Activation-induced cytidine deaminase targets DNA at sites of RNA polymerase II stalling by interaction with Spt5

Abstract

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. We performed an shRNA screen to identify factors required for class switch recombination (CSR) of antibody loci. We found that Spt5, a factor associated with stalled RNA polymerase II (Pol II) and single stranded DNA (ssDNA), is required for CSR. Spt5 interacts with AID, it facilitates association between AID and Pol II, and AID recruitment to its Ig and non-Ig targets. ChIP-seq experiments reveal that Spt5 colocalizes 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.

Figure 1. Lentiviral-based shRNA screen in CH12 cells

CSR is sensitive to AID depletion. Flow cytometry plots of CH12 cells infected with five unique shRNAs to AID (shAID1-5) and empty vector control. Numbers indicate the percentage of IgA positive cells. (B) AID protein levels in whole cells extracts from the same cells shown in (A). Western blots were probed with an anti-AID antibody and anti-tubulin as a loading control. (C) Representative flow cytometry plots of CH12 cells infected with shRNAs against genes involved in CSR: Nfkb1 (NFκB p50 subunit), Prkdc (DNAPKcs catalytic subunit), Irf4, Runx3 and Tgfbr1 (TFGβ receptor 1) (Table S3). (D) Schematic of the experimental approach used for the screen. (E) Representative data from a single plate of shRNAs analyzed in triplicate. Error bars show the standard deviation obtained from the three replicate plates for %IgA+ cells (X axis) and cell numbers (Y axis). Negative (LacZ, GFP and RFP) and positive (AID) controls shRNAs are indicated. The dotted red line shows the position corresponding to 50% of the averaged negative control CSR value. Two sets of clones with <50% CSR are boxed. The upper box consists of viable clones that are considered as positive hits. The lower box contains clones that were discarded due to poor viability. (F) Pie chart showing the distribution of 181 selected hits as a function of the number of shRNAs per gene and their effect on CSR as calculated based on the percentage reduction of CSR compared to the averaged negative control values as shown in (E). See also Tables S2 and S3.

Figure 2. Spt5 is required for CSR and switch region mutation in CH12 cells

(A) Upper panel shows representative flow cytometry plots of CH12 infected with two unique shRNAs to Spt5 (shSpt5-1 and shSpt5-2) and controls (shLacZ and shAID) and stimulated to undergo CSR. Numbers indicate percentage of IgA positive cells. The graph in the lower panel summarizes the data from 4–6 independent experiments. (B) Decreased switch region mutation after Spt5 knockdown in CH12 cells. Upper panel represents the mutation frequency and corresponding p value from control (shLacZ) and shSpt5-1 infected cells stimulated to undergo CSR for 48 hrs. The table in the lower panel summarizes the mutation analysis (represented as unique mutations/nucleotides sequenced). (C) Graphs show Q-PCR analysis for Spt5, AID, Igα and Igμ germline (GLT) mRNA levels in activated CH12 cells infected as in (A) with the indicated shRNAs. The data summarizes three independent experiments with standard deviation indicated as error bars. In all cases, shLacZ was assigned an arbitrary value of 1.0. (D) Western blot analysis of Spt5 and AID protein levels in WCEs from activated CH12 cells infected with the indicated shRNAs. Threefold serial dilutions of WCEs were loaded. β-Actin was used as a loading control. Numbers below the blots represent normalized band intensities for Spt5 and AID with the shLacZ lanes assigned an arbitrary value of 1. (E) Spt5 is required for CSR in primary B cells. Representative flow cytometry plots of B cells stimulated with LPS + IL-4 and infected with retroviruses expressing shSpt5-1, shSpt5-2 or LMP vector alone. Efficiency of switching was determined by gating on GFP-positive cells. Numbers indicate percentage of IgG1 positive cells. The graph in the lower panel summarizes the data from three independent experiments. See also Figures S2 and S3.

Figure 3. Spt5 interacts with AID in fibroblasts and primary B cells

(A) Anti-Flag immunoprecipitates from whole cell extracts (WCEs) from 293T cells transfected with Flag-tagged AID (F-AID), or Flag-tagged Apobec2 (F-Apo2) or pMX vector probed with anti-Flag or anti-Spt5 antibodies as indicated. (B) Anti-Spt5 immunoprecipitates from WCEs from 293T cells transfected as in (A). Blots were probed as in (A). Anti-Blnk was used as an isotype control. (C) Anti-Flag immunoprecipitates from WCEs from cultured splenic AID^F/F B cells. Blots were probed as in (A). E1 and E2 represent first and second elutions with Flag peptide respectively. (D) Bacterially expressed GST-AID, GST-APOBEC2 (GST-Apo2) or GST alone were bound to glutathione sepharose beads and incubated with purified recombinant Spt5-Spt4 heterodimer (DSIF). Bound material was eluted with glutathione and analyzed by SDS-PAGE and blotted using antibodies against Spt5 and GST. The input lane for DSIF represents 1% of the amount used in the reaction. (E) Anti-Flag immunoprecipitates from WCEs of CH12 cells transfected with F-AID and depleted of Spt5 by shSpt5-1. shLacZ is used as a control. Blots were probed as in (A) and with anti-Pol II. (F) ChIP analysis for AID occupancy in Sμ regions of CH12 cells infected with shSpt5-1 or shLacZ control. Data represents a total of 7 experiments using two different anti-AID antibodies (Chaudhuri et al., 2004; McBride et al., 2006). For each experiment, shLacZ was assigned an arbitrary value of 1. The p value is indicated. See also Figures S4 and S5.

Figure 4. ChIP-Seq analysis of Spt5 genomic occupancy

(A) Venn diagram showing overlap between genes recruiting Spt5 and Pol II using ChIP-Seq data from LPS+IL4 activated B cells (Table S4). There was a significant association between the presence of Spt5 and Pol II at genes (Pearson’s Chi-square test; P < 0.0005). (B) Correlation between Spt5 and Pol II density per gene. For each gene that recruited above-background amounts of Pol II and Spt5, the number of sequence tags aligning between -1 Kb upstream of the transcriptional start site to its transcriptional termination site were normalized per gene length (in Kb), per million aligned reads (reads per Kb per million, RPKM) and shown as a hexagonal binning plot. Spearman’s correlation coefficient (ρ) is indicated. (C) Spt5 profile at all Spt5+ genes from −2 Kb to +5 Kb of the TSS. Data was normalized as reads per million per nucleotide. Dots represent densities at individual nucleotides and the line a 10 nucleotide moving average. (C) Correlation between the stalling index calculated based on Pol II or Spt5 occupancy (see Material and Methods). Spearman’s correlation coefficient (ρ) is indicated. (E) Comparative analysis of transcript levels (determined by mRNA-Seq, (Kuchen et al., 2010)) and Spt5 recruitment at all Spt5+ genes. Spearman’s correlation coefficient (ρ) is indicated. See also Tables S4 and S5

Figure 5. ChIP-seq profiles of Spt5 on selected genes

(A, B and C) Pol II and Spt5 reads per million plotted in 100 bp windows across (A) the Igμ locus, (B) Spt5^hi and (C) Spt5^lo genes. The axes scales are identical for all histograms. Tag mappability (shown below) was calculated based on the percentage of 36 nt sequences that uniquely aligned to the genomic site with a 10 bp window resolution. Only windows with a significant enrichment compared to a random background model are shown. The location of the TSS for each gene is indicated. The histograms cover the length of the gene. Whsc1 and Tnfaip3 were previously sequenced (Robbiani et. al., 2009). All profiles were generated using the UCSC genome browser. See also Tables S4 and S5.

Figure 6. Spt5 occupancy is predictive of AID-dependent somatic mutations

(A) Graphical representation of somatic mutation analysis for Spt^5hi and Spt^5lo genes from IgκAID and AID^−/− splenic B cells (see Figure 5B and 5C). Mutations in the AID^−/− control is subtracted in each case (see Figure S6) and mutation frequencies indicated. (B) Correlation between Spt5 and AID read density per gene. For each gene that recruited above-background amounts of AID and Spt5, the number of sequence tags aligning between -1 Kb upstream of the transcriptional start site to its transcriptional termination site were normalized per gene length (in Kb), per million aligned reads (reads per Kb per million, RPKM) and shown as a hexagonal binning plot. The Spearman’s correlation coefficient (ρ) is indicated. (C) ChIP analysis for AID occupancy at the Gas5 gene in CH12 cells infected with shSpt5-1 or shLacZ control. Data represents a total of 4 experiments using two different anti-AID antibodies. For each experiment, shLacZ was assigned an arbitrary value of 1. The p value is indicated. See also Figure S6.