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区域相关的复杂遗传性疾病，其中基因组印记起着重要作用。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率的基因突变，要么是两个印记簇（IC 1和IC 2）中的主要部分表观遗传改变。后者包括最近由其他人和我们在家族性BWS病例中描述的母体H19/IGF 2印迹控制区（ICR 1）的微缺失。这些微缺失与IGF 2的印记丢失（LOI）和转录增加有关。然而，我们能够证明这一LOI不足以导致BWS。基于我们对先前研究期间ICR 1微缺失家族成员的研究结果，并在一般表观遗传学数据的背景下，我们提出了一个模型，解释了ICR 1如何依赖于DNA-蛋白质-和增强子-启动子-相互作用调节H19和IGF 2的转录。通过我们的提议，我们希望通过定义IC 1中表观遗传元件的组合和相互作用来测试这个模型，这些表观遗传元件能够导致BWS和相关肿瘤。我们计划使用细胞模型分析，修改下拉和染色质构象捕获技术。我们还将通过异种移植方法检验BWS/WT发生的关键IGF 2阈值剂量的假设。由于印迹可能演变为一种一般机制，我们平行搜索印迹介导的反式因子，能够结合明确的调控区ASSRO在Angelman综合征（AS）患者的15号染色体上，测试他们的能力，以介导的AS区域以及在BWS相关的基因组区域的印迹。最初的实验揭示了两种有希望的候选蛋白质。这些AS-SRO结合推定的转因子将进一步表征其印迹调节能力。这些努力应该导致进一步定义特定的BWS引起机制和描述的DNA相关的一般表观遗传调控以及蛋白质因子，结合到他们。

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.