Sayeed Syed Khund, Zhao Jianfei, Sathyanarayana Bangalore K, Golla Jaya Prakash, Vinson Charles
Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Room 3128, Building 37, Bethesda, MD 20892, United States.
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Room 5120, Building 37, Bethesda, MD 20892, United States.
Biochim Biophys Acta. 2015 Jun;1849(6):583-9. doi: 10.1016/j.bbagrm.2015.03.002. Epub 2015 Mar 13.
During mammalian development, some methylated cytosines (5mC) in CG dinucleotides are iteratively oxidized by TET dioxygenases to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). The effect of these cytosine oxidative products on the sequence-specific DNA binding of transcription factors is being actively investigated. Here, we used the electrophoretic mobility shift assay (EMSA) to examine C/EBPα and C/EBPβ homodimers binding to all 25 chemical forms of a CG dinucleotide for two DNA sequences: the canonical C/EBP 8-mer TTGC|GCAA and the chimeric C/EBP|CRE 8-mer TTGC|GTCA. 5hmC in the CG dinucleotide in the C/EBP|CRE motif 8-mer TGAC|GCAA inhibits binding of C/EBPβ but not C/EBPα. Binding was increased by 5mC, 5fC and 5caC. Circular dichroism monitored thermal denaturations for C/EBPβ bound to the C/EBP|CRE motif confirmed the EMSA. The structural differences between C/EBPα and C/EBPβ that may account for the difference in binding 5hmC in the 8-mer TGAC|GCAA are explored.
在哺乳动物发育过程中，CG二核苷酸中的一些甲基化胞嘧啶（5mC）被TET双加氧酶反复氧化为5-羟甲基胞嘧啶（5hmC）、5-甲酰基胞嘧啶（5fC）和5-羧基胞嘧啶（5caC）。目前正在积极研究这些胞嘧啶氧化产物对转录因子序列特异性DNA结合的影响。在这里，我们使用电泳迁移率变动分析（EMSA）来检测C/EBPα和C/EBPβ同型二聚体与两个DNA序列的CG二核苷酸的所有25种化学形式的结合：经典的C/EBP 8聚体TTGC|GCAA和嵌合的C/EBP|CRE 8聚体TTGC|GTCA。C/EBP|CRE基序8聚体TGAC|GCAA中CG二核苷酸的5hmC抑制C/EBPβ的结合，但不抑制C/EBPα的结合。5mC、5fC和5caC可增加结合。圆二色性监测与C/EBP|CRE基序结合的C/EBPβ的热变性，证实了EMSA结果。我们还探讨了C/EBPα和C/EBPβ之间的结构差异，这些差异可能解释了在8聚体TGAC|GCAA中结合5hmC的差异。
