Spatial organization of transcribed genes in mammalian cells

目前的拨款申请是我以前在SPP 2202的研究项目的延续，该项目的重点是转录真核基因的空间组织。在过去的一段时间里，我们研究了几个长期高表达的小鼠基因作为模型，并证明了转录基因从其窝藏位点扩展，形成一个开放的转录环（TL）与聚合酶移动沿着环和携带新生RNA。值得注意的是，TL可以跨越微米，这与最近关于间期染色质是凝胶还是固体的主张相矛盾。TL的延伸和形状表明其固有的刚度，我们归因于密集的装饰的高表达基因与多个大量的新生核糖核蛋白（nRNP）。刚度假设成功地测试实验和表达基因的聚合物建模。总之，我们的数据与流行的转录工厂模型相矛盾，并表明尽管显微镜下可分辨的TL对长时间高度表达的基因具有特异性，但其形成机制可能代表真核转录的一般方面。这项研究发表在Nature Cell Biology（2022）上。虽然之前的工作带来了关于表达基因的空间组织的明确信息，但它提出了关于真核转录的新的有趣问题，我将在新的资助期内解决这些问题。(1)关于剪接的经典观点是，它严格地与内含子共转录地发生，内含子在它们被通读后不久被切除。使用Tg基因作为模型，我计划研究在大量转录的情况下共转录剪接的速度。(2)以前的TL观察是在固定的组织或细胞中利用FISH进行的。通过靶向nRNAs的Ttn和Cald 1基因通过茎环/外壳蛋白系统，我计划活细胞观察的时空动态的TL和转录爆发。(3)由于nRNP和mRNP的尺寸很小，因此对它们的结构知之甚少。基于我们的初步EM数据，我计划使用相关显微镜（cryo-CLEM）和cryo-EM断层扫描来研究肌管中Ttn形成的TLs的nRNP和3D结构。(4)教科书上的知识，染色体领域的间期核的主要特征是不支持我们以前的工作表明，领域性很可能是最后一次有丝分裂的结果。为了解决这个问题，我计划评估领土的基础上olipainting的小鼠染色体在细胞中的有丝分裂期的持续时间不同。(5)最后，我计划完成对Tg基因的研究，作为转录高度和稳健上调的模型。特别是，我计划测试Tg的昼夜节律和内含子保留在Tg成绩单作为可能的机制分离外分泌（甲状腺球蛋白分泌）和内分泌（激素生产）活动在甲状腺细胞。

The current grant proposal is a continuation of my previous research project within SPP2202, which was focused on spatial organization of transcribed eukaryotic genes. In the course of the past period, we studied several long highly expressed mouse genes as models and demonstrated that a transcribed gene expands from its harboring locus and forms an open-ended transcription loop (TL) with polymerases moving along the loop and carrying nascent RNAs. Remarkably, TLs can span across microns arguing against recent propositions that interphase chromatin is a gel or a solid. Extension and shape of TLs suggest their intrinsic stiffness, which we attribute to dense decoration of highly expressed genes with multiple voluminous nascent ribonucleoproteins (nRNPs). The stiffness hypothesis was successfully tested experimentally and by polymer modeling of expressed genes. In summary, our data contradict the popular model of transcription factories and suggest that although microscopically resolvable TLs are specific for long highly expressed genes, the mechanisms underlying their formation could represent a general aspect of eukaryotic transcription. The work is now published in the journal Nature Cell Biology (2022). Whereas the previous work brought a clear message about spatial organization of expressed genes, it raised new intriguing questions concerning eukaryotic transcription, which I am going to address in the new funding period. (1) The canonical view on splicing is that it occurs strictly co-transcriptionally with introns being excised shortly after they are read through. Using the Tg gene as a model, I plan to study rapidity of co-transcriptional splicing in case of massive transcription. (2) The previous observations of TLs were performed utilizing FISH in fixed tissues or cells. By targeting nRNAs of the Ttn and Cald1 genes via the stem-loop/coat protein system, I plan live-cell observations of spatiotemporal dynamics of TLs and transcriptional bursting. (3) Very little is known about structure of nRNPs and mRNPs owing to their small size. Based on our preliminary EM data, I plan to study nRNPs and 3D structure of TLs formed by Ttn in myotubes, using correlative microscopy (cryo-CLEM) and cryo-EM tomography. (4) The textbook knowledge that chromosome territories are the major feature of an interphase nucleus is not supported by our previous works indicating that territoriality is likely a mere consequence of the last mitosis. To address this question, I plan to evaluate territoriality based on oligopainting of mouse chromosomes in cells that vary by duration of their postmitotic period. (5) And finally, I plan to complete the study of the Tg gene as a model for high and robust upregulation of transcription. In particular, I plan to test Tg circadian rhythmicity and intron retention in Tg transcripts as possible mechanisms for separation of exocrine (thyroglobulin secretion) and endocrine (hormone production) activities in thyrocytes.