Mai Bernard, Miles Shawna, Breeden Linda L
Fred Hutchinson Cancer Research Center, Division of Basic Sciences, Seattle, Washington 98109-1024, USA.
Mol Cell Biol. 2002 Jan;22(2):430-41. doi: 10.1128/MCB.22.2.430-441.2002.
The transcription factor Mcm1 is regulated by adjacent binding of a variety of different factors regulating the expression of cell-type-specific, cell cycle-specific, and metabolic genes. In this work, we investigate a new class of Mcm1-regulated promoters that are cell cycle regulated and peak in late M-early G(1) phase of the cell cycle via a promoter element referred to as an early cell cycle box (ECB). Gel filtration experiments indicate that the ECB-specific DNA binding complex is over 200 kDa in size and includes Mcm1 and at least one additional protein. Using DNase I footprinting in vitro, we have observed protection of the ECB elements from the CLN3, SWI4, CDC6, and CDC47 promoters, which includes protection of the 16-bp palindrome to which Mcm1 dimers are known to bind as well as protection of extended flanking sequences. These flanking sequences influence the stability and the variety of complexes that form on the ECB elements, and base substitutions in the protected flank affect transcriptional activity of the element. Chromatin immunoprecipitations show that Mcm1 binds in vivo to ECB elements throughout the cell cycle and that binding is sensitive to carbon source changes.
转录因子Mcm1受多种不同因子的毗邻结合调控，这些因子调控细胞类型特异性、细胞周期特异性和代谢基因的表达。在本研究中，我们研究了一类新的受Mcm1调控的启动子，它们受细胞周期调控，并通过一种称为早期细胞周期框（ECB）的启动子元件在细胞周期的M期末期至G1期早期达到峰值。凝胶过滤实验表明，特异性结合ECB的DNA复合物大小超过200 kDa，包含Mcm1和至少一种其他蛋白质。利用体外DNase I足迹法，我们观察到CLN3、SWI4、CDC6和CDC47启动子的ECB元件受到保护，其中包括已知Mcm1二聚体结合的16 bp回文序列以及侧翼延伸序列的保护。这些侧翼序列影响在ECB元件上形成的复合物的稳定性和多样性，并且受保护侧翼中的碱基替换会影响该元件的转录活性。染色质免疫沉淀表明，Mcm1在整个细胞周期中均在体内与ECB元件结合，且这种结合对碳源变化敏感。
