Spatial organization of transcribed genes in mammalian cells

目前的拨款提案是我之前在SPP2202的研究项目的延续，该项目专注于真核生物转录基因的空间组织。在过去的一段时间里，我们研究了几个长时间高表达的小鼠基因作为模型，并证明了转录基因从其隐藏位点扩展并形成开放式转录环（TL），聚合酶沿着环移动并携带新生rna。值得注意的是，tl可以跨越微米，这反驳了最近关于间期染色质是凝胶或固体的主张。TLs的延伸和形状表明它们的内在刚性，我们将其归因于高表达基因与多个体积新生核糖核蛋白（nRNPs）的密集装饰。通过实验和表达基因的聚合物建模成功地验证了刚度假设。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解的TLs是长时间高表达基因的特异性，但它们形成的机制可能代表真核生物转录的一般方面。这项研究成果发表在《自然细胞生物学》（Nature Cell Biology, 2022）杂志上。虽然先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生物转录的新的有趣问题，我将在新的资助期解决这些问题。(1)关于剪接的规范观点是，它严格地发生在共转录过程中，内含子在读完后不久就被切除。我计划以Tg基因为模型，研究在大量转录情况下共转录剪接的速度。(2)之前对TLs的观察是利用FISH在固定组织或细胞中进行的。通过茎环/外壳蛋白系统靶向Ttn和Cald1基因的nRNAs，我计划在活细胞中观察TLs的时空动态和转录爆发。(3)由于nRNPs和mRNPs的体积小，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断层扫描研究肌管中Ttn形成的nrnp和TLs的三维结构。(4)教科书上关于染色体区域是间期细胞核的主要特征的知识，并没有得到我们之前的工作的支持，表明区域可能仅仅是最后一次有丝分裂的结果。为了解决这个问题，我计划根据小鼠细胞中染色体的寡涂来评估领土性，这些染色体的寡涂随有丝分裂后时期的持续时间而变化。(5)最后，我计划完成Tg基因作为转录高稳健上调模型的研究。特别是，我计划测试Tg昼夜节律性和Tg转录本中的内含子保留，作为分离甲状腺细胞外分泌（甲状腺球蛋白分泌）和内分泌（激素产生）活动的可能机制。

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.