Fig. 3: BbYY1 effects on precise TIR-dependent DNA recombination mediated by bbRAG1L/2L ex vivo. | Nature Communications

Fig. 3: BbYY1 effects on precise TIR-dependent DNA recombination mediated by bbRAG1L/2L ex vivo.

From: Functional regulation of an ancestral RAG transposon ProtoRAG by a trans-acting factor YY1 in lancelet

Fig. 3

a The TR5 element on ProtoRAG TIR is overlapped by a YY1-binding motif. The rainbow indicates the YY1-binding motif in the JASPAR database. Orange indicates critical base pairs in the YY1-binding motif. b Schematic diagram of the detection of TIR-dependent DNA recombination through FACS. Briefly, the pTIRG8 construct and indicated bbRAG1L/bbRAG2L expression vectors were co-transfected into 293T or 293TshYY1 cells. After recombination, the polyadenylation poly(A) terminal signal would be excised off and the green fluorescent protein (GFP) would be expressed. The GFP signal could be detected by FACS. c Schematic diagram of TIR elements containing mutation plasmids pTIRG8, pTIRG10, pCptG, and pTIR104. The pTIRG8 and pTIR104 are WT TIR-containing constructs. The pTIRG10 is a substrate carrying deletion of both TR5 elements on TIRs. The pCptG is a construct with the mutated TR5 element on the 3′ TIR. CAT, chloramphenicol acetyltransferase  gene; CMV, CMV promoter; Lac, Lac promoter. d Quantification of GFP-positive cells by flow cytometry showed that the TR5 element of ProtoRAG was important for effective TIR-dependent recombination ex vivo. pTIRG8, pTIRG10, and pCptG are as indicated in c. See also Supplementary Fig. 4A. e The flow cytometry assay showed that bbYY1 participated in TIR-dependent recombination. Quantification of GFP-positive cells by flow cytometry after transfection of pTIRG8 substrate and bbRAG1L/2L expression vectors into 293T cells (or 293TshYY1 cells) with or without bbYY1 expression constructs. See also Supplementary Fig. 4B. f Diagrammatic sketch of precise and imprecise HDJ products in 293TshYY1 cells. See also Supplementary Fig. 5A. g Percentage of precise and imprecise HDJ recombination in 293T and 293TshYY1 cells. The values of d and e are the means ± s.d. with n = 3 biologically independent experiments. A two-tailed, unpaired Student’s t test was used for the comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant. For d–g, source data are provided as Source Data file.

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