Large Scale Chromatin Structure

大尺度染色质结构是染色质在几百个碱基的尺度上的高阶折叠。人们对这种折叠知之甚少，但这种结构肯定会影响DNA的转录、复制和修复。为了更多地了解这些结构，我们以前用光学显微镜检查了400-2000kb的转录活性结构域。我们发现，在光学显微镜图像中，几个不同的结构域显示出相似的结构，即一系列直径约0.5微米的相邻点状或“珠子”。当这些相同的结构域在转录上不活跃时，只检测到一个标点或“珠子”。因此，转录诱导了从单珠状结构到多珠状结构的解凝聚。我们现在已经使用转录因子迁移率项目中描述的串联阵列系统研究了多聚酶II与这些珠状结构的关联。我们发现，聚合酶也形成与上述染色质珠子相关联的“珠子”，但不直接覆盖在上面描述的染色质珠子上。虽然以前还没有报道将聚合酶与染色质结构进行直接比较，但聚合酶聚集的趋势已经被广泛报道，并导致了这样一种模型，即在这些聚合体或“工厂”中，转录是通过DNA在工厂中的移动而发生的，而不是沿着聚合酶的DNA移动。这个模型预测聚合酶工厂应该被最近转录的DNA包围。我们通过开发一种在细胞中保存脆弱DNA结构的新方法来测试这一点，并发现与串联阵列相关的聚合酶工厂实际上被来自串联阵列的DNA包围。工厂周围的DNA似乎是最近转录的，因为它的标志是与最近转录的DNA相关的某种组蛋白修饰。这些观察结果为转录通过固定聚合酶的DNA移动而发生的假设提供了新的支持。

Large-scale chromatin structure is the higher-order folding of chromatin on the scale of several hundred kilobases. Little is known about this folding, but the structures must influence transcription, replication and repair of DNA. To learn more about these structures, we previously examined transcriptionally active domains of 400 - 2000 kb by light microscopy. We found that several different domains exhibit similar structures in light microscope images, namely a series of adjacent puncta or "beads" approximately 0.5 microns in diameter. When these same domains are transcriptionally inactive, only one punctum or "bead" is detected. Thus transcription induces a decondensation from single beaded to multi-beaded structures. We have now examined the association of polyermase II with these beaded structures using the tandem array system described in the transcription factor mobility project. We find that the polymerase also forms "beads" that associate with, but do not directly overlay the chromatin beads described above. Although a direct comparison of polymerase with chromatin structure has not been reported before, the tendency of polymerase to cluster has been widely reported, and has led to the model that transcription occurs in these clusters or "factories" by movement of the DNA through the factory, rather than movement along the DNA of the polymerase. This model predicts polymerase factories should be surrounded by recently transcribed DNA. We have tested this by developing a new method for preserving fragile DNA structures in cells and found that the polymerase factories associated with the tandem array are in fact surrounded by DNA from the tandem array. This DNA surrounding the factory seems to have been recently transcribed, since it is marked by a certain histone modification associated with recently transcribed DNA. These observations provide new support to the hypothesis that transcription occurs by movement of DNA through a stationary polymerase.