Beckwith-Wiedemann syndrome and associated tumors - Identification of factors essential for imprinting mechanisms

Beckwith-Wiedemann综合征（BWS）是一种与染色体11p15.5区相关的复杂遗传疾病，基因组印记在其中起着重要作用。迄今为止，已经描述了许多致病的分子机制，这些机制要么是低频基因突变，要么是两个印迹簇（IC1和IC2）的主要表观遗传改变。后者包括我们和其他人最近在家族性BWS病例中描述的母体H19/IGF2印记控制区（ICR1）的微缺失。这些微缺失与印迹缺失（LOI）和IGF2转录增加有关。然而，我们能够证明这种意向书不足以引起BWS。基于我们对先前研究时期ICR1微缺失家族成员的研究结果，以及在一般表观遗传学数据的背景下，我们提出了一个模型，该模型解释了ICR1如何依赖于dna -蛋白质和增强子-启动子相互作用来调节H19和IGF2的转录。根据我们的建议，我们希望通过定义IC1中能够引起BWS和相关肿瘤的表观遗传元件的组合和相互作用来测试该模型。我们计划使用细胞模型分析，改进的下拉和染色质构象捕获技术。我们还将通过异种移植物方法检验IGF2临界阈剂量对BWS/WT发生的假设。由于印迹可能是作为一种普遍的机制进化而来，我们同时寻找印迹介导的反式因子，这些反式因子能够结合Angelman综合征（as）患者15号染色体上明确的调节区域ASSRO，以测试它们在as区域以及BWS相关基因组区域介导印迹的能力。最初的实验揭示了两种有希望的候选蛋白质。这些AS-SRO结合的推定转录因子将进一步表征其印迹调节能力。这些努力将导致进一步定义特定的BWS引起机制，并描述与一般表观遗传调控相关的dna元件以及与它们结合的蛋白质因子。

Beckwith-Wiedemann syndrome (BWS) is a complex genetic disorder associated with chromosomal region 11p15.5 in which genomic imprinting plays an essential role. To date a number of causative molecular mechanisms have been described, that either are at a low frequency gene mutations, or for the main part epigenetic alterations in two imprinting clusters (IC1 and IC2). The latter includes microdeletions in the maternal H19/IGF2 imprinting control region (ICR1) recently described by others and us in familial BWS cases. These microdeletions are associated with loss of imprinting (LOI) and transcriptional increase of IGF2. Yet we were able to demonstrate that this LOI is not sufficient to cause BWS. Based on our results from the study of family members with microdeletions in ICR1 from the previous research period and in the context with general epigenetic data, we propose a model that explains how ICR1 regulates transcription of H19 and IGF2 relying on DNA-protein- and enhancer-promoter-interactions. With our proposal we want to test this model by defining combinations and interactions of epigenetic elements in IC1, that are able to cause BWS and associated tumors. We plan to use cellular model assays, modified pull-down and chromatin conformation capture techniques. We will also test the hypothesis of a critical IGF2 threshold dosage for BWS/WT genesis by a xenograft approach. Since imprinting probably evolved as a general mechanism, we searched in parallel for imprinting mediating trans-factors that are able to bind the well defined regulatory region ASSRO in Angelman syndrome (AS) patients on chromosome 15, to test them for the ability to mediate imprinting in the AS area as well as in the BWS associated genomic region. Initial experiments revealed two promising candidate proteins. These AS-SRO binding putative transfactors will be further characterized in respect to its imprinting regulatory ability. The efforts should result in further definition of specific BWS causing mechanisms and the description of DNA-elements relevant for general epigenetic regulation as well as protein factors that bind to them.