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

我们研究的长期目标是研究表观遗传学 染色质结构的改变会促进人类发育障碍和疾病，如漂浮- 海港综合征(FHS)。最近发现SRCAP(SNF2相关CBP激活剂)突变 蛋白质)基因导致FHS，这是一种罕见的显性疾病，其特征是与 面部特征畸形，骨骼成熟延迟，语言发育迟缓。然而， 该病的分子基础仍有待阐明。SRCAP是SNF2家族的成员 依赖于ATP的染色质重塑酶，并形成一个12亚基的大蛋白复合体。所有SRCAP FHS患者的突变是杂合性截断等位基因，紧密聚集在最后的第33和34位 外显子，表明SRCAP的C-末端结构域对SRCAP的功能至关重要。重要的是，它是 报道称，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缺失是单效率的。SRCAP综合体需要用于 将组蛋白变异体H_2A.Z掺入核小体。H_2A.Z沉积在启动子近端 核小体，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。 这项拟议研究的总体目标是描述SRCAP复合体的特征，并确定如何 SRCAP突变改变了表观遗传的染色质结构和功能，从而导致FHS。我们的整体 这项建议的策略是利用生化、生物物理和基因组学的强大组合 剖析SRCAP复合体调节H2A.Z沉积的分子机制的技术 而截断的SRCAP导致FHS。在目标1中，我们将剖析H_2A.Z沉积的机理 SRCAP复合体。SRCAP络合物催化H2A.Z沉积的分子机制为 这在很大程度上是未知的，主要是由于蛋白质的可获得性有限，因为SRCAP形成了一个大型的多蛋白质复合体。 为了解决这个问题，我们成功地从单个、重组的 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，我们还将研究SRCAP在小鼠体内的功能 胚胎干细胞(ESCs)，因为H_2A.Z是ESC分化所必需的。我们将进行基因组学研究 分析SRCAP如何在ESC分化过程中调节H_2A.Z的表观遗传格局。在……里面 目的2，我们将确定浮港综合征突变对SRCAP复合体的影响。尽管所有人 FHS患者的SRCAP突变是杂合性截短等位基因，目前尚不清楚截短的原因 SRCAP产生显性的负效应。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，并分析它是如何影响组装的 和SRCAP复合体的功能。然后，我们将检验两个假设，以确定 通过使用各种生物物理和基因组学方法截断SRCAP。 好了！

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. !