Transcription factor occupancy can mediate active turnover of DNA methylation at regulatory regions.
Transcription factor occupancy can mediate active turnover of DNA methylation at regulatory regions.
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DOI：
10.1371/journal.pgen.1003994
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发表时间：
2013
期刊：
影响因子：
4.5
通讯作者：
Schübeler D
中科院分区：
文献类型：
--
作者：
Feldmann A;Ivanek R;Murr R;Gaidatzis D;Burger L;Schübeler D
远端调控元件，包括增强子，在调控基因活性中起关键作用。转录因子与这些元件的结合与低甲基化区域（LMR）在一个过程中，这是知之甚少。在这里，我们问是否以及如何实际占用的DNA结合因子与DNA甲基化在单个分子的水平。以CTCF为例，我们观察到结合频率与去甲基化状态的可能性相关，并且CTCF基序内的低占据位点显示异质性DNA甲基化。结合和DNA甲基化周转的动态模型，我们发现，5-羟甲基胞嘧啶（5 hmC），形成的活性去甲基化的中间状态，在干细胞和体细胞的LMR富集。此外，在分化过程中，5 hmC的显著变化发生在这些区域，这表明转录因子活性可能是主动去甲基化的关键驱动因素。由于CTCF的缺失对胚胎干细胞是致命的，我们使用REST的遗传缺失作为另一个与LMR形成有关的DNA结合因子来验证这一假设。REST的缺乏导致羟甲基化的减少和伴随的DNA甲基化在其结合位点的增加。这些数据支持一种模型，其中DNA结合因子可以介导DNA甲基化的周转，作为调控区的维持和重编程的一个组成部分。细胞身份由差异基因表达决定，而差异基因表达又受近端和远端调控元件（如增强子）的组合活性控制。活性增强子元件内的DNA由于转录因子（TF）结合而被标记为低甲基化状态。在这里，使用CTCF作为DNA结合因子的例子，我们通过富集CTCF占据的DNA，在单分子水平上探索结合和DNA甲基化之间的关系。令我们惊讶的是，CTCF结合的分子的甲基化与全基因组亚硫酸氢盐测序确定的结合位点的平均甲基化水平没有差异。我们发现结合强度与CTCF基序内的DNA甲基化呈负相关，在低占有率位点具有异源甲基化水平，这表明CTCF可以与具有不同甲基化状态的分子结合。此外，我们观察到富集的5-羟甲基胞嘧啶在组成和细胞类型特异性TF结合位点指示一个积极的去甲基化过程。为了测试TF结合对所观察到的羟甲基化的要求，并且由于CTCF缺失与胚胎干细胞的存活不相容，我们使用了其中REST（先前显示参与LMR形成的因子）被遗传缺失的细胞。这种缺失导致其结合位点的羟甲基化丧失，表明结合是周转所必需的。我们的数据支持一个模型，其中TF占用介导的DNA甲基化的持续营业额在维护和形成的活性调控区。
Distal regulatory elements, including enhancers, play a critical role in regulating gene activity. Transcription factor binding to these elements correlates with Low Methylated Regions (LMRs) in a process that is poorly understood. Here we ask whether and how actual occupancy of DNA-binding factors is linked to DNA methylation at the level of individual molecules. Using CTCF as an example, we observe that frequency of binding correlates with the likelihood of a demethylated state and sites of low occupancy display heterogeneous DNA methylation within the CTCF motif. In line with a dynamic model of binding and DNA methylation turnover, we find that 5-hydroxymethylcytosine (5hmC), formed as an intermediate state of active demethylation, is enriched at LMRs in stem and somatic cells. Moreover, a significant fraction of changes in 5hmC during differentiation occurs at these regions, suggesting that transcription factor activity could be a key driver for active demethylation. Since deletion of CTCF is lethal for embryonic stem cells, we used genetic deletion of REST as another DNA-binding factor implicated in LMR formation to test this hypothesis. The absence of REST leads to a decrease of hydroxymethylation and a concomitant increase of DNA methylation at its binding sites. These data support a model where DNA-binding factors can mediate turnover of DNA methylation as an integral part of maintenance and reprogramming of regulatory regions. Cell identity is determined by differential gene expression, which in turn is controlled by the combined activity of proximal and distal regulatory elements such as enhancers. DNA within active enhancer elements is marked by a hypomethylated state as a result of transcription factor (TF) binding. Here, using CTCF as an example for a DNA-binding factor, we explore the relationship between binding and DNA methylation at the level of single molecules by enriching for CTCF occupied DNA. To our surprise, methylation at molecules which are bound by CTCF does not differ from the average methylation levels at the binding sites defined by whole-genome bisulfite sequencing. We find that binding strength inversely correlates with DNA methylation within the CTCF motif with heterogenic methylation levels at low occupancy sites, suggesting that CTCF can bind to molecules with different methylation states. Moreover, we observed enrichment of 5-hydroxymethylcytosines at constitutive and cell-type specific TF binding sites indicative of an active demethylation process. To test the requirement of TF binding for the observed hydroxymethylation, and as CTCF deletion is incompatible with the survival of embryonic stem cells, we made use of cells in which REST – a factor which was previously shown to be involved in LMR formation - was genetically deleted. This deletion leads to loss of hydroxymethylation at its binding sites, suggesting that binding is necessary for turnover. Our data support a model in which TF occupancy mediates a continuous turnover of DNA methylation during maintenance and formation of active regulatory regions.
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影响因子：
64.8
作者：
Heintzman, Nathaniel D.;Hon, Gary C.;Hawkins, R. David;Kheradpour, Pouya;Stark, Alexander;Harp, Lindsey F.;Ye, Zhen;Lee, Leonard K.;Stuart, Rhona K.;Ching, Christina W.;Ching, Keith A.;Antosiewicz-Bourget, Jessica E.;Liu, Hui;Zhang, Xinmin;Green, Roland D.;Lobanenkov, Victor V.;Stewart, Ron;Thomson, James A.;Crawford, Gregory E.;Kellis, Manolis;Ren, Bing
通讯作者：
Ren, Bing
影响因子：
14.9
作者：
Burger L;Gaidatzis D;Schübeler D;Stadler MB
通讯作者：
Stadler MB
影响因子：
7
作者：
Arnold P;Schöler A;Pachkov M;Balwierz PJ;Jørgensen H;Stadler MB;van Nimwegen E;Schübeler D
通讯作者：
Schübeler D
影响因子：
64.5
作者：
Bhutani N;Burns DM;Blau HM
通讯作者：
Blau HM
影响因子：
3.7
作者：
Globisch D;Münzel M;Müller M;Michalakis S;Wagner M;Koch S;Brückl T;Biel M;Carell T
通讯作者：
Carell T