Genomic codes governing enhancer RNA fate

  1. Chi Wai Yip
  2. Callum Parr
  3. Hazuki Takahashi
  4. Kayoko Yasuzawa
  5. Matthew Valentine
  6. Hiromi Nishiyori-Sueki
  7. Camilla Ugolini
  8. Valeria Ranzani
  9. Mitsuyoshi Murata
  10. Masaki Kato
  11. Wenjing Kang
  12. Giulia Fois
  13. Wing Hin Yip
  14. Youtaro Shibayama
  15. Andre Darah Sim
  16. Ying Chen
  17. Xufeng Shu
  18. Jonathan Moody
  19. Ramzan Umarov
  20. Manli Yang
  21. Alice Lambolez
  22. Jen-Chien Chang
  23. Luca Pandolfini
  24. Tsugumi Kawashima
  25. Michihira Tagami
  26. Tomoe Nobusada
  27. Tsukasa Kouno
  28. Carlos Alfonso-Gonzalez
  29. Rodrigo Pracana
  30. Raquel Sofia Silva
  31. Roberto Albanese
  32. Francesco Dossena
  33. Nejc Haberman
  34. Kokoro Ozaki
  35. Takeya Kasukawa
  36. Boris Lenhard
  37. Martin Frith
  38. Beatrice Bodega
  39. Francesco Nicassio
  40. Lorenzo Calviello
  41. Magda Bienko
  42. Ivano Legnini
  43. Valérie Hilgers
  44. Stefano Gustincich
  45. Jonathan Göke
  46. Charles-Henri Lecellier
  47. Jay W. Shin
  48. Chung-Chau Hon
  49. Piero Carninci
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Abstract

Long-read sequencing has transformed transcriptome profiling, yet capturing full-length, non-polyadenylated transcripts like enhancer RNAs (eRNAs) remains challenging. Here, we introduce CFC-seq, combining cap-trapping and in vitro poly(A)-tailing to sequence poly(A) and non-poly(A) RNAs with precise transcription start site. Paired with our assembler, SALA, we identified 39,425 novel transcriptional units, including ∼24,000 eRNAs. Our data reveal a distinct genomic code governing eRNA fate dictated by core promoter architecture. CpG-island enhancers show high chromatin connectivity but yield short, exosome-sensitive RNAs. Conversely, TATA-box enhancers systematically co-opt LTR retrotransposons to inherit structural motifs that produce long, stable, and spliced RNAs. Mechanistically, the pioneer factor NF-Y activates these viral elements to license transcription, balanced by TEAD4 activity across a dual-gear regulatory axis. Finally, non-poly(A) eRNAs terminate via exosome-associated processing at structural-depleted cleavage zones. This comprehensive annotation links enhancer sequence architecture to RNA fate, providing a new transformative framework for decoding the functional human genome.

Highlights

  • Expanded genomic architecture: CFC-seq unmasks a hidden layer of human transcriptome, identifying 39,425 novel transcriptional units with high-confidence TSS support, including ∼24,000 eRNAs.

  • TSS-first assembler: We introduce SALA, a specialized long-read assembler that prioritizes authentic 5’ Cap-trapped ends to accurately reconstruct the TSS-resolved transcript models.

  • Genomic code of eRNA fate: CGI enhancers drive short and exosome-sensitive transcripts associated with repressive H3K27me3 mark and high chromatin connectivity. TATA-box enhancers produce cell-type-specific, long, stable, and frequently spliced eRNAs.

  • Evolutionary co-option of retrotransposons: A major fraction of TATA-box eRNAs originate from LTR retrotransposons, providing a direct mechanism for integration of viral elements into the human regulatory landscape.

  • A dual-gear pioneering axis: The pioneer factor NF-Y activates unprimed LTR-TATA enhancers to license transcription independent of histone acetylation cascades, operating in parallel with TEAD4-mediated activation.

  • Structural determinants of eRNA termination: Non-poly(A) eRNA TES features a secondary structure depletion zone that coordinates pol II termination and calibrates exosome-mediated turnover.

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  1. Version published to 10.1101/2024.10.31.620483 on bioRxiv