Epigenetics-mediated transcription regulation in mammals

描述（由申请人提供）： DNA甲基化是一种重要的表观遗传修饰，在多种生物学过程中起着至关重要的作用。技术的进步，特别是各种基于深度测序的技术，使得以单碱基分辨率监测DNA甲基化组变化成为可能。然而，如何解释由快速积累的甲基化组数据编码的表观遗传信息仍然具有挑战性。传统上认为DNA甲基化破坏了转录因子（TF）与顺式调控区之间的相互作用，从而沉默下游靶基因的表达。然而，最近的研究，特别是我们最近的发现，表明许多TF和辅因子优先结合甲基化的DNA基序，并在某些情况下，反式激活下游基因表达，挑战目前的DNA甲基化在转录调控的范式。全面的功能甲基化位点及其相互作用的合作伙伴的识别将大大扩大蛋白质-DNA相互作用的景观在一个新的方向，并承诺在DNA甲基化的生物学作用的理解显着的进步。为了实现这些目标，我们在R 01应用程序中提出了四个具体目标。首先，我们将调查所有可能的8个碱基的DNA序列组合，以确定甲基化的序列，可以识别的人TF。将在人TF蛋白微阵列上探测甲基化DNA基序的池，并回收由微阵列上的蛋白捕获的DNA片段，并用深度测序确定其序列。其次，我们将预测这些甲基化基序中哪些可能在基因调控中发挥作用并与蛋白质相互作用。在回收群体中统计学富集的那些8聚体序列将被映射到可用的甲基化组，并检查它们是否与已知的调控区重叠。将合成合格的基序，并在蛋白质微阵列上单独探测，以鉴定其结合配偶体。第三，我们将预测负责甲基化DNA结合的蛋白质结构域。将比较来自相同TF亚家族的序列，并且可以最好地分离具有和不具有甲基化结合活性的蛋白质的位置将是甲基化结合的候选者。将通过定点诱变结合凝胶位移和基于细胞的荧光素酶测定来测试预测。最后，我们将使用体外和体内哺乳动物轴突再生模型来研究新发现的mCpG依赖性TF-DNA相互作用的生理作用。该目的提供的积极结果不仅将揭示哺乳动物轴突再生的新表观遗传机制，而且还提供了概念验证证据，即mCpG依赖性TF-DNA相互作用是基因表达的生理调节剂。

DESCRIPTION (provided by applicant): DNA methylation is an important epigenetic modification that plays crucial roles in multiple biological processes. Technical advance, especially a variety of deep sequencing-based techniques, have made it possible to monitor DNA methylome changes at a single-base resolution. However, how to interpret the epigenetic information encoded by the fast accumulating methylome data is still challenging. DNA methylation is traditionally considered to disrupt the interactions between transcription factors (TFs) and cis-regulatory regions and thus, silence the expression of downstream target genes. However, recent studies, especially our recent discoveries, suggest that many TFs and co-factors preferentially bind to methylated DNA motifs and, in some cases, transactivate downstream gene expression, challenging the current paradigm of DNA methylation in transcription regulation. Identification of comprehensive sets of functional methylation sites and their interacting partners will greatly expand the protein-DNA interaction landscape in a new direction and promise significant advances in the understanding of the biological roles of DNA methylation. To achieve these goals, we propose four specific aims in this R01 application. First, we will survey all possible 8-base DNA sequence combinations to identify methylated sequences that can be recognized by human TFs. A pool of methylated DNA motifs will be probed on the human TF protein microarrays and the DNA fragments that are captured by the proteins on the microarrays will be recovered and their sequences determined with deep-sequencing. Second, we will predict which of these methylated motifs are likely to play a role in gene regulation and interact with proteins. Those 8-mer sequences that are statistically enriched in the recovered population will be mapped to the available methylomes and examine whether they overlap with the known regulatory regions. The qualified motifs will be synthesized and individually probed on the protein microarrays to identify their binding partners. Third, we will predict the protein domains that are responsible fo methylated DNA binding. The sequences from the same TF subfamilies will be compared and the positions that can best separated the proteins with and without methylated binding activities will be the candidates for methylation binding. The prediction will be tested by site-directed mutagenesis coupled with gel shift and cell-based luciferase assays. Finally, we will use both in vitro and in vivo models of mammalian axon regeneration to investigate the physiological roles of newly identified mCpG-dependent TF-DNA interactions. The positive results provided by this Aim will not only reveal novel epigenetic mechanisms of mammalian axon regeneration, but also provide proof-of-concept evidence that mCpG-dependent TF-DNA interactions are physiological regulators of gene expression.