The Mina and Everard Goodman Faculty of Life Sciences and Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, Israel.
Biochemistry Laboratory, Samson Assuta Ashdod University Hospital, Ashdod, Israel.
Histochem Cell Biol. 2024 Jul;162(1-2):91-107. doi: 10.1007/s00418-024-02294-w. Epub 2024 May 19.
Super-enhancers are unique gene expression regulators widely involved in cancer development. Spread over large DNA segments, they tend to be found next to oncogenes. The super-enhancer c-MYC locus forms long-range chromatin looping with nearby genes, which brings the enhancer and the genes into proximity, to promote gene activation. The colon cancer-associated transcript 1 (CCAT1) gene, which is part of the MYC locus, transcribes a lncRNA that is overexpressed in colon cancer cells through activation by MYC. Comparing different types of cancer cell lines using RNA fluorescence in situ hybridization (RNA FISH), we detected very prominent CCAT1 expression in HeLa cells, observed as several large CCAT1 nuclear foci. We found that dozens of CCAT1 transcripts accumulate on the gene locus, in addition to active transcription occurring from the gene. The accumulating transcripts are released from the chromatin during cell division. Examination of CCAT1 lncRNA expression patterns on the single-RNA level showed that unspliced CCAT1 transcripts are released from the gene into the nucleoplasm. Most of these unspliced transcripts were observed in proximity to the active gene but were not associated with nuclear speckles in which unspliced RNAs usually accumulate. At larger distances from the gene, the CCAT1 transcripts appeared spliced, implying that most CCAT1 transcripts undergo post-transcriptional splicing in the zone of the active gene. Finally, we show that unspliced CCAT1 transcripts can be detected in the cytoplasm during splicing inhibition, which suggests that there are several CCAT1 variants, spliced and unspliced, that the cell can recognize as suitable for export.
超级增强子是广泛参与癌症发展的独特基因表达调控因子。它们分布在大片段 DNA 上，往往位于癌基因附近。超级增强子 c-MYC 基因座与附近基因形成长程染色质环，将增强子和基因拉近，促进基因激活。结肠癌相关转录物 1（CCAT1）基因是 MYC 基因座的一部分，通过 MYC 的激活转录出一种在结肠癌细胞中过表达的 lncRNA。通过 RNA 荧光原位杂交（RNA FISH）比较不同类型的癌细胞系，我们在 HeLa 细胞中检测到非常明显的 CCAT1 表达，表现为几个大的 CCAT1 核焦点。我们发现，除了基因活跃转录外，还有数十个 CCAT1 转录本在基因座上积累。在细胞分裂过程中，积累的转录本从染色质中释放出来。在单个 RNA 水平上检查 CCAT1 lncRNA 表达模式，发现未剪接的 CCAT1 转录本从基因释放到核质中。这些未剪接的转录本大部分观察到与基因活性接近，但不与核斑点（unspliced RNAs 通常聚集在核斑点中）相关。在离基因更远的地方，CCAT1 转录本出现剪接，这意味着大多数 CCAT1 转录本在活性基因区域经历转录后剪接。最后，我们表明，在剪接抑制期间可以在细胞质中检测到未剪接的 CCAT1 转录本，这表明细胞可以识别出几种适合输出的未剪接和剪接的 CCAT1 变体。
