The MBD7 complex promotes expression of methylated transgenes without significantly altering their methylation status (mRNA-seq). Arabidopsis thaliana

DNA methylation is a chromatin modification that is associated with gene silencing in eukaryotic organisms. Although pathways controlling the establishment, maintenance and removal of DNA methylation have been identified, relatively little is understood about how DNA methylation influences gene expression. Using complementary genetic and biochemical approaches we identified a protein complex that antagonizes the transcriptional gene silencing of two LUCIFERASE (LUC) reporters in a manner that requires DNA methylation. At its core, this complex contains LOW IN LUCIFERASE EXPRESSION (LIL), an α-crystallin domain protein, and METHYL-CpG-BINDING DOMAIN 7 (MBD7), a protein previously associated with DNA methylation. At the LUC reporters, loss of MBD7 or LIL resulted in decreased LUC expression concomitant with modest, but reproducible increases in DNA methylation that can be phenocopied by DNA demethylase mutants. These findings are consistent with other reports and reveal a genetic connection between MBD7, LIL and DNA demethylation. However, we found that the hyper-methylation and gene expression phenotypes at a LUC reporter can be genetically uncoupled, demonstrating that changes in DNA methylation alone are not sufficient to silence LUC expression, and suggesting a role for the MBD7-LIL complex downstream of DNA methylation. Consistent with this hypothesis, our more extensive analysis of DNA methylation in mbd7 and lil mutants revealed only a small number of hyper-methylated loci genome wide. Furthermore, these loci displayed minimal overlap with demethylase targets, suggesting that, in general, the DNA demethylation machinery does not function in a manner dependent on the MBD7-LIL complex. Taken together, our findings place the MBD7-LIL complex amongst a small number of factors that regulate gene expression without causing significant changes in DNA methylation. This complex, however, is unique in that it functions to suppress, rather than enforce the silencing effects of DNA methylation, enabling gene expression of several transgene reporters despite high levels of promoter methylation. Overall design: Examination of global RNA expression in 14-day-old Col, YJ , mbd7-4, mbd7-3, LUCH lil-2, LUCH, YJ lil-1 seedlings.

DNA甲基化（DNA methylation）是真核生物中与基因沉默相关的染色质修饰方式。尽管学界已明确调控DNA甲基化建立、维持与去除的通路，但对于DNA甲基化如何影响基因表达的机制仍知之甚少。本研究通过互补的遗传与生化研究手段，鉴定出一种蛋白复合物，该复合物可通过依赖DNA甲基化的方式，拮抗两株LUCIFERASE（LUC）报告基因的转录基因沉默。该复合物的核心组分包含低LUC表达蛋白（LOW IN LUCIFERASE EXPRESSION, LIL）——一种α-晶状体蛋白结构域蛋白，以及甲基-CpG结合结构域7（METHYL-CpG-BINDING DOMAIN 7, MBD7）——一种此前被证实与DNA甲基化相关的蛋白。在LUC报告基因位点处，敲除MBD7或LIL会导致LUC表达量下降，同时伴随DNA甲基化水平的适度但可重复的升高，该表型可由DNA去甲基化酶突变体模拟。上述研究结果与其他文献报道一致，并揭示了MBD7、LIL与DNA去甲基化之间的遗传关联。然而本研究发现，LUC报告基因位点处的高甲基化与基因表达表型可发生遗传解偶联，这表明仅靠DNA甲基化水平的变化并不足以沉默LUC的表达，并提示MBD7-LIL复合物在DNA甲基化下游发挥调控功能。与该假说相符，我们对mbd7和lil突变体开展全基因组DNA甲基化的深度分析后发现，仅存在少量全基因组高甲基化位点。此外，这些高甲基化位点与去甲基化酶的靶位点仅存在极少的重叠，这提示总体而言DNA去甲基化系统的功能并不依赖于MBD7-LIL复合物。综上，本研究的发现将MBD7-LIL复合物归入为数不多的一类可调控基因表达但不会引发DNA甲基化显著变化的因子之列。不过该复合物的独特之处在于，其功能是抑制而非强化DNA甲基化的沉默效应，即使靶基因启动子区域存在高水平甲基化，仍可保障多个转基因报告基因的表达。实验整体设计：对14日龄的哥伦比亚生态型（Columbia, Col）、YJ、mbd7-4、mbd7-3、LUCH lil-2、LUCH、YJ lil-1幼苗进行全基因组RNA表达谱分析。