Synthetic reversed sequence reveals default chromatin states [Yeast_ATAC-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.绝大多数人类基因组序列均可检测到转录迹象，但已注释的转录本仅占其中不足5%。造成这一显著差异的分子机制尚不明确，目前仍无法确定过量转录究竟具有生物学功能，抑或仅仅是广泛存在的非特异性RNA聚合酶（RNA polymerase）结合与转录起始的副产物。若要明确观测到的广泛基因组转录活性是否具有功能，厘清基因组的默认状态将极具参考价值。现存生物的基因组均经历了数十亿年的演化，这使得我们无法判断观测到的任何基因组活性（或缺失活性）是否曾被自然选择所保留。为此，我们向两种真核生物——酿酒酵母（S. cerevisiae）与小家鼠（M. musculus）的基因组中引入了一段全新的101 kb基因座，并通过染色质可及性、染色质状态及转录水平分析对该位点的基因组活性进行表征。该基因座的设计思路为反向转录人类HPRT1基因座序列但不进行互补，同时包含上下游各约30 kb的调控区域，如此可保留重复序列频率与GC含量等序列特征，同时消除编码信息与转录因子结合位点。我们还在两种生物体系中，将该反向基因座与天然人类HPRT1基因座的合成版本进行了对比。无论是合成型HPRT1基因座还是其反向版本，均未演化出酵母启动子序列。然而，我们在酵母中观测到了两个基因座的广泛转录活性，且无论该位点以附加体形式存在还是整合至染色体中，均能观测到这一广泛活性。在后一种情形下，该位点的染色质可及性与转录起始水平显著高于其侧翼的天然酵母基因组序列。与之形成鲜明对比的是，当该基因座整合至小鼠基因组时，合成型HPRT1基因座仅在HPRT1编码序列区域呈现对应转录活性，而反向位点则完全无转录活性，反而被多梳蛋白复合体（Polycomb machinery）主动沉默。综上，上述结果表明，不含编码信息的新型基因组序列在酵母中具有转录活性，但在小鼠干细胞中会被沉默，这揭示了这两种亲缘关系较远的真核生物在默认基因组状态上存在显著差异，该发现对于理解广泛存在的转录现象与新基因的起源具有重要意义。