FSH-Patient Derived IPS Cells to Study the Developmental Regulation of DUX4 Trans

项目4 FSH患者来源的IPS细胞研究DUX4转录的发育调控 因此，可能形成了具有多个拷贝的无内含子DUX4基因的D4Z4重复阵列 在胚胎干细胞中表达的祖先基因的逆转录转座。我们最近的出版物和初步数据 显示D4Z4的转录本在ES细胞中很容易被检测到，使它们成为 DUX4转录调控的研究。我们的初步数据与DUX4是 在ES细胞中组成性表达，其调控在很大程度上是通过转录沉默作为细胞实现的 分化为其他组织类型。我们的假设是FSHD是由D4Z4的序列变异引起的 这会对肌肉细胞产生独特的毒性，从而破坏这一沉默过程。我们的长期目标是定义 发育过程中的正常和致病转录模式，并确定负责 FSHD基因表达模式的改变。在目标1中，我们描述了开发和 鉴定来自对照组和受FSHD影响的患者的IPS细胞，并确定表观遗传标记是否 在ES细胞中看到的D4Z4调节在IPS细胞中重现。在目标2中，实验被描述为 识别转录增强子和沉默区域，推测它们是发育的目标 因此，D4Z4基因的调控将成为治疗干预的重要靶点。我们会 确定当这些区域被移除或使用人类基因突变时转录调控如何改变 和小鼠发育模型。在目标3中，我们将修改受FSHD影响的患者的细胞以产生 具有纯合子D4Z4基因的同基因克隆和量化转录差异 这些修改的后果。在这样做的过程中，我们将从FSHD患者中产生成肌细胞，其中包含 只有非致病性的，随着IPS细胞发育的进步，才能用于自体移植 移植。

PROJECT 4 FSH-Patient Derived IPS Cells to Study the Developmental Regulation of DUX4 Transcription The D4Z4 repeat array with multiple copies of the intronless DUX4 gene likely developed as a consequence of retrotransposition of an ancestral gene expressed in ES cells. Our recent publication and preliminary data shows that transcripts from D4Z4 are readily detectable in ES cells making them an important cell type for the study of DUX4 transcriptional regulation. Our preliminary data are consistent with the idea that DUX4 is expressed constitutively in ES cells and its regulation is largely achieved by transcriptional silencing as cells differentiate into other tissue types. Our hypothesis is that FSHD is caused by sequence variations in D4Z4 that disrupt this silencing process with unique toxicity to muscle cells. Our long term goal is to define the normal and pathogenic transcription patterns during development, and identify sequences responsible for the altered pattern of gene expression in FSHD. In Aim 1 we describe experiments to develop and characterize IPS cells from controls and FSHD-affected patients, and determine if epigenetic markers of D4Z4 regulation seen in ES cells are recapitulated in IPS cells. In Aim 2, experiments are described to identify transcriptional enhancer and silencing regions that are presumably the targets of developmental regulation of the D4Z4 locus, and therefore will be important targets for therapeutic intervention. We will determine how transcriptional regulation is altered when these areas are removed or mutated using human and mouse developmental models. In Aim 3 we will modify cells from FSHD-affected patients to produce isogenic clones with homozygous D4Z4 genotypes and quantify transcription differences that result as a consequence of these modifications. In so doing we will produce myoblasts from FSHD patients that contain only non-pathogenic an-ays and with advances in IPS cell development, could be used for autologous transplantation.