Identification of H4K20me3- and H3K4me3-associated RNAs using CARIP-Seq expands the transcriptional and epigenetic networks of embryonic stem cells

J Biol Chem. 2018 Sep 28;293(39):15120-15135. doi: 10.1074/jbc.RA118.004974. Epub 2018 Aug 16.


RNA has been shown to interact with various proteins to regulate chromatin dynamics and gene expression. However, it is unknown whether RNAs associate with epigenetic marks such as post-translational modifications of histones, including histone 4 lysine 20 trimethylation (H4K20me3) or trimethylated histone 3 lysine 4 (H3K4me3), to regulate chromatin and gene expression. Here, we used chromatin-associated RNA immunoprecipitation (CARIP) followed by next-generation sequencing (CARIP-Seq) to survey RNAs associated with H4K20me3- and H3K4me3-marked chromatin on a global scale in embryonic stem (ES) cells. We identified thousands of mRNAs and noncoding RNAs that associate with H4K20me3- and H3K4me3-marked chromatin. H4K20me3- and H3K4me3-interacting RNAs are involved in chromatin organization and modification and RNA processing, whereas H4K20me3-only RNAs are involved in cell motility and differentiation, and H3K4me3-only RNAs are involved in metabolic processes and RNA processing. Expression of H3K4me3-associated RNAs is enriched in ES cells, whereas expression of H4K20me3-associated RNAs is enriched in ES cells and differentiated cells. H4K20me3- and H3K4me3-interacting RNAs originate from genes that co-localize with features of active chromatin, including transcriptional machinery and active promoter regions, and the histone modification H3K36me3 in gene body regions. We also found that H4K20me3 and H3K4me3 are associated with distinct gene features including transcripts of greater length and exon number relative to unoccupied transcripts. H4K20me3- and H3K4me3-marked chromatin is also associated with processed RNAs (exon transcripts) relative to unspliced pre-mRNA and ncRNA transcripts. In summary, our results provide evidence that H4K20me3- and H3K4me3-associated RNAs represent a distinct subnetwork of the ES cell transcriptional repertoire.

Keywords: CARIP-Seq; H3K4me3; H4K20me3; RNA; chromatin; embryonic stem cell; epigenetics; histone modification.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromatin / genetics
  • Chromatin / metabolism
  • Embryonic Stem Cells / metabolism*
  • Epigenesis, Genetic / genetics*
  • Histone Code / genetics*
  • Histone-Lysine N-Methyltransferase / chemistry
  • Histone-Lysine N-Methyltransferase / genetics
  • Histones / chemistry
  • Histones / genetics
  • Methylation
  • Mice
  • RNA, Messenger / genetics
  • Transcription, Genetic*


  • Chromatin
  • Histones
  • RNA, Messenger
  • histone H3 trimethyl Lys4
  • Histone-Lysine N-Methyltransferase