Activation-Induced Cytidine Deaminase Targets DNA at Sites of RNA Polymerase II Stalling by Interaction with Spt5

Abstract

Summary Activation induced cytidine deaminase (AID) initiates antibody gene diversification by creating U:G mismatches. However, AID is not specific for antibody genes. Off-target lesions can activate oncogenes or cause chromosome translocations. Despite its importance in these transactions little is known about how AID finds its targets. To address this, we performed an shRNA screen. We found that Spt5, a factor associated with stalled RNA polymerase II (Pol II) and single stranded DNA (ssDNA), is required for class switch recombination (CSR). Spt5 interacts with AID, it is required for the association of AID and Pol II, and for AID recruitment to Ig switch regions and non-Ig targets. ChIP-seq experiments reveal that Spt5 co-localizes with AID and stalled Pol II. Further, Spt5 accumulation at sites of Pol II stalling is predictive of AID-induced mutation. We propose that AID is targeted to sites of Pol II stalling in part via its association with Spt5.

Histone H3.3 promotes IgV gene diversification by enhancing formation of AID ‐accessible single‐stranded DNA

“…However, pausing of RNAPII during transcription can lead to more extensive exposure of ssDNA in the transcription bubble (Gómez‐González & Aguilera, 2007) and a number of studies have suggested that transcriptional pausing is also a key mechanism to attract AID to the IgV mutation domain (Kenter, 2012). Supporting this idea, AID has been physically linked to RNAPII through its ability to binding the RNAPII‐associated processivity factor Spt5 (Pavri et al , 2010), the PAF complex (Willmann et al , 2012) and components of the exosome (Basu et al , 2011), the latter observation providing a potentially elegant solution to the problem of AID targeting both strands of DNA, as the exosome could facilitate exposure of the template strand in addition to the coding strand.…”

Accumulation of the FACT complex, as well as histone H3.3, serves as a target marker for somatic hypermutation

“…It has been proposed that Spt5, the component of another elongation factor DSIF, promotes SHM (9). To test whether SSRP1 knockdown affects Spt5 loading, we tested the Spt5 occupancy with SSRP1-depleted cells.…”

“…Spt5 binding is almost proportional to the level of RNAPII at genetic loci (9,17). Similarly, H3K4me3 serves as a general marker of active chromatin (18), and the Paf1 complex acts as a scaffold for the H3K4 trimethylase complex and is required for the elongation-promoting activity of the DSIF complex (19).…”

“…Studies by many groups including ourselves have revealed that several transcription elongation factors play important roles in the antibody diversification processes. Pavri et al (9) proposed that suppressor of Ty 5 homolog (Spt5), the large subunit of elongation factor DSIF (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole sensitivity-inducing factor), promotes both CSR and SHM by recruiting AID through physical interaction and by inducing RNAPII stalling. We previously showed that Spt5 is involved in the generation of H3K4me3 marking on chromatin, which is required for DNA cleavage by AID (11).…”

“…In addition to DNA structure, recent studies revealed the importance of transcription elongation in SHM targeting (8,9). Transcription elongation by RNA polymerase II (RNAPII) is a dynamic process that is controlled by a group of molecules called transcription elongation factors (10).…”