Characterization of DNA methylation reader proteins of Arabidopsis thaliana

In plants, cytosine DNA methylation (mC) is largely associated with transcriptional repression of transposable elements, but it can also be found in the body of expressed genes, referred to as gene body methylation (GbM). GbM is correlated with ubiquitously expressed genes, however its function, or absence thereof, is highly debated. The different output that mC can have raises questions as to how it is interpreted - or read - differently in these sequence and genomic contexts. To screen for potential mC binding proteins, we performed an unbiased DNA affinity pull-down assay combined with quantitative mass spectrometry using methylated DNA probes for each DNA sequence context. All mC readers known to date were found to preferentially bind to the methylated probes, along with a range of new mC binding protein candidates. Functional characterization of these mC readers, focused on the MBD and SUVH families, was undertaken by ChIP-seq mapping of genome-wide binding sites, their protein interactors, and the impact of high-order mutations on transcriptomic and epigenomic profiles. Together, this highlighted specific context preferences for these proteins, and in particular the ability of MBD2 to bind specifically to GbM. This comprehensive analysis of Arabidopsis mC readers emphasizes the complexity and interconnectivity between DNA methylation and chromatin remodelling processes in plants.

在植物中，胞嘧啶DNA甲基化（mC）主要与转座因子的转录沉默相关，但也可存在于表达基因的基因体区域，这类修饰被称为基因体甲基化（GbM）。基因体甲基化（GbM）与广谱表达基因呈正相关，然而其功能或其缺失的生物学意义仍存在广泛争议。mC所介导的不同生物学效应引发了一个核心问题：在不同的序列与基因组背景下，细胞如何对其进行差异化识别（或称"读取"）。为筛选潜在的mC结合蛋白，我们针对每一种DNA序列背景设计甲基化DNA探针，结合无偏倚DNA亲和下拉实验与定量质谱技术开展了筛选。实验不仅检测到所有目前已报道的mC读取蛋白均优先结合甲基化探针，还筛选得到一批全新的mC结合蛋白候选分子。我们针对MBD家族与SUVH家族的mC读取蛋白开展了功能表征：通过染色质免疫共沉淀测序（ChIP-seq）绘制其全基因组结合位点图谱、鉴定其蛋白质互作伴侣，并分析高阶突变对转录组与表观基因组谱的影响。综合上述实验结果，我们明确了这些蛋白对不同甲基化背景的特异性偏好，其中尤为关键的是MBD2可特异性结合基因体甲基化（GbM）区域。本次针对拟南芥mC读取蛋白的系统性分析，揭示了植物中DNA甲基化与染色质重塑过程之间的复杂性与相互关联性。