Effects of Cytosine Modifications on Transcription Factor Binding

胞嘧啶修饰对转录因子结合的影响

• 批准号: 10262776
• 负责人: Stewart Durell
• 金额: $ 3.58万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10262776
• 关键词: Affect; Amino Acid Sequence; Binding; Binding Proteins; Brain; CCAAT-Enhancer-Binding Proteins; Cellular biology; Consensus; CpG dinucleotide; Cyclic AMP-Responsive DNA-Binding Protein; Cytosine; DNA; DNA Binding; DNA Sequence; DNA-Directed DNA Polymerase; Dimerization; Dioxygenases; Epigenetic Process; Family; Family member; Foundations; Gene Expression; Genes; Goals; Helix-Turn-Helix Motifs; Leucine Zippers; Mediating; Methylation; Modification; N-terminal; Oxides; Property; Response Elements; Single-Stranded DNA; Site; Specificity; Structure; TCF7L2 gene; Thymine DNA Glycosylase; Tissues; USF1 gene; Variant; Work; ZIP protein; activating transcription factor 1; cell type; demethylation; ds-DNA; in vivo; stem cells; transcription factor

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.

转录因子 (TF) 的序列特异性 DNA 结合是调控基因表达的基础。最近的两项观察结果拓宽了哺乳动物转录因子序列特异性 DNA 结合的词汇：第一个是甲基化胞嘧啶 (5mC) 被双加氧酶的 10-11 易位家族反复氧化为 5-羟甲基胞嘧啶 (5hmC)、5-甲酰胞嘧啶(5fC) 和 5-羧基胞嘧啶 (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，发现 5hmC 在多种组织中积累，表明它可能具有调节功能。第二个发现是，5mC 可以发生在 CG 二核苷酸之外，最初在干细胞中观察到，后来在大脑中观察到。 CG 二核苷酸中的 5mC 已知可抑制或增强 B-ZIP 蛋白的 DNA 结合。 cAMP 反应元件结合蛋白 (CREB1) 通过与其共有 CRE 序列 (TGAC'GTCA) 及其变体（包括 CRE/CEBP 嵌合位点 (TGAC'GCAA)）结合来调节细胞基因的表达。 CRE 基序中心 CG 二核苷酸的双链胞嘧啶甲基化会抑制 CREB1 结合，从而抑制基因表达。相反，C/EBP 基序 TTGC'GCAA 中中央 CG 二核苷酸的甲基化增加了 C/EBP 家族成员的 DNA 结合。虽然已经探讨了 CG 二核苷酸中的 5mC 和 5hmC 对转录因子的序列特异性 DNA 结合的影响，但尚未检查它们在 CG 二核苷酸之外对 DNA 结合的影响。 B-ZIP 转录因子家族具有二分结构，亮氨酸拉链区域介导二聚化，N 端碱性区域介导序列特异性 DNA 结合。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已得到详细研究。然而，B-ZIP 碱性区域的氨基酸序列和它们结合的 DNA 序列（包括那些带有修饰胞嘧啶的 DNA 序列）之间的关系尚未被探索。当双链单链DNA时，T7 DNA聚合酶可以有效地将5mC和5hmC掺入DNA中。我们利用这一特性，使用 5mC 或 5hmC 在安捷伦微阵列上形成双链单链 DNA 60 聚体，创建包含 5mC 和 5hmC 不对称分布的双链 DNA 序列，模拟体内多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，在第二条链上含有 5mC 或 5hmC，我们开始检查许多 B-ZIP 和螺旋环的序列特异性结合。除 CREB1 之外的螺旋转录因子：即 ATF1 C/EBP、JUND、TCF4、USF1 和 ZTA。