Fig. 1: Identification of cis-acting elements and YY1-binding motif of ProtoRAG. | Nature Communications

Fig. 1: Identification of cis-acting elements and YY1-binding motif of ProtoRAG.

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

Fig. 1

a Genomic architecture of ProtoRAG on BAC73 clone from B. belcheri BAC libraries and constructs generated for luciferase reporter assay. Potential TSSs are indicated in red. b Luciferase reporter experiments showed that both full-length 5′ and 3′ TIRs (TIR-FL) of ProtoRAG contain cis-acting elements that may drive the transcription of bbRAG1L and bbRAG2L. See also Supplementary Fig. 1A–D. c The 3′ TIR-FL of ProtoRAG can enhance the transcriptional activity of 5′ TIR-FL by luciferase reporter experiments. d The luciferase reporter assay indicated that the transcriptional activity of TIR-FL is comparable to that from the SV40 promoter but lower than that from the CMV promoter. e Cis-acting elements in both 5′ and 3′ TIR-FLs have bidirectional transcription activities according to the analysis of data from luciferase reporter assays. The reporter assays were performed in 293T cells with TIR-containing plasmids, as shown in a. f Schematic diagram of pdTIR and pdTIR-Mu constructs. pTIR-Mu indicates the construct with mutated conserved 9-bp TR5 elements. The 5′-GCCATCTTG-3′ element was mutated into 5′-GAACGCTTG-3′. g Homologs of trans factors that bind to TIRs with high match score by JASPAR prediction. h The luciferase reporter assay indicated that hsYY1 had the most significant transcriptional suppression of pdTIR among the high scored trans factors predicted by JASPAR. i Mini core cis-acting elements for TIR transcriptional activity, probes generated for EMSA and TIR elements for TIR-containing plasmids are indicated. j Representative gels of EMSA indicated that hsYY1 could bind to the YY1-binding motif-containing probes. The results are representatives from three independent experiments, with similar results. See also Supplementary Fig. 1F. The values of b–e and h are the means ± s.d. with n = 3 biologically independent experiments. A two-tailed, unpaired Student’s t-test was used for the comparisons in c–e and h. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant. For b–e, h, and j, source data are provided as Source Data file.

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