Hydroxy-Sensitive Cut Counting (HSCC); simultaneous, genome-wide mapping of 5-methylcytosine and 5-hydroxymethylcytosine in mammals

从一个受精卵细胞开始，人类基因组包含了创造人类所必需的所有指令（基因）。虽然在所有细胞中都存在相同的基因组序列，但在人体中存在数百种明显不同的细胞类型，例如脑（神经元）、肾和肝细胞。每种细胞类型必须“开启”不同的基因子集，才能使每种组织类型正常运作。细胞标记基因开启或关闭的主要机制被称为“表观遗传学”。表观遗传学是指基因表达的可遗传变化，这种变化不是由潜在DNA序列的突变引起的。哺乳动物中研究最全面的表观遗传标记是DNA甲基化，它涉及到一个被称为“甲基”的标签分子附着在胞嘧啶上，产生5-甲基胞嘧啶（5mC）。它通常发生在CpG二核苷酸的背景下，尽管非CpG甲基化发生在胚胎干细胞中，但在哺乳动物中，60-90%的CpG甲基化。DNA甲基化与基因沉默有关，对正常发育至关重要。基因组印迹、x染色体失活、重复元件抑制、基因表达稳定性和致癌等关键过程依赖或涉及DNA甲基化模式的动态变化。从这个角度来看，了解修饰DNA在基因组中的位置是很重要的。我们对哺乳动物DNA甲基化的了解大多是基于一套可以区分5mC和未甲基化胞嘧啶(C)的技术。这些方法包括使用对甲基敏感的限制性内切酶（可以切割未甲基化但不能甲基化的DNA）和亚硫酸酯测序，这种方法可以同时精确量化几个相邻胞嘧啶的甲基化水平。这一分析被一种新型修饰DNA——5-羟甲基胞嘧啶（5hmC）的鉴定所混淆，这种DNA在哺乳动物组织中含量很高。事实上，5hmC在小鼠大脑样本中的含量是5mC的40%。尽管对DNA甲基化进行了近30年的深入研究，但5hmC在哺乳动物组织中的存在一直被忽视，特别是大多数用于鉴定甲基化的技术都不能区分5mC和5hmC。我们提出了一种激进的新技术，称为羟基敏感切割计数（HSCC），以同时分析在小鼠和人类基因组中分别存在的150 - 230万个CCGG序列中的5mC， 5hmC和C。HSCC是基于观察到的限制性内切酶MspI，如果其内部C是5羟甲基胞嘧啶，则可以切割其靶位点CCGG，但如果它是β -葡萄糖基-5羟甲基胞嘧啶（ghmC）则不能。使用一种特性良好且市售的T4噬菌体β -葡萄糖基转移酶，我们将把基因组中的所有5hmC转化为ghmC，并在MspI处理前后消化样品。然后使用下一代测序对每个MspI位点周围的序列进行测序。每个MspI位点处理过的和未处理过的样品中序列的数量是对存在的5羟甲基胞嘧啶量的测量，允许全基因组5hmC水平的半定量。得到的5hmC图谱将代表组织的“标识符”；一种条形码，可以读出正常组织状态。HSCC代表了对现有的“全基因组”技术的巨大改进，该技术使用抗体收集含有5hmC的DNA。这种“亲和”技术只能测定含有5hmC的基因组区域，并且经常表现出强烈的序列-上下文偏差。相比之下，无论5hmC水平如何，HSCC都会检测MspI位点。这是至关重要的，因为如果我们要了解这个令人兴奋的新标记在哺乳动物生物学中的作用，知道5hmC在哪里耗尽与知道它在哪里富集同样重要。

The human genome contains all the instructions (genes) necessary to create a human being, beginning from a single fertilised egg cell. Although the same genome sequence is present in all cells, there are hundreds of markedly different cell types, such as brain (neurons), kidney, and liver cells, in the human body. Each cell type must 'turn on' a different subset of genes for the correct functioning of each tissue type. A primary mechanism by which cells mark genes to be turned on or off is called, 'epigenetics'. Epigenetics refers to heritable changes in gene expression that are not caused by mutations in the underlying DNA sequence. The most comprehensively studied epigenetic mark in mammals is DNA methylation which involves the attachment of a tag-molecule called a 'methyl group' to cytosines to give 5-methylcytosine (5mC). It typically occurs in a CpG dinucleotide context, although non-CpG methylation occurs in embryonic stem cells, between 60-90% of all CpGs are methylated in mammals. DNA methylation is associated with gene silencing and is essential for normal development. Key processes including genomic imprinting, X-chromosome inactivation, suppression of repetitive elements, stability of gene expression and carcinogenesis either depend or involve dynamic changes in DNA methylation patterns. From this perspective it is important to know where modified DNA resides in the genome.Much of what we know about DNA methylation in mammals is based on a set of techniques which can distinguish between 5mC and an unmethylated cytosine (C). These include the use of methyl-sensitive restriction enzymes (which can cut unmethylated, but not methylated DNA) and bisulfite sequencing, a method that allows accurate quantification of methylation levels at several neighbouring cytosines simultaneously. This analysis has been confounded by the identification of a new type of modified DNA, 5-hydroxymethylcytosine (5hmC) that is present at high levels in mammalian tissues. Indeed, 5hmC is 40% as common as 5mC in mouse brain samples. Despite intense study of DNA methylation for the last 30 years, the presence of 5hmC in mammalian tissues had been missed especially as most the techniques used to identify methylation, do not differentiate between 5mC and 5hmC. We propose a radical, new technique termed Hydroxy Sensitive Cut Counting (HSCC) to simultaneously analyse for 5mC, 5hmC and C at the 1.5 -2.3 million CCGG sequences present throughout the mouse and human genomes respectively. HSCC is based on the observation that the restriction enzyme MspI, can cut its target site, CCGG, if the internal C is 5hydroxymethylcytosine, but not if it is beta-glucosyl-5-hydroxymethylcytosine (ghmC). Using a well characterised and commercially available enzyme T4 Phage beta-glucosyltransferase, we will convert all the 5hmC in the genome to ghmC, and digest the sample before and after treatment with MspI. The sequences surrounding each MspI site will then be sequenced using next generation sequencing. The number of sequences in the treated versus untreated samples for each MspI site is a measure of the amount of 5hydroxymethylcytosine present, allowing for semi-quantification of genome-wide 5hmC levels. The resulting 5hmC profiles will represent tissue 'identifier'; a barcode that will be a read-out of a normal tissue state. HSCC represents a dramatic improvement on the existing 'genome-wide' techniques that use antibodies to harvest DNA containing 5hmC. Such 'affinity' techniques can only assay regions of the genome which contain 5hmC and often exhibit strong sequence-context biases. In contrast, HSCC will assay MspI sites regardless of 5hmC level. This is vital, as knowing where 5hmC is depleted is as important as knowing where it is enriched if we are to understand the role of this exciting new mark in mammalian biology.