pNET-seq in Arabidopsis after flavopiridol (FLA) treatment

Eukaryotic euchromatin is the less compact chromatin and modified by many histone modifications such as H3 Lysine 36 methylation (H3K36me). Here we report a new chromatin state, Transcription Resistive, which is differentiated from activation and silencing. Transcription resistive is stamped by H3K36me with almost undetectable transcription activity but open chromatin state, and occupies most documented plant essential genes. Mutating SDG8, previously known as the major H3K36 methyltransferase in Arabidopsis, surprisingly elevates 78.7% H3K36me3 marked resistive loci, which count for 39.4% of the coding genome. Genetically, SDG8 prevents H3K36me activity of SDG4 at short and intron-less genes to secure plant fertility, while SDG8 collaborates with other H3K36me methyltransferases on long and intron-rich genes. Together, our results reveal SDG8 is the primary sensor to suppress excessive H3K36me, and uncovered the resistive is the conserved H3K36me-stamped novel transcription state in plants, highlighting the regulatory diversities and biological significances of H3K36 methylation in eukaryotes. To test the possibility that the transcription rate of some genes is too rapid to be effectively captured by pNET-seq, we conducted the flavopiridol (FLA, 10 μM) treatment (4 hours) with the 7-day seedlings to slow down the PolII elongation rate and then performed the PolII-Ser2p pNET-seq.