Synthetic reversed sequence reveals default chromatin states [Yeast_ChIP-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均可能表现出转录活性证据，但目前已注释的转录本占比尚不足5%。这种显著差异的成因尚不明确，目前仍无法确定过量转录究竟是具有生物学功能的过程，还是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，有助于判断观测到的广泛转录活性是否具备功能性。所有现存生物的基因组均经历了数十亿年的进化历程，因此难以确定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。为解答这一问题，本研究将一段全新的101 kb基因座（locus）分别引入两种真核生物——酿酒酵母（Saccharomyces cerevisiae，S. cerevisiae）和小家鼠（Mus musculus，M. musculus）的基因组中，并基于染色质可及性、染色质状态及转录水平对该基因座的基因组活性进行表征。该基因座的设计思路为：将人类HPRT1基因座的序列反向排列但不进行互补，同时保留上下游各约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，又能消除编码信息与转录因子（transcription factor）结合位点。本研究还在两种生物体系中，分别将该反向基因座与正常人类HPRT1基因座的合成版本进行了对比。无论是合成型HPRT1基因座还是其反向版本，均未进化出酿酒酵母的启动子（promoter）。尽管如此，本研究在酿酒酵母中仍观测到两个基因座均存在广泛的转录活性，且这种广泛活性在基因座以游离型质粒（episome）存在以及染色体整合两种情况下均有出现。在染色体整合的情况下，其染色质可及性与转录起始水平均显著高于侧翼的本土酵母基因组序列。与之形成鲜明对比的是，当合成型HPRT1基因座整合至小鼠基因组中时，其转录活性恰好对应HPRT1的编码序列；而反向基因座则完全无转录活性，反而被多梳蛋白（Polycomb）调控系统主动沉默。综合以上结果可知，缺乏编码信息的全新基因组序列在酿酒酵母中具备转录活性，但在小鼠干细胞中则被沉默，这表明这两种亲缘关系较远的真核生物的基因组默认状态存在显著差异，该发现对于理解广泛转录现象与新基因的起源具有重要意义。