A role for the RNA pol II–associated PAF complex in AID-induced immune diversification

Abstract

Antibody diversification requires the DNA deaminase AID to induce DNA instability at immunoglobulin (Ig) loci upon B cell stimulation. For efficient cytosine deamination, AID requires single-stranded DNA and needs to gain access to Ig loci, with RNA pol II transcription possibly providing both aspects. To understand these mechanisms, we isolated and characterized endogenous AID-containing protein complexes from the chromatin of diversifying B cells. The majority of proteins associated with AID belonged to RNA polymerase II elongation and chromatin modification complexes. Besides the two core polymerase subunits, members of the PAF complex, SUPT5H, SUPT6H, and FACT complex associated with AID. We show that AID associates with RNA polymerase-associated factor 1 (PAF1) through its N-terminal domain, that depletion of PAF complex members inhibits AID-induced immune diversification, and that the PAF complex can serve as a binding platform for AID on chromatin. A model is emerging of how RNA polymerase II elongation and pausing induce and resolve AID lesions.

APOBEC3A and APOBEC3B Preferentially Deaminate the Lagging Strand Template during DNA Replication

“…AID's specificity for transcription-associated ssDNA appears to be facilitated by interactions with multiple proteins present in the transcription apparatus (Chaudhuri et al, 2004, Pavri et al, 2010, Willmann et al, 2012) as well as long non-coding RNAs transcribed from the immunoglobulin switch regions (Zheng et al, 2015). In the absence of direct recruitment to a specific ssDNA substrate, the activity of the majority of APOBECs may be dictated by the abundance and persistence of the ssDNA intermediate.…”

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.…”

Mutations, kataegis and translocations in B cells: understanding AID promiscuous activity

“…Other factors that associate with AID and might facilitate its recruitment to super-enhancers are the RNA Pol II complex 53 , the Pol II-associated factors SPT5 50 and SPT6 54 , the Pol II elongation PAF complex 55 , and chromatin modifiers, such as KAP1 56 . As discussed in detail in the next two subsections, the RNA exosome complex 57 and the splicing machinery might also play a role 58, 59 .…”