Accelerated DNA Methylation Alterations in Hutchinson-Gilford Progeria Syndrome

项目总结/摘要 Hutchinson-Gilford早老综合征（HGPS）是一种罕见的遗传性疾病， 从童年开始的过早衰老。这种情况是由核纤层蛋白LMNA突变引起的 该基因导致产生一种称为早老蛋白的显性功能获得性同种型。 早老蛋白引起核形态和功能的重大破坏，包括核蛋白输出和核蛋白质合成。 有丝分裂，复制应力，并有助于增加DNA损伤以及异染色质的损失 和其他表观遗传标记的去甲基化，包括胞嘧啶-5DNA甲基化。我们最近提供了 直接的实验证据表明，衰老相关的DNA甲基化损失在核板层连接区域 基因组的分裂是细胞分裂相关DNA复制的直接结果。我们利用这一原则， 开发了一个表观遗传有丝分裂时钟，称为dTali，它提供了细胞复制的可靠估计。 历史我们假设HGPS患者DNA甲基化模式的改变可能是HGPS患者DNA甲基化模式改变的积极因素。 与HGPS患者中观察到的严重加速老化有关。在这里，我们建议进行高- 在连续培养的HGPS细胞中进行DNA甲基化改变的分辨率分析，并研究是否 我们可以通过操纵DNA甲基化模式来延长HGPS模型系统的寿命。 在具体目标1中，我们将定义从早期到晚期连续培养的HGPS成纤维细胞中的DNA甲基化动力学。 使用具有成本效益的Infinium DNA甲基化阵列通过复制衰老。 在具体目标2中，我们将在HGPS的关键阶段进行高分辨率单细胞甲基化组分析 成纤维细胞培养以检测出现的畸变并描绘群体转变。我们已经开发出一种 单细胞全基因组亚硫酸氢盐测序（sc-WGBS）方法，其提供远为上级的基因组覆盖率 任何其他已发表的sc-WGBS方法。 在具体目标3中，我们将研究DNA甲基化操作是否可以延长HGPS的寿命。 模型在Aim 3A中，我们将测试DNA甲基化写入器和擦除器的过表达是否会增加DNA甲基化水平。 HGPS成纤维细胞的复制寿命。在目标3B中，我们将靶向DNA甲基转移酶过表达， HGPS小鼠模型中的动脉平滑肌细胞，以研究这是否会降低动脉平滑肌细胞的活性。 肌肉损失和延长寿命。我们将使用一种新的方法监测这种小鼠模型中DNA甲基化的变化。 具有成本效益的DNA甲基化阵列。 我们提出了广泛和令人信服的初步数据，证明了可行性和相关性 提出的目标。这项研究的结果可能对我们的研究产生重要影响。 了解DNA甲基化改变对HGPS表型的贡献，可能会打开 治疗早衰症的新疗法的途径。此外，这项研究可以揭示类似的 在正常老化中以较长的时间尺度运作的机制。

PROJECT SUMMARY / ABSTRACT Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder characterized by the rapid onset of premature aging beginning in childhood. The condition is caused by a mutation in the nuclear lamin LMNA gene, which leads to the production of a dominant gain-of-function isoform of the protein, called progerin. Progerin causes major disruptions to nuclear morphology and function, including nuclear protein export and mitosis, replication stress, and contributes to increased DNA damage as well as the loss of heterochromatin and dysregylation of other epigenetic marks, including cytosine-5 DNA methylation. We have recently provided direct experimental evidence that aging-associated loss of DNA methylation at nuclear lamina-attached regions of the genome is a direct consequence of cell division-associated DNA replication. We used this principle to develop an epigenetic mitotic clock, called RepliTali, which provides a reliable estimate of cellular replicative history. We hypothesize that altered DNA methylation patterns in patients with HGPS may contribute actively to the severely accelerated aging observed in patients with HGPS. Here, we propose to conduct high- resolution analyses of DNA methylation alterations in serially cultured HGPS cells and to investigate whether we can extend the lifespan of HGPS model systems by manipulating DNA methylation patterns. In Specific Aim 1 we will define DNA methylation dynamics in serially cultured HGPS fibroblasts from early passage through replicative senescence using cost-effective Infinium DNA methylation arrays. In Specific Aim 2 we will conduct high-resolution single-cell methylome analyses at key stages of HGPS fibroblast culture to detect arising aberrations and delineate population transitions. We have developed a single-cell whole-genome bisulfite sequencing (sc-WGBS) method that delivers genomic coverage far superior to any other published sc-WGBS methods. In Specific Aim 3 we will investigate whether DNA methylation manipulation can extend lifespan in HGPS models. In Aim 3A we will test whether overexpression of DNA methylation writers and erasers increases the replicative lifespan of HGPS fibroblasts. In Aim 3B, we will target DNA methyltransferase overexpression to arterial smooth muscle cells in an HGPS mouse model to investigate whether this reduces arterial smooth muscle loss and extends lifespan. We will monitor DNA methylation changes in this mouse model using a new cost-effective DNA methylation array. We present extensive and compelling preliminary data that demonstrates both the feasibility and relevance of the proposed aims. The outcome of this proposed research could have important impacts on our understanding of the contribution of DNA methylation alterations to HGPS phenotypes, potentially opening avenues for new therapeutic approaches to treat progeria. In addition, this study could shed light on similar mechanisms operating at a longer timescale in normal aging.