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

基本信息

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
项目类别：
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

项目摘要

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学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，例如浮动 - 港口综合征（FHS）。最近发现SRCAP的突变（与SNF2相关的CBP激活剂蛋白质）基因引起FHS，这是一种罕见的显性障碍畸形的面部特征，延迟的骨成熟和延迟的语音发展。但是，该疾病基础的分子碱仍有待阐明。 SRCAP是SNF2家族的成员依赖于ATP的染色质重塑酶，并形成12个亚基大蛋白质复合物。所有SRCAP 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和第34位。外显子表明SRCAP的C末端结构域对于SRCAP函数至关重要。重要的是，是报道说携带含有srcap基因的染色体区域缺失的个体没有报道的表型，表明SRCAP缺失是单倍体的。 SRCAP复合物是需要的将组蛋白变体H2A.Z掺入核小体中。 H2A.Z沉积在启动子proximal中核小体，并在转录，基因组稳定性和正确的干细胞分化中起着重要作用。这项拟议研究的总体目的是表征SRCAP复合物，并确定如何 SRCAP突变改变了表观遗传染色质的结构和功能，从而导致FHS。我们的整体该提案中的策略是利用生化，生物物理和基因组学的强大组合剖析SRCAP复合物调节H2A.Z沉积的分子机制的技术截短的SRCAP导致FHS。在AIM 1中，我们将通过 SRCAP复合物。 SRCAP复合物催化H2A.Z沉积的分子机制是在很大程度上未知，这主要是由于蛋白质的可用性有限，因为SRCAP形成了大型多蛋白质复合物。为了解决这个问题，我们成功地重组了个人重组的整个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，特别是SRCAP复合物的组蛋白交换活性。我们将采用各种染色质重塑包括基于FRET的测定法。此外，我们将研究鼠标中SRCAP的功能胚胎干细胞（ESC），因为H2A.Z对于ESC分化是必需的。我们将执行基因组学分析以剖析SRCAP如何调节ESC分化过程中H2A.Z的表观遗传景观。在 AIM 2，我们将定义浮动 - 哈伯综合征突变对SRCAP复合物的影响。虽然全部 FHS患者的SRCAP突变是杂合的截断等位基因，尚不清楚截断如何 SRCAP产生主要的负面影响。为了定义FHS突变对SRCAP函数的影响，我们将重建包含FHS突变的SRCAP复合物，并剖析其如何影响组件 SRCAP复合物的功能。然后，我们将检验两个假设的主要负面影响通过采用各种生物物理和基因组学方法来截断SRCAP。呢