Synthetic reversed sequence reveals default chromatin states [Yeast_RNA-seq]

Most of human genome may show evidence of transcription, yet annotated transcripts account for less than 5%. The basis for this major discrepancy is not clear, and it remains uncertain whether excess transcription is functional, or simply a byproduct of pervasive, non-specific RNA polymerase binding and transcription initiation. Understanding the default state of the genome would be informative in determining whether the observed pervasive activity is functional. The genome of any extant organism has undergone billions of years of evolution, making it unclear whether any observed genomic activity, or lack thereof, has been selected for. We sought to address this question by introducing a completely novel 101 kb locus into the genomes of two eukaryotic organisms, S. cerevisiae and M. musculus, and characterizing its genomic activity based on chromatin accessibility, chromatin states, and transcription. The locus was designed by reversing, but not complementing, the sequence of the human HPRT1 locus, including ~30 kb of both upstream and downstream regulatory regions, allowing retention of sequence features like repeat frequency and GC content but ablating coding information and transcription factor binding sites. We also compared this reversed locus with a synthetic version of the normal human HPRT1 locus in both organismal contexts. Neither the synthetic HPRT1 locus nor its reversed version evolved to harbor yeast promoters. Nevertheless, we observed widespread transcriptional activity of both loci in yeast, and this pervasive activity was observed both when the loci were present as episomes and when chromosomally integrated. In the latter case, it was obvious that the accessibility and level of transcription initiation substantially exceeded that of the flanking native yeast genome sequences. In contrast, when integrated in the mouse genome, the synthetic HPRT1 locus showed transcriptional activity corresponding precisely to the HPRT1 coding sequence, whereas the reverse locus displayed no activity at all, but was instead actively repressed by Polycomb machinery. Together, these results show that novel genomic sequences lacking coding information are active in yeast, but repressed in mouse stem cells, indicating a major difference in default genomic states between these two divergent eukaryotes, with implications for understanding pervasive transcription and the birth of new genes.人类基因组的绝大多数区域都可能存在转录证据，但目前被注释的转录本占比尚不足5%。这一显著差异的成因尚不明确，我们仍无法确定过量转录究竟是具备生物学功能的，还是仅为广泛存在的非特异性RNA聚合酶（RNA polymerase）结合与转录起始所产生的副产物。解析基因组的默认状态，将有助于判断观测到的广泛基因组活性是否具备功能。现存生物的基因组均经历了数十亿年的演化，这使得我们无法明确，观测到的基因组活性或活性缺失是否为自然选择所保留。我们尝试通过向两种真核生物——酿酒酵母（S. cerevisiae）与小家鼠（M. musculus）——的基因组中引入一段全新的101 kb基因座（locus），并基于染色质可及性、染色质状态与转录情况对其基因组活性进行表征，以解答这一问题。该基因座的设计思路为：将人类次黄嘌呤磷酸核糖转移酶1（HPRT1）基因座的序列进行反转，但不进行互补，同时保留其上下游各约30 kb的调控区域，这样既能保留重复序列频率、GC含量等序列特征，又可消除编码信息与转录因子结合位点。我们还在两种生物宿主环境中，将该反转基因座与正常人类HPRT1基因座的合成版本进行了对比。无论是合成的HPRT1基因座还是其反转版本，均未演化出酵母启动子。尽管如此，我们在酵母中观测到两个基因座均呈现广泛的转录活性，且无论基因座以附加体（episome）形式存在，或是被染色体整合时，均能观测到该广泛活性。在染色体整合的情况下，其染色质可及性与转录起始水平显著高于侧翼的天然酵母基因组序列，这一点十分明确。与之形成对比的是，当合成的HPRT1基因座被整合至小鼠基因组中时，其转录活性与HPRT1编码序列完全对应；而反转基因座则完全无转录活性，反而被多梳蛋白复合物（Polycomb machinery）主动沉默。综合来看，这些结果表明：缺乏编码信息的全新基因组序列在酵母中具有活性，但在小鼠干细胞中则被沉默，这说明这两种分化差异显著的真核生物在基因组默认状态上存在巨大差异，该发现对于理解广泛存在的转录现象与新基因的诞生具有重要参考价值。