Poly(A)-Selected Intergenic Transcripts in In Vivo Developed Bovine Oocytes and Pre-Implantation Embryos

Abstract

Intergenic transcription, either failure to terminate at the transcription end site (TES), or transcription initiation at other intergenic regions, is present in cultured cells and enhanced in the presence of stressors such as viral infection. Such intergenic transcription has not been characterized in natural biological samples such as pre-implantation embryos which express more than 10,000 genes and undergo drastic changes in DNA methylation. Using Automatic Readthrough Transcription Detection (ARTDeco) and poly(A)-selected RNA-seq libraries from in vivo developed bovine oocytes and embryos, we found abundant intergenic transcripts that we termed as read-outs (transcribed from 5 to 15 kb after TES) and read-ins (transcribed 1 kb upstream of reference genes, extending up to 15 kb upstream). Read-throughs (continued transcription from TES of expressed reference genes, 4–15 kb in length), however, were much fewer. For example, the numbers of read-outs and read-ins ranged from 3084 to 6565 or 33.36% to 66.67% of expressed reference genes at different stages of embryo development. The less copious read-throughs were at an average of 10% and significantly correlated with reference gene expression (p < 0.05). Interestingly, intergenic transcription did not seem to be random because many intergenic transcripts (1504 read-outs, 1045 read-ins, and 1021 read-throughs) were associated with common reference genes across all stages of pre-implantation development. Their expression also seemed to be regulated by developmental stages because many were differentially expressed (log₂ fold change ≥ 2, p < 0.05). Additionally, while gradual but un-patterned decreases in DNA methylation densities 10 kb both up- and downstream of the intergenic transcribed regions were observed, the correlation between intergenic transcription and DNA methylation was insignificant. Finally, transcription factor binding motifs and polyadenylation signals were found in 27.2% and 12.15% of intergenic transcripts, respectively, suggesting considerable novel transcription initiation and RNA processing. In summary, in vivo developed oocytes and pre-implantation embryos express large numbers of intergenic transcripts, which are not related to the overall DNA methylation profiles either up- or downstream.