New embryological perspectives on imprinting disease

哺乳动物细胞通常有两组染色体，分别从母亲和父亲那里遗传来的。在大多数情况下，两个亲本基因(等位基因)具有相似的活性，但在重要的少数(约占总数的0.5%)中，从一个亲本继承的等位基因相对于从另一个亲本继承的对应等位基因是活跃的。这些基因被认为是印记的，它们的表达平衡对胚胎存活和避免疾病至关重要。产生印记基因表达的染色质标记可以采取相对稳定的基因组DNA甲基化形式，已经知道了大约30年。最近，人们发现包装基因组DNA的组蛋白也携带似乎只在植入前发育期间持续的印记；在此之后，每个亲本等位基因的表达变得相等。我们最近通过结合亲本歧视单个胚胎基因组甲基化和基因表达分析，在小鼠中记录了这两种印记类型。这项工作证实了特征良好的印记，但表明存在多个以前没有报道的新印记区域，包括71个新的印记基因nBiX(用于新的胚泡-印记表达)。小鼠nBiX基因被修饰的组蛋白(组蛋白3中的赖氨酸27的三甲基化，H3K27me3)标记，并在植入前的囊胚期单亲表达，但在之后不久表达相同：它们是瞬时印记的。nBiX基因在发育中的大脑中高表达，几乎所有的人类同源基因都与疾病相关：许多基因参与代谢和膜调节，因此它们的失调在不同的病理中表现出来。已知印记基因的中断是致癌的，将印记基因调控与癌症联系在一起，越来越多的证据表明，早期胚胎中的表观遗传失调会导致成人疾病：例子包括核移植克隆(导致肥胖和其他病理)和代际表观遗传，在这些遗传中，父母获得的疾病特征变得可遗传。本提案旨在测试未经仔细审查的假设，即在植入前单亲表达窗口期间印记(特别是nBiX)基因表达的瞬时中断会导致疾病。我们将在高分辨率下确定当nBiX表达水平在植入前发育期间短暂中断时，哪些性状受到失调。这将通过我们开发的胚胎学工具来实现，在小鼠着床前发育的特定阶段增加或减少nBiX转录活性。从初步分析来看，这种nBiX表达的瞬时中断阻碍了胚胎发生。我们将利用分子、细胞和生理方法对nBiX中断的病理后果进行更全面的分析，因为它表现在植入前和植入后、围生期发育和成年的所有阶段。小鼠的研究将通过深入分析人类囊胚中父母特定的基因组活动，使我们能够将小鼠在疾病中的nBiX失调映射到人类的印记上。目前还没有替代可逆地提出的实验策略来消除瞬时印记基因的表达，并揭示与疾病的联系。这种印记现象是短暂而微妙的：H3K27me3可能在疾病显现之前很久就被取代了，需要新的视角来研究它。这项工作有望揭示表观遗传调控的致癌机制，并建立一种新的、易于处理的胚胎模型。更广泛地说，它将展示胚胎发育的最早事件如何影响疾病特征的表观遗传，向ART提供信息，以便它可能被调整以最大限度地减少易患疾病的表观遗传贡献。

Mammalian cells typically have two sets of chromosomes, one each inherited from the mother and the father. In most cases, both parental genes (alleles) have similar activities, but in an important minority (~0.5% of the total), the allele inherited from one parent is active relative to the corresponding allele from the other. These genes are said to be imprinted, and the balance of their expression is critical for embryonic viability and the avoidance of disease.The chromatin marks that engender imprinted gene expression can take the comparatively stable form of genomic DNA methylation and have been known for some thirty years. Recently, it has been found that histones, which package genomic DNA, also carry imprints that seem only to last during preimplantation development; after this, expression from each parental allele becomes equivalent.We recently documented both imprint types in the mouse by combining parent-discriminatory single embryo genome methylation and gene expression analyses. This work confirmed well-characterised imprints, but suggested that there exist multiple new imprinted regions that had not previously been reported, including 71 new imprinted genes called nBiX (for novel blastocyst-imprinted expressed). Mouse nBiX genes were marked by the modified histone (trimethylation of lysine 27 in histone 3, H3K27me3) and uniparentally expressed at the blastocyst stage before implantation, but equivalently expressed shortly thereafter: they are transiently imprinted.nBiX genes are highly expressed in the developing brain and almost all have human counterparts that are associated with disease: many are involved in metabolic and membrane regulation, so their dysregulation manifests in diverse pathologies. Disruption of known imprinted genes is oncogenic, linking imprinted gene regulation and cancer, and there is increasing evidence that epigenetic dysregulation in early embryos results in adult disease: examples include nuclear transfer cloning (which produces obesity and other pathologies) and inter-generational epigenetic inheritance, in which parentally-acquired disease traits become heritable.The present proposal seeks to test the unscrutinised hypothesis that transient disruption of imprinted (particularly nBiX) gene expression during the preimplantation window of uniparental expression causes disease. We will determine at high resolution which traits are dysregulated when nBiX expression levels are briefly disrupted during preimplantation development. This will be accomplished using embryological tools developed by us to increase or reduce nBiX transcript activity at specified stages in mouse preimplantation development.From preliminary analysis, such transient disruption of nBiX expression impedes embryogenesis. We will extend this to a fuller analysis of the pathological consequences of nBiX disruption using molecular, cellular and physiological approaches, as it manifests at all stages pre- and post-implantation, peri-natal development and into adulthood. The mouse studies will be informed by in-depth analysis of parent-specific genome activity in human blastocysts, allowing us to map mouse nBiX dysregulation in disease onto human imprints.There is currently no alternative experimental strategy to the one proposed reversibly to abrogate transient imprinted gene expression and reveal links to disease. The imprinting phenomenon is transient and subtle: H3K27me3 may be displaced long before disease is manifest, requiring new perspectives to study it. The work promises to reveal epigenetically-regulated mechanisms predisposing to cancer, and to establish a new and tractable embryonic model. More broadly it will show how the earliest events in embryonic development can effect epigenetic inheritance of disease traits, informing ART so that it may be adapted to minimise epigenetic contributions that predispose to disease.