DOT1L activity limits transcription elongation velocity and favors RNAPII pausing to facilitate mutagenesis by AID. [RNA-Seq]

Activation-induced deaminase (AID) initiates class switch recombination (CSR) and somatic hypermutation of immunoglobulin genes, which are required for efficient antibody responses. Collaterally, AID can also mutate a set of off-target genes with oncogenic consequences. The mechanisms that make such loci susceptible to AID are not fully understood. We find that the H3K79 histone methyltransferase DOT1L is located close to AID in the nucleus. Not only does DOT1L activity facilitate CSR, but it also facilitates AID off-target activity, including Igh-cMyc translocations. Genes mutated by AID display high DOT1L activity. DOT1L deficiency causes mostly increased nascent transcription, accompanied by a modest reduction in RNAPII occupancy. By integrating different genomic approaches, we discovered that DOT1L restricts the velocity of transcription elongation proportionally to the level of higher order H3K79 methylation (H3K79me2/3) and favors RNAPII pausing. These transcriptional conditions enhance AID occupancy and, consequently, its activity. Our data indicate a role for DOT1L and H3K79me2/3 in limiting transcription and highlight stronger RNAPII pausing and attenuated RNAPII elongation velocity as critical steps for productive AID association and mutagenesis at target loci. Furthermore, increased transcription elongation velocity could explain both upregulation and downregulation of genes in DOT1L-deficient cells. Overall design: To investige gene expression changes upon Dot1l depletion, CH12F3-G Dot1l knockout cells were generated by CRIPSR. We then perform paired-end RNA-seq of unstimulated WT and Dot1l-/- CH12F3 cells, using Kc167 cells as spike-in controls. Differential gene expression for WT vs Dot1l-/- CH12F3-G cells. Normalized counts, log2 fold changes and DESeq2 padj values are in the supplementary file "norm_counts.txt".