Impact of Floating-Harbor syndrome mutations on chromatin remodeling by the SRCAP complex

浮港综合征突变对 SRCAP 复合体染色质重塑的影响

• 批准号: 9975861
• 负责人: Shinya Watanabe
• 金额: $ 8.38万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-11 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9975861
• 关键词: Address; Affect; Alleles; Baculovirus Expression System; Biochemical; Biological Assay; Biophysics; C-terminal; Chromatin Structure; Chromatin Structure Alteration; Deposition; Disease; Dominant-Negative Mutation; Enzymes; Epigenetic Process; Exons; Face; Family; Floating-Harbor syndrome; Fluorescence Resonance Energy Transfer; Gene Expression; Gene Proteins; Genes; Genetic Transcription; Genome; Genome Stability; Genomic approach; Genomics; Goals; Histones; Human; Individual; Lead; Molecular; Multiprotein Complexes; Mus; Mutation; Nucleosomes; Patients; Phenotype; Play; Property; Proteins; Recombinants; Reporting; Research; Role; Speech Delay; Speech Development; Techniques; Testing; Variant; base; biophysical techniques; chromatin remodeling; developmental disease; embryonic stem cell; genomic locus; member; novel therapeutic intervention; promoter; protein complex; protein function; reconstitution; recruit; stem cell differentiation

The long-term goal of our research is to investigate the molecular mechanisms by which epigenetic alterations of chromatin structure promote human developmental disorders and diseases such as Floating- Harbor syndrome (FHS). It was recently found that mutations of the SRCAP (SNF2-related CBP activator protein) gene cause FHS, which is a rare dominant disorder characterized by proportionate short stature with dysmorphic facial features, delayed osseous maturation, and delayed speech development. However, the molecular bases underlying the disease remain to be elucidated. SRCAP is a member of the SNF2 family of ATP-dependent chromatin remodeling enzymes, and forms a 12-subunit, large protein complex. All of SRCAP mutations in FHS patients are heterozygous truncating alleles, tightly clustered within the final 33th and 34th exons, suggesting that the C-terminal domain of SRCAP is crucial for the SRCAP function. Importantly, it was reported that individuals carrying a deletion of a chromosomal region containing the SRCAP gene have no reported phenotype, suggesting that SRCAP deletion is haplosufficient. The SRCAP complex is required for the incorporation of histone variant H2A.Z into nucleosomes. H2A.Z is deposited within promoter-proximal nucleosomes, and plays essential roles in transcription, genome stability, and proper stem cell differentiation. The overall objective of this proposed research is to characterize the SRCAP complex, and determine how SRCAP mutations alter epigenetic chromatin structure and function, thus resulting in FHS. Our overall strategy in this proposal is to exploit a powerful combination of biochemical, biophysical, and genomics techniques to dissect the molecular mechanisms by which the SRCAP complex regulates H2A.Z deposition and the truncated SRCAP causes FHS. In Aim 1, we will dissect the mechanism of H2A.Z deposition by the SRCAP complex. The molecular mechanism by which the SRCAP complex catalyzes H2A.Z deposition is largely unknown, mainly due to the limited protein availability, as SRCAP forms a large multi-protein complex. To address this, we have successfully reconstituted the whole SRCAP complex from individual, recombinant subunits using the Multibac baculovirus expression system. We will define the detailed biochemical properties, especially histone exchange activity, of the SRCAP complex. We will employ various chromatin remodeling assays including FRET-based assays. Furthermore, we will investigate the function of SRCAP in mouse embryonic stem cells (ESCs), since H2A.Z is necessary for ESC differentiation. We will perform genomics analyses to dissect how SRCAP regulates the epigenetic landscape of H2A.Z during ESC differentiation. In Aim 2, we will define the effects of Floating-Harbor syndrome mutations on the SRCAP complex. Although all SRCAP mutations in FHS patients are heterozygous truncating alleles, it remains unclear how the truncated SRCAP produces a dominant negative effect. To define the effects of FHS mutations on the SRCAP function, we will reconstitute the SRCAP complex containing a FHS mutation, and dissect how it affects the assembly and function of the SRCAP complex. Then, we will test two hypotheses for the dominant negative effect of the truncated SRCAP by employing various biophysical and genomics approaches. !

我们研究的长期目标是研究表观遗传的分子机制 染色质结构的改变会促进人类发育障碍和疾病，例如漂浮- 港口综合症（FHS）。最近发现SRCAP（SNF2相关CBP激活剂）的突变 蛋白）基因导致 FHS，这是一种罕见的显性疾病，其特征是身材矮小，伴有 面部特征畸形、骨质成熟延迟和言语发育延迟。然而， 该疾病的分子基础仍有待阐明。 SRCAP 是 SNF2 家族的成员 ATP 依赖性染色质重塑酶，并形成 12 个亚基的大型蛋白质复合物。 SRCAP 的全部内容 FHS 患者的突变是杂合的截短等位基因，紧密聚集在最后的第 33 个和第 34 个等位基因中 外显子，表明 SRCAP 的 C 端结构域对于 SRCAP 功能至关重要。重要的是，这是 据报道，携带含有 SRCAP 基因的染色体区域缺失的个体没有 报道的表型，表明 SRCAP 缺失是单倍充足的。需要 SRCAP 复合体 组蛋白变体 H2A.Z 并入核小体。 H2A.Z 沉积在启动子近端 核小体，并在转录、基因组稳定性和适当的干细胞分化中发挥重要作用。 这项研究的总体目标是描述 SRCAP 复合体的特征，并确定如何 SRCAP 突变改变表观遗传染色质结构和功能，从而导致 FHS。我们的整体 该提案中的策略是利用生物化学、生物物理和基因组学的强大组合 剖析 SRCAP 复合物调节 H2A.Z 沉积分子机制的技术 截断的 SRCAP 会导致 FHS。在目标 1 中，我们将通过以下方式剖析 H2A.Z 沉积的机制： SRCAP 复合体。 SRCAP复合物催化H2A.Z沉积的分子机制是 很大程度上未知，主要是由于蛋白质的可用性有限，因为 SRCAP 形成了一个大型的多蛋白质复合物。 为了解决这个问题，我们成功地从单个重组体中重建了整个 SRCAP 复合物 使用 Multibac 杆状病毒表达系统的亚基。我们将定义详细的生化特性， 尤其是 SRCAP 复合体的组蛋白交换活性。我们将采用各种染色质重塑 检测包括基于 FRET 的检测。此外，我们将研究 SRCAP 在小鼠中的功能 胚胎干细胞 (ESC)，因为 H2A.Z 对于 ESC 分化是必需的。我们将进行基因组学 分析 SRCAP 如何在 ESC 分化过程中调节 H2A.Z 的表观遗传景观。在 目标 2，我们将定义浮港综合征突变对 SRCAP 复合体的影响。虽然所有 FHS患者的SRCAP突变是杂合截短等位基因，目前尚不清楚截短是如何发生的 SRCAP 产生显着的负面影响。为了确定 FHS 突变对 SRCAP 功能的影响， 我们将重建包含 FHS 突变的 SRCAP 复合体，并剖析它如何影响组装 SRCAP 复合体的功能和功能。然后，我们将检验两个假设，以了解 通过采用各种生物物理和基因组学方法截断了 SRCAP。 ！