Molecular mechanisms for the establishment of transcriptional memory are poorly understood. 5,6-dichloro-1-D-ribofuranosyl-benzimidazole (DRB) is a P-TEFb kinase inhibitor that artificially induces the poised RNA polymerase II (RNAPII), thereby manifesting intermediate steps for the establishment of transcriptional activation. Here, using genetics and DRB, we show that mammalian Absent, small, or homeotic discs 1-like (Ash1l), a member of the trithorax group proteins, methylates Lys36 of histone H3 to promote the establishment of Hox gene expression by counteracting Polycomb silencing. Importantly, we found that Ash1l-dependent Lys36 di-, tri-methylation of histone H3 in a coding region and exclusion of Polycomb group proteins occur independently of transcriptional elongation in embryonic stem (ES) cells, although both were previously thought to be consequences of transcription. Genome-wide analyses of histone H3 Lys36 methylation under DRB treatment have suggested that binding of the retinoic acid receptor (RAR) to a certain genomic region promotes trimethylation in the RAR-associated gene independent of its ongoing transcription. Moreover, DRB treatment unveils a parallel response between Lys36 methylation of histone H3 and occupancy of either Tip60 or Mof in a region-dependent manner. We also found that Brg1 is another key player involved in the response. Our results uncover a novel regulatory cascade orchestrated by Ash1l with RAR and provide insights into mechanisms underlying the establishment of the transcriptional activation that counteracts Polycomb silencing.
Transcriptional mechanisms in eukaryotes are composed of numerous consecutive steps, including chromatin modification and remodeling. Recent reports using yeast genetics have revealed that Lys36 methylation of histone H3, a hallmark of the active gene, is a consequence of transcriptional elongation. Similarly, a report using Drosophila genetics showed that exclusion of the Polycomb repressive complexes, general repressor complexes that regulate development and cellular differentiation, is another consequence of transcription. Here, we provide evidence that these causal relationships are not really general. By ceasing ongoing transcription at a certain step using an inhibitor in combination with mouse genetics, we have identified novel intermediate steps of transcription: Ash1l-mediated Lys36 methylation of histone H3 and subsequent exclusion of the Polycomb complexes that occur independently of transcriptional elongation. Furthermore, we show that binding of a nuclear receptor may promote trimethylation of Lys36 in its associated gene independent of its ongoing transcription. In this paper, we detail previously unknown key machineries orchestrated against Polycomb silencing, providing an innovative view of the mechanisms involved in the establishment of transcriptional memory.