Alternative promoter usage involved in the regulation of transcription, splicing, and translation contributes to proteome diversity and is involved in a large number of diseases, in particular, cancer. Epigenetic mechanisms and cis regulatory elements are involved in alternative promoter activity. Multiple transcript isoforms can be produced from a gene, due to the initiation of transcription at different transcription start sites (TSS). These transcripts may not have regions that allow discrimination during RT-qPCR, making quantification technically challenging. This study presents a general method for the relative quantification of a transcript synthesized from a particular TSS that we called AP-TSS (analysis of particular TSS). AP-TSS is based on the specific elongation of the cDNA of interest, followed by its quantification by qPCR. As proof of principle, AP-TSS was applied to two non-coding RNA: telomeric repeat-containing RNAs (TERRA) from a particular subtelomeric TSS, and Alu transcripts. The treatment of cells with a DNA methylation inhibitor was associated with a global increase of the total TERRA level, but the TERRA expression from the TSS of interest did not change in HT1080 cells, and only modestly increased in HeLa cells. This result suggests that TERRA upregulation induced by global demethylation of the genome is mainly due to activation from sites other than this particular TSS. For Alu RNA, the signal obtained by AP-TSS is specific for the RNA Polymerase III-dependent Alu transcript. In summary, our method provides a tool to study regulation of gene expression from a given transcription start site, in different conditions that could be applied to many genes. In particular, AP-TSS can be used to investigate the epigenetic regulation of alternative TSS usage that is of importance for the development of epigenetic-targeted therapies.

中文翻译：

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AP-TSS：一种分析特定和具有挑战性的转录起始位点RNA表达的新方法。

参与转录，剪接和翻译调控的其他启动子的使用有助于蛋白质组的多样性，并涉及多种疾病，尤其是癌症。表观遗传机制和顺式调控元件参与替代启动子活性。由于在不同转录起始位点（TSS）处转录的起始，因此可以从一个基因产生多种转录同工型。这些转录本可能没有允许在RT-qPCR期间进行区分的区域，这使得定量分析在技术上具有挑战性。这项研究提出了一种相对定量从特定TSS合成的转录本的相对方法，我们称之为AP-TSS（特定TSS的分析）。AP-TSS基于目标cDNA的特异性延伸，然后通过qPCR进行定量。作为原理证明，将AP-TSS应用于两种非编码RNA：来自特定亚端粒TSS的端粒重复序列含RNA（TERRA）和Alu转录本。用DNA甲基化抑制剂处理细胞与总TERRA水平的总体增加有关，但是感兴趣的TSS的TERRA表达在HT1080细胞中没有变化，而在HeLa细胞中仅适度增加。该结果表明，由基因组的整体去甲基化诱导的TERRA上调主要是由于来自该特定TSS以外的位点的激活。对于Alu RNA，通过AP-TSS获得的信号对RNA聚合酶III依赖的Alu转录物具有特异性。总之，我们的方法提供了一种工具，可以研究在给定转录起始位点下基因表达的调控情况，这些条件可以应用于许多基因。