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， 染色质结构的改变促进人类发育障碍和疾病，如漂浮- Harbor综合征（FHS）。最近发现，SRCAP（SNF 2相关CBP激活剂）的突变可导致 FHS是一种罕见的显性疾病，其特征是身材矮小， 畸形的面部特征、延迟的骨成熟和延迟的语言发育。但 这种疾病的分子基础仍有待阐明。SRCAP是SNF 2家族的成员， ATP依赖性染色质重塑酶，并形成一个12亚基的大蛋白复合物。关于SRCAP FHS患者的突变是杂合截短等位基因，紧密聚集在最后33和34位， 外显子，表明SRCAP的C-末端结构域对于SRCAP功能至关重要。重要的是 报道，携带含有SRCAP基因的染色体区域缺失的个体没有 报告的表型，表明SRCAP缺失是单倍充分的。需要SRCAP复合体， 将组蛋白变体H2A.Z掺入核小体。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表征SRCAP复合物，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 该建议中战略是利用生物化学、生物物理学和基因组学的强大组合 技术来剖析SRCAP复合物调节H2A.Z沉积的分子机制 截短的SRCAP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络合物催化H2A.Z沉积的分子机理是 这在很大程度上是未知的，主要是由于有限的蛋白质可用性，因为SRCAP形成了一个大的多蛋白质复合物。 为了解决这一问题，我们成功地从单个重组SRCAP复合物中重建了整个SRCAP复合物， 使用Multibac杆状病毒表达系统，我们将详细定义其生化特性， 尤其是组蛋白交换活性。我们将采用各种染色质重塑 包括基于FRET的测定。此外，我们还将研究SRCAP在小鼠体内的功能 胚胎干细胞（ESC），因为H2A.Z是ESC分化所必需的。我们将进行基因组学 分析以剖析SRCAP如何在ESC分化期间调节H2A.Z的表观遗传景观。在 目的2，我们将确定浮港综合征突变对SRCAP复合物的影响。尽管所有 FHS患者中的SRCAP突变是杂合的截短等位基因，目前尚不清楚截短的等位基因是如何表达的。 SRCAP产生显性负效应。为了确定FHS突变对SRCAP功能的影响， 我们将重组含有FHS突变的SRCAP复合物，并分析它如何影响装配， 和SRCAP复合物的功能。然后，我们将测试两个假设的显性负面影响的 通过采用各种生物物理学和基因组学方法来截短SRCAP。 !