TdIF1 recognizes a specific DNA sequence through its Helix-Turn-Helix and AT-hook motifs to regulate gene transcription

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TdIF1 promotes transcription in a luciferase reporter assay.

<p>(A) TdIF1 promotes transcription. The pGL3-promoter-TdIF1-binding sequence and pRL-TK were co-transfected with pEGFP or pEGFP-TdIF1 into 293T cells, and the luciferase activity was assayed. The relative luciferase activity was normalized to the value obtained with the pGL3-promoter and pEGFP. Error bars represent S.E.M. Samples significantly different from the control are indicated by asterisks (p<0.01). (B) TdIF1 promotes transcription by directly binding to DNA. The pGL3-promoter-TdIF1-binding sequence and pRL-TK were co-transfected with pEGFP-TdIF1 truncated and point mutants, and the luciferase activity was assayed. Error bars represent S.E.M.</p

Identification of the DNA sequences recognized by TdIF1 by SELEX.

<p>(A) Flow chart of the SELEX experiment. (B) EMSA results in the 7th cycle. DNA was detected by EtBr staining. The arrow indicates the TdIF1/oligoDNA complex. (C) Oligonucleotides selected by SELEX aligned around the identified consensus 5′-GNTGCATG-3′, underlined in each case. Sequences of six or more continuous As or Ts are in red. Flanking linker sequence shown in italics. (D) Conserved nucleotides drawn as a sequence logo <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066710#pone.0066710-Crooks1" target="_blank">[20]</a>.</p

TdIF1 preferentially recognizes 5′-GNTGCATG-3′ following an AT-tract.

<p>(A) Sequences of the biotin-labelled AT-rich probe and the competitors used. Asterisks indicate replaced residues in the AT-tract or 5′-GNTGCATG-3′ motifs. (B) Competitive EMSA. All reaction mixtures contained 5 pmol of biotin-labelled AT-rich probe. Purified His-TdIF1 (100 ng) was incubated in the reaction mixture alone or with competitors (5, 15, or 45 pmol) as indicated (lanes 2–14). (C) Competitive EMSA using TdIF1mtHTH2, which has mutations in the HTH. TdIF1mtHTH2 (200 ng) or wild-type TdIF1 (100 ng) was incubated with biotin-labelled AT-rich probe alone or with competitors (15 or 45 pmol), as indicated.</p

Ubiquitylation of Terminal Deoxynucleotidyltransferase Inhibits Its Activity

<div><p>Terminal deoxynucleotidyltransferase (TdT), which template-independently synthesizes DNA during V(D)J recombination in lymphoid cells, is ubiquitylated by a BPOZ-2/Cul3 complex, as the ubiquitin ligase, and then degraded by the 26 S proteasome. We show here that TdT is ubiquitylated by the Cul3-based ubiquitylation system <em>in vitro</em>. Because TdT could also be ubiquitylated in the absence of Cul/BPOZ-2, we determined that it could also be directly ubiquitylated by the E2 proteins UbcH5a/b/c and UbcH6, E3-independently. Furthermore, the ubiquitylated TdT inhibited its nucleotidyltransferase activity.</p> </div

UbcH5a and UbcH6 promote TdT ubiquitylation.

<p>(A) UbcH5a enhances TdT ubiquitylation in 293 T cells. 293 T cells were transfected with plasmids encoding His-Ub (lanes 1, 3 to 5), Myc-TdT (lanes 2 to 5), and/or Flag-UbcH5a (lanes 1, 2, 4 and 5) in the indicated combinations. After a 24 h incubation, the cells were treated with 10 µM MG132 for another 12 h, lysed under denaturing conditions, and the ubiquitylated proteins were affinity-purified and separated by SDS-PAGE. Ubiquitylated Myc-TdT was detected by immunoblotting with an anti-Myc antibody. Myc-TdT and Flag-UbcH5a in the lysate were detected using an anti-Myc or anti-Flag antibody. (B) UbcH6 enhances TdT ubiquitylation in 293 T cells. 293 T cells were transfected with plasmids encoding His-Ub (lanes 1, 3 to 5), Flag-TdT (lanes 2 to 5), and/or Myc-UbcH6 (lanes 1, 2, 4 and 5) in the indicated combinations. After incubation for 24 h, the cells were treated with 10 µM MG132 for another 12 h. The cells were lysed under denaturing conditions, and the ubiquitylated proteins were affinity-purified and separated by SDS-PAGE. Ubiquitylated TdT was detected by immunoblotting using an anti-Flag antibody. Flag-TdT and Myc-UbcH6 in the lysate were detected using an anti-Flag or anti-Myc antibody. (C) UbcH7 does not enhance TdT ubiquitylation in 293 T cells. 293 T cells were transfected with plasmids encoding His-Ub (lanes 1, 3 to 5), Flag-TdT (lanes 2 to 5), and/or Myc-UbcH7 (lanes 1, 2, 4 and 5) in the indicated combinations. After a 24 h incubation, the cells were treated with 10 µM MG132 for another 12 h, lysed under denaturing conditions, and the ubiquitylated proteins were affinity-purified and separated by SDS-PAGE. Ubiquitylated TdT was detected by immunoblotting using an anti-Flag antibody. Flag-TdT and Myc-UbcH7 in the lysate were detected using an anti-Flag or anti-Myc antibody.</p