Imaging local and global chromatin structure as a 3D continuum within the nucleus

 描述(申请人提供)：我们基因组的生物学功能和活性不是仅仅由线性DNA序列信息决定的。为了适应细胞核，DNA与核小体组装形成染色质，染色质卷曲到空间定义的区域，通过鲜为人知的机制确定基因是活跃的还是沉默的。染色质的局部和全球组织整合在细胞核中，以确定基因活性和基因组功能。为了在完整细胞中显示基因组功能的不同结构尺度，ChromEM已经开发出一种利用细胞可渗透的荧光小分子与DNA特异结合，并在激发时用一种可以用电子显微镜(EM)观察到的嗜Os聚合物在染色质表面涂抹的技术。通过将ChromEM与多倾斜电磁层析成像(EMT)的新进展相结合，染色质的超微结构和3D组织可以在核小体分辨率下通过前所未有的核体积被可视化为一个连续体。在目标1中，ChromEM将被用于在核小体分辨率和基因组尺度上可视化人类胚胎干细胞和分化的肺上皮细胞的染色质结构-功能。我们将定性和定量地分析腺病毒感染和HDAC/DNA甲基转移酶抑制剂对染色质结构和组织的大规模变化。我们将使用连续截面EMT和连续块Face EM来扩大规模和3D核体积。一个主要的目标将是在中期细胞中重建完整的姐妹染色单体对，以确定它们是否具有相同的染色体结构和染色质组织。在目标2中，将开发“多色”荧光的EM等价物。我们将利用mini SOG和ChromEM的顺序激发循环来实现‘多色EM’，以光氧化DAB的不同金属络合物，这些络合物可以通过电子能量损失谱(EELS)的元素映射来区分。多色电子显微镜将用于显示病毒癌蛋白和PML小体与染色质的结构相互作用，染色质调节p53和抗病毒基因的沉默和激活。此外，工作流程将被扩展到包括实时成像，以可视化PML和染色质相关相互作用的时空动态，这些相互作用被原代细胞中的野生型和变异型腺病毒感染所破坏。在目标3中，将开发新的探针，使其能够在保持天然超微结构和序列背景的同时，在细胞核内的大量染色质中识别单一拷贝基因。合成的自组装纳米颗粒将被设计成具有不同的结构、大小、金属和荧光特性，以便可以使用实时成像、X射线显微镜和高分辨率3D EM来标记和可视化多个基因。这些标记将使绿色荧光蛋白标记的基因座能够通过EMT与使用dCAS9融合的内源基因座一起可视化。端粒和关键生长调节基因的染色质超微结构将使用实时成像和3D EM在细胞周期和病毒感染的动态中可视化。这些研究将改变人们对细胞核和染色质结构-功能的理解。

 DESCRIPTION (provided by applicant): The biological functions and activity of our genomes is not determined by linear DNA sequence information alone. To fit within the nucleus, DNA assembles with nucleosomes to form chromatin that coils into spatially defined territories that determine if genes are active or silent through poorly understood mechanisms. The local and global organization of chromatin are integrated in the nucleus to determine gene activity and genome function. To visualize the different structural scales of genome function in intact cells, ChromEM has been developed that exploits a cell permeable fluorescent small molecule that binds specifically to DNA and upon excitation paints the surface of chromatin with an osmiophillic polymer that can be visualized using electron microscopy (EM). By combining ChromEM with new advances in multi-tilt EM tomography (EMT), chromatin ultrastructure and 3D organization can be visualized at nucleosome resolutions as a continuum through unprecedented nuclear volumes. In Aim 1, ChromEM will be used to visualize chromatin structure-function in human embryonic stem cells and differentiated lung epithelial cells at nucleosome resolutions and genomic scales. We will qualitatively and quantitatively analyze the large-scale changes in chromatin structure and organization in response to Adenovirus infection and HDAC/DNA methyl transferase inhibitors. We will expand the scale and 3D nuclear volumes using serial section EMT and serial block face EM. A major goal will be to reconstruct entire sister chromatid pairs in metaphase cells to determine if they have identical chromosome architectures and chromatin organization. In Aim 2, the EM equivalent of `multi-color' fluorescence will be developed. We will implement `multi-color EM' by using sequential excitation cycles of miniSOG and ChromEM to photo-oxidize different metal chelates of DAB that can be discriminated by elemental mapping using electron energy loss spectroscopy (EELS). Multi-color EM will be used to visualize the structural interactions of viral oncoprotein and PML bodies with chromatin that regulate he silencing and activation of p53 and anti-viral genes. Also, workflow will be extended to incorporate live imaging to visualize the spatiotemporal dynamics of PML and chromatin associated interactions that are disrupted by wild type and mutant adenovirus infections in primary cells. In Aim 3, novel probes will be developed that enable a single copy gene to be identified in a sea of chromatin within the nucleus while preserving native ultrastructure and sequence context. Synthetic self- assembling nanoparticles will be engineered with different architectures, sizes, metal and fluorescent properties so that multiple genes can be labeled and visualized using live imaging, X-Ray microscopy and high resolution 3D EM. These labels will enable GFP-tagged loci to be visualized by EMT together with endogenous gene loci using dCAS9 fusions. The chromatin ultrastructure of telomeres and critical growth regulatory genes will be visualized using live imaging and 3D EM in the dynamics of the cell cycle and viral infection. These studies will change the understanding of the nucleus and chromatin structure-function.