Genomic analysis of a synthetic reversed sequence reveals default chromatin states in yeast and mammalian cells

Up to 93% of the human genome may show evidence of transcription, yet annotated transcripts account for less than 5%. It is unclear what makes up this major discrepancy, and to what extent the excess transcription has a definable biological function, or is just a pervasive byproduct of 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 has a definable function. The genome of any modern 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 100-kb locus into the genomes of two eukaryotic organisms, S. cerevisiae and M. musculus, and characterizing its genomic activity based on chromatin accessibility 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. Despite neither the synthetic HPRT1 locus nor its reverse version coding for any promoters evolved for gene expression in yeast, we observed widespread transcriptional activity of both loci. This activity was observed both when the loci were present as episomes and when chromosomally integrated, although it did not correspond to any of the known HPRT1 functional regulatory elements. In contrast, when integrated in the mouse genome, the synthetic HPRT1 locus showed transcriptional activity corresponding precisely to the HPRT1 coding sequence, while the reverse locus displayed no activity at all. Together, these results show that genomic sequences with no coding information are active in yeast, but relatively inactive in mouse, indicating a potentially major difference in "default genomic states" between these two divergent eukaryotes.