Cremer Marion, Schmid Volker J, Kraus Felix, Markaki Yolanda, Hellmann Ines, Maiser Andreas, Leonhardt Heinrich, John Sam, Stamatoyannopoulos John, Cremer Thomas
LMU Biocenter, Department Biology II, Ludwig Maximilians-Universität (LMU Munich), Grosshadernerstr. 2, 82152, Martinsried, Germany.
BioImaging Group, Department of Statistics, Ludwig Maximilians-Universität (LMU Munich), Munich, Germany.
Epigenetics Chromatin. 2017 Aug 7;10(1):39. doi: 10.1186/s13072-017-0146-0.
The association of active transcription regulatory elements (TREs) with DNAse I hypersensitivity (DHS[+]) and an 'open' local chromatin configuration has long been known. However, the 3D topography of TREs within the nuclear landscape of individual cells in relation to their active or inactive status has remained elusive. Here, we explored the 3D nuclear topography of active and inactive TREs in the context of a recently proposed model for a functionally defined nuclear architecture, where an active and an inactive nuclear compartment (ANC-INC) form two spatially co-aligned and functionally interacting networks.
Using 3D structured illumination microscopy, we performed 3D FISH with differently labeled DNA probe sets targeting either sites with DHS[+], apparently active TREs, or DHS[-] sites harboring inactive TREs. Using an in-house image analysis tool, DNA targets were quantitatively mapped on chromatin compaction shaped 3D nuclear landscapes. Our analyses present evidence for a radial 3D organization of chromatin domain clusters (CDCs) with layers of increasing chromatin compaction from the periphery to the CDC core. Segments harboring active TREs are significantly enriched at the decondensed periphery of CDCs with loops penetrating into interchromatin compartment channels, constituting the ANC. In contrast, segments lacking active TREs (DHS[-]) are enriched toward the compacted interior of CDCs (INC).
Our results add further evidence in support of the ANC-INC network model. The different 3D topographies of DHS[+] and DHS[-] sites suggest positional changes of TREs between the ANC and INC depending on their functional state, which might provide additional protection against an inappropriate activation. Our finding of a structural organization of CDCs based on radially arranged layers of different chromatin compaction levels indicates a complex higher-order chromatin organization beyond a dichotomic classification of chromatin into an 'open,' active and 'closed,' inactive state.
活性转录调控元件（TREs）与DNA酶I超敏位点（DHS[+]）以及“开放”的局部染色质构象之间的关联早已为人所知。然而，在单个细胞的核格局中，TREs的三维拓扑结构与其活性或非活性状态的关系仍不清楚。在此，我们在最近提出的一个功能定义的核架构模型背景下，探索了活性和非活性TREs的三维核拓扑结构，其中活性和非活性核区室（ANC-INC）形成两个空间上共对齐且功能上相互作用的网络。
使用三维结构照明显微镜，我们用不同标记的DNA探针组进行了三维荧光原位杂交，这些探针组靶向具有DHS[+]的位点（明显为活性TREs）或含有非活性TREs的DHS[-]位点。使用内部图像分析工具，将DNA靶点定量映射到染色质压缩形成的三维核格局上。我们的分析提供了证据，表明染色质结构域簇（CDC）呈径向三维组织，从外周到CDC核心染色质压缩程度逐渐增加。含有活性TREs的片段在CDC的解压缩外周显著富集，其环延伸到染色质间区室通道中，构成ANC。相反，缺乏活性TREs（DHS[-]）的片段向CDC的压缩内部（INC）富集。
我们的结果为ANC-INC网络模型提供了进一步的支持证据。DHS[+]和DHS[-]位点不同的三维拓扑结构表明，TREs在ANC和INC之间的位置会根据其功能状态发生变化，这可能为防止不适当激活提供额外保护。我们发现基于不同染色质压缩水平的径向排列层的CDC结构组织，表明染色质存在复杂的高阶组织，超越了将染色质简单分为“开放”、活性和“封闭”、非活性状态的二分法分类。
