Kühnel Theresa, Heinz Helena Sophie Barbara, Utz Nadja, Božić Tanja, Horsthemke Bernhard, Steenpass Laura
Institute of Human Genetics, University Hospital Essen, University Duisburg-Essen, Hufelandstr 55, 45147 Essen, Germany.
Present address: Institute of Neuropathology, Justus Liebig University Giessen, Aulweg 128, 35392 Giessen, Germany.
Heliyon. 2020 Jan 29;6(1):e03261. doi: 10.1016/j.heliyon.2020.e03261. eCollection 2020 Jan.
Transcriptional interference and transcription through regulatory elements (transcriptional read-through) are implicated in gene silencing and the establishment of DNA methylation. Transcriptional read-through is needed to seed DNA methylation at imprinted genes in the germ line and can lead to aberrant gene silencing by DNA methylation in human disease. To enable the study of parameters and factors influencing transcriptional interference and transcriptional read-through at human promoters, we established a somatic cell culture system. At two promoters of imprinted genes ( and ) and two promoters shown to be silenced by aberrant transcriptional read-through in human disease ( and ) we tested, if transcriptional read-through is sufficient for gene repression and the acquisition of DNA methylation. Induction of transcriptional read-through from the doxycycline-inducible CMV promoter resulted in consistent repression of all downstream promoters, independent of promoter type and orientation. Repression was dependent on ongoing transcription, since withdrawal of induction resulted in reactivation. DNA methylation was not acquired at any of the promoters. Overexpression of DNMT3A and DNMT3L, factors needed for DNA methylation establishment in oocytes, was still not sufficient for the induction of DNA methylation. This indicates that induction of DNA methylation has more complex requirements than transcriptional read-through and the presence of de novo DNA methyltransferases.
转录干扰以及通过调控元件的转录（转录通读）与基因沉默和DNA甲基化的建立有关。转录通读是在生殖系中印记基因上启动DNA甲基化所必需的，并且在人类疾病中可能通过DNA甲基化导致异常的基因沉默。为了能够研究影响人类启动子处转录干扰和转录通读的参数及因素，我们建立了一种体细胞培养系统。在印记基因的两个启动子（和）以及在人类疾病中显示因异常转录通读而沉默的两个启动子（和）处，我们测试了转录通读是否足以导致基因抑制和DNA甲基化的获得。从强力霉素诱导型CMV启动子诱导转录通读导致所有下游启动子持续受到抑制，这与启动子类型和方向无关。抑制依赖于持续的转录，因为诱导的撤除会导致重新激活。在任何启动子处均未获得DNA甲基化。卵母细胞中建立DNA甲基化所需的因子DNMT3A和DNMT3L的过表达，仍不足以诱导DNA甲基化。这表明DNA甲基化的诱导比转录通读和从头DNA甲基转移酶的存在具有更复杂的要求。
