Figure 4

Details of the bidirectional, sense/antisense and combination-type mechanisms at Klf7 , Syn2/ AK038749 and Celf2 . (A) Close-up of the Klf7 downstream integration cluster. Three integrations are located in this cluster including one determined by RNA-seq in tumor 324 and two from the BALB/c mouse screen, one of which is positioned in the opposite orientation (604). RACE products obtained from tumors 324 and 2110 in the activated unannotated region are shown. At the 3′-LTR of the provirus in tumor 324 transcription proceeds in a direction from viral to mouse sequence (red arrow) showing that transcription is activated by promoter insertion at the integration site. (B) Close-up of the sense/antisense activation mechanism of AK038749 and Syn2. The proviruses are located in an H3K4Me1-enriched enhancer region in the Syn2 EPU [25], located ~11 kb from the Syn2 promoter (H3K4Me1/H3K4Me3-enriched region). The proximity (~200 bp) of Syn2 and AK038749 suggest activation through a bidirectional promoter. AK038749 expression was also confirmed by RACE. (C) The Celf2 intronic integration cluster contains five integrations in tumors from either RNA-seq (410) or the BALB/c mouse screen. In 410 the provirus is integrated ~25 kb from the other proviruses including the provirus in tumor 128 that was subjected to qPCR analysis (described in the main text). (D) The figure shows an alignment resulting from remapping of chimeric reads. Activation of Celf2 involves a combination-type mechanism including promoter insertion, enhancer activation and alternative (chimeric) splicing. The non-chimeric upstream transcript variant, and initiation of transcription in the 3′-LTR, was confirmed by 5′-RACE. The stippled angled lines indicate shortening of the intron. The curved bold arrow illustrates retroviral enhancer activation. Intronic splice donor (iSD). Transcription start site (TSS). In A-D, the proviruses identified in RNA-seq are shown in black, and angled arrows denote the 5′-LTR. In A-C, tumor numbers are indicated and the approx. integration positions.