(6):449-459. doi: 10.1093/jb/mvae065.
Changes in the absolute protein amounts of transcription factors are important for regulating gene expression during cell differentiation and in responses to changes in the cellular and extracellular environment. However, few studies have focused on the absolute quantification of mammalian transcription factors. In this study, we established an absolute quantification method for the transcription factors BACH1 and BACH2, which are expressed in B cells and regulated by direct heme binding. The method used purified recombinant proteins as controls in western blotting and was applied to mouse naïve B cells in the spleen, as well as activated B cells and plasma cells. BACH1 was present in naïve B cells at approximately half the levels of BACH2. In activated B cells, BACH1 decreased compared to naïve B cells, whilst BACH2 increased. In plasma cells, BACH1 increased back to the same extent as in naïve B cells, whilst BACH2 was not detected. Their target genes, Prdm1 and Hmox1, were highly induced in plasma cells. BACH1 was found to undergo degradation with lower concentrations of heme than BACH2. Therefore, BACH1 and BACH2 are similarly abundant in B cells but differ in heme sensitivity, potentially regulating gene expression differently depending on their heme responsiveness.
转录因子绝对蛋白量的变化对于细胞分化过程中以及对细胞内和细胞外环境变化作出反应时的基因表达调控至关重要。然而，很少有研究聚焦于哺乳动物转录因子的绝对定量。在本研究中，我们建立了一种针对转录因子BACH1和BACH2的绝对定量方法，这两种转录因子在B细胞中表达并受直接血红素结合调控。该方法在蛋白质印迹中使用纯化的重组蛋白作为对照，并应用于小鼠脾脏中的初始B细胞、活化B细胞和浆细胞。BACH1在初始B细胞中的含量约为BACH2的一半。在活化B细胞中，与初始B细胞相比，BACH1减少，而BACH2增加。在浆细胞中，BACH1回升至与初始B细胞相同的水平，而未检测到BACH2。它们的靶基因Prdm1和Hmox1在浆细胞中被高度诱导。发现BACH1在比BACH2更低的血红素浓度下发生降解。因此，BACH1和BACH2在B细胞中的丰度相似，但在血红素敏感性方面存在差异，可能根据它们对血红素的反应性不同地调控基因表达。
