Effects of Cytosine Modifications on Transcription Factor Binding

转录因子序列特异性DNA结合是调控基因表达的基础。最近的两个观察拓宽了哺乳动物TFs序列特异性DNA结合的词典：第一个是甲基化胞嘧啶（5mC）被10 - 11易位双加氧酶家族迭代氧化为5-羟甲基胞嘧啶（5hmC）， 5-甲酰胞嘧啶（5fC）和5-羧胞嘧啶（5caC）。5fC和5caC被胸腺嘧啶DNA糖基化酶去除，完成DNA去甲基化循环。然而，在一些组织中发现了5hmC的积累，这表明它可能具有调节功能。第二个发现是5mC可以出现在CG二核苷酸之外，最初在干细胞中被观察到，后来在大脑中被观察到。已知CG二核苷酸中的5mC可抑制或增强B-ZIP蛋白的DNA结合。cAMP反应元件结合蛋白（CREB1）通过结合其共识CRE序列（TGAC'GTCA）及其变体，包括CRE/CEBP嵌合位点（TGAC'GCAA），调控细胞基因的表达。CRE基序中心的CG二核苷酸的双链胞嘧啶甲基化抑制CREB1结合，导致基因表达抑制。相反，C/EBP基序TTGC'GCAA中中心CG二核苷酸的甲基化增加了C/EBP家族成员的DNA结合。虽然已经探索了CG二核苷酸中5mC和5hmC对转录因子序列特异性DNA结合的影响，但尚未研究它们在CG二核苷酸外对DNA结合的影响。B-ZIP家族转录因子具有两部分结构，亮氨酸拉链区介导二聚化，n端碱性区介导序列特异性DNA结合。详细研究了亮氨酸拉链结构域介导的B-ZIP二聚化特异性和稳定性的结构决定因素。然而，B-ZIP碱基区氨基酸序列与其结合的DNA序列之间的关系，包括那些带有修饰胞嘧啶的DNA序列，尚未被探索。T7 DNA聚合酶可以在双链单链DNA时有效地将5mC和5hmC结合到DNA中。我们利用这一特性，在Agilent微阵列上使用5mC或5hmC对双链单链DNA进行60-mers处理，创建了包含5mC和5hmC不对称分布的双链DNA序列，模拟了体内几种细胞类型中发生的情况。使用这些蛋白质结合微阵列，在两条DNA链上都含有胞嘧啶，或者在一条链上含有胞嘧啶与另一条链上的5mC或5hmC的组合，我们开始检测除CREB1外，许多B-ZIP和螺旋环-螺旋转录因子的序列特异性结合：即ATF1 C/EBP， JUND, TCF4， USF1和ZTA。

Sequence-specific DNA binding of transcription factors (TFs) is the foundation of regulated gene expression. Two recent observations have broadened the lexicon of sequence-specific DNA binding of mammalian TFs: the first is that methylated cytosines (5mC) are iteratively oxidized by the ten-eleven-translocation family of dioxygenases to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). 5fC and 5caC are removed by thymine DNA glycosylase, completing the DNA demethylation cycle. However, 5hmC is found accumulatesd in several tissues suggesting it may have a regulatory function. The second finding is that 5mC can occur outside of CG dinucleotides, initially being observed in stem cells and later in the brain. 5mC in CG dinucleotides is known to inhibit or enhance the DNA binding of B-ZIP proteins. The cAMP response element-binding protein (CREB1) regulates expression of cellular genes by binding to its consensus CRE sequence (TGAC'GTCA) and its variants, including the CRE/CEBP chimeric site (TGAC'GCAA). Double-stranded cytosine methylation of the CG dinucleotide at the center of the CRE motif inhibits CREB1 binding, leading to suppression of gene expression. In contrast, methylation of the central CG dinucleotide in the C/EBP motif TTGC'GCAA increases DNA binding of C/EBP family members. While the effect of 5mC and 5hmC in CG dinucleotides on sequence-specific DNA binding of transcription factors has been explored, their effect outside of CG dinucleotides on DNA binding has not been examined. The B-ZIP family of transcription factors have a bipartite structure with the leucine zipper region mediating dimerization and the N-terminal basic region mediating sequence specific DNA binding. The structural determinants of leucine zipper domain mediated B-ZIP dimerization specificity and stability have been investigated in detail. However, the relationship between the amino acid sequence of the B-ZIP basic region and the DNA sequences they bind, including those with modified cytosines, has not been explored. T7 DNA polymerase can efficiently incorporate 5mC and 5hmC into DNA when double-stranding single-stranded DNA. We exploited this property to double-strand single-stranded DNA 60-mers on an Agilent microarray using 5mC or 5hmC, creating double-stranded DNA sequences containing an asymmetric distribution of 5mC and 5hmC that mimics what occurs in several cell types in vivo. Using these protein binding microarrays, containing either cytosines on both DNA strands or a combination of cytosines on one strand and 5mC or 5hmC on the second strand, we started to examined the sequence specific binding of a number of B-ZIP and helix-loop-helix transcription factors in addition to CREB1: i.e., ATF1 C/EBP, JUND, TCF4, USF1 and ZTA.