Stable unmethylated DNA demarcates expressed genes and their cis-regulatory space in plant genomes. Stable unmethylated DNA demarcates expressed genes and their cis-regulatory space in plant genomes

The genomic sequences of diverse varieties of many crop species continue to be produced at a frenetic pace. However, it remains challenging to develop complete annotations of functional genes and regulatory elements in these genomes. Here, we explore the potential to use DNA methylation profiles to develop more complete and refined annotations. Using leaf tissue in maize, we define ~100,000 unmethylated regions (UMRs) that account for 5.8% of the genome; 33,375 UMRs (1.3% of the genome) are found greater than 2 kilobase pairs from genes. UMRs are highly stable in multiple vegetative tissues and they capture the vast majority of accessible chromatin regions from leaf tissue. However, many UMRs are not accessible in leaf (leaf-iUMRs) and these represent a set of genomic regions with potential to become accessible in specific cell types or developmental stages. Leaf-iUMRs often occur near genes that are expressed in other tissues and are enriched for transcription factor (TF) binding sites of TFs that are also not expressed in leaf tissue. The leaf-iUMRs exhibit unique chromatin modification patterns and are enriched for chromatin interactions with nearby genes. The total UMRs space in four additional monocots ranges from 80-120 megabases, which is remarkably similar considering the range in genome size of 271 megabases to 4.8 gigabases. In summary, based on the profile from a single tissue, DNA methylation signatures pinpoint both accessible regions and regions poised to become accessible or expressed in other tissues. Thus, UMRs can provide powerful filters to distill large genomes down to the small fraction putative functional elements and facilitate the discovery of tens of thousands of novel candidate regulatory regions. Overall design: WGBS for 6 maize B73 leaf samples.

众多作物物种的不同品种的基因组序列正以空前迅猛的速度持续产出。然而，对这些基因组中的功能基因与调控元件进行完整注释仍是一项极具挑战性的工作。
本研究探索了利用DNA甲基化图谱（DNA methylation profile）构建更完整、精细的基因组注释的可行性。本研究以玉米叶片组织为实验材料，鉴定得到约10万个未甲基化区域（unmethylated regions, UMRs），这些区域占基因组总长度的5.8%；其中33375个UMRs（占基因组总长度的1.3%）位于距离基因2千碱基对以外的区域。
UMRs在多种营养组织中均表现出高度稳定性，且可覆盖叶片组织中绝大多数的可及染色质区域（accessible chromatin regions）。然而，部分UMRs在叶片组织中无法被检测到可及性，此类区域被命名为叶片不可及UMRs（leaf-iUMRs），它们代表了一类有望在特定细胞类型或发育阶段获得可及性的基因组区域。
叶片不可及UMRs多富集于在其他组织中表达的基因附近，且富集有同样不在叶片组织中表达的转录因子（transcription factor, TF）的结合位点。此类叶片不可及UMRs展现出独特的染色质修饰模式，且与邻近基因存在富集的染色质相互作用（chromatin interaction）。
另外4种单子叶植物的总UMRs区域跨度为80~120兆碱基（megabases, Mb）；尽管其基因组大小范围从271 Mb到4.8吉碱基（gigabases, Gb）不等，但该数值仍表现出惊人的一致性。
综上，仅基于单个组织的DNA甲基化特征，即可精准定位可及染色质区域，以及有望在其他组织中获得可及性或实现表达的基因组区域。因此，UMRs可作为高效的筛选工具，将庞大的基因组精简至仅占极小比例的推定功能元件范畴，同时助力数以万计的新型候选调控区域的发现。
实验整体设计：对6份玉米B73叶片样本开展全基因组亚硫酸氢盐测序（whole-genome bisulfite sequencing, WGBS）。