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Genes to Cells (2006) 11, 47-57. doi:10.1111/j.1365-2443.2005.00916.x
© 2006 Blackwell Publishing or its licensors
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Direct binding of TReP-132 with TdT results in reduction of TdT activity

Seiichiro Fujisaki, Asami Sato, Tsuguri Toyomoto, Takahide Hayano, Maki Sugai, Takashi Kubota and Osamu Koiwai*

Faculty of Science & Technology, Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
N regions at the junction of V, D and J DNA segments are synthesized with large protein complexes including terminal deoxynucleotidyltransferase (TdT) during V(D)J recombination in B- or T-cells. TdT directly binds to TdIF1, TdIF2, PCNA and the Ku70/86 heterodimer. Using a yeast two-hybrid system, we isolated a cDNA clone encoding the gene for TReP-132, which is involved in P450scc gene expression in steroid-hormone-producing cells or lymphoid cells. Interaction between TReP-132 and TdIF1 was confirmed by pull-down assay and immunoprecipitation assay using specific antibodies against TReP-132 both in vitro and in vivo. TdT also directly bound to TReP-132 through its confined N-terminal region. Furthermore, the co-expression of TdIF1 and TReP-132 or TdT and TReP-132 in COS7 cells showed that these proteins are co-localized within the nucleus. TReP-132 reduces TdT activity to 2.5% of its maximum value in the in vitro assay system using double-stranded DNA with a 3' protrusion as a primer. These findings suggest that TdT synthesizes N region under a negative control of TReP-132 during V(D)J recombination.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
V(D)J recombination is initiated by the recognition of a recombination signal sequence (RSS) in T-cell receptor (TcR) or immunoglobulin (Ig) genes using recombination activation genes 1 (RAG1) and 2 (RAG2). RAG1 and RAG2 together induce the double-strand cleavage between the coding sequence and RSS to produce hairpin-coding ends. The generated hairpin-coding ends are opened by Artemis and DNA-PKcs (Ma et al. 2002), and terminal deoxynucleotidyltransferase (TdT) synthesizes deoxynucleotides (N nucleotides; N region) with Ku70, Ku86 and DNA-PKcs between V and D or between D and J DNA segments to increase the repertoires for TcR or Ig (Gottlieb & Jackson 1993; Bogue et al. 1997; Gu et al. 1997; Mahajan et al. 1999; Mickelsen et al. 1999; Purugganan et al. 2001). Finally, XRCC4 and DNA ligase IV connect N nucleotides with coding ends (Li et al. 1995; Grawunder et al. 1997; Mahajan et al. 2002).

To clarify the detailed mechanism of N region synthesis with TdT, we attempted to isolate genes with products that directly bind to TdT using yeast two-hybrid screening. We isolated the genes coding for proliferating cell nuclear antigen (PCNA) (Ibe et al. 2001), TdT interacting factor 1 (Dnttip1; TdIF1, Yamashita et al. 2001) and TdIF2 (Fujita et al. 2003). TdIF1 is a 37 kDa DNA-binding protein and has a high homology to the oncofetal protein p65 of the nuclear receptor superfamily (Hanausek et al. 1996). Compared with a typical member of the nuclear receptor superfamily, TdIF1 lacks the A/B domain (AF-1), which is found in the N-terminal region of the superfamily and works as a ligand-independent activator of transcription (Nagpal et al. 1993). However, the homologous region of TdIF1 with p65 extends from the rear half of the C4-type zinc finger to the C-terminal end, indicating that TdIF1 contains functional domains for hormone binding, DNA binding, dimerization, and the ligand-dependent transcriptional activation of AF-2. The proteins of the nuclear receptor superfamily generally bind to coactivator(s) or corepressor(s) to induce chromatin remodeling during transcriptional gene expression. Thus, we speculated that the chromatin structure is remodeled for easy access of TdT by forming a protein complex of TdIF1 and coactivator(s) or corepressor(s). Therefore, we attempted to isolate genes with products that directly bind to TdIF1 using the yeast two-hybrid system. Here, we show that TdIF1 directly binds to TReP-132, which is thought to function as a gene expression coactivator in hormone-producing or lymphoid cells (Gizard et al. 2001).

TReP-132 is a transcriptional coactivator bound to the upstream region of the gene coding for P450scc, which catalyzes the conversion of cholesterol to pregnenolone, the first step in the synthesis of steroid hormones (Pikuleva & Waterman 1999; Gizard et al. 2001). TReP-132 is expressed in many steroidogenic tissues, such as the adrenal cortex, prostate and testis and in some non-steroidogenic tissues, such as the lungs, skin, kidneys, uterus and thymus (Gizard et al. 2001, 2002a; Duguay et al. 2003). TReP-132 contains three C2H2 zinc fingers as well as elements frequently found in transcriptional-activating or chromosome-remodeling proteins, such as those in a glutamine-rich region, a proline-rich region, an ELM2 domain, a SANT domain and an acidic-amino acid-rich region (Klug & Schwabe 1995; Aasland et al. 1996; Solari et al. 1999). In addition, it contains two LXXLL motifs that are found in coactivators for nuclear receptors and assumed to bind to other nuclear receptors (Heery et al. 1997; McInerney et al. 1998). TReP-132 directly binds to CBP/p300 (Chrivia et al. 1993; Eckner et al. 1994; Lundblad et al. 1995; Bannister & Kouzarides 1996) and steroidogenic factor-1 (SF-1), which is an orphan nuclear receptor and regulates the gene expression for human P450scc. SF-1 also directly binds to CBP/p300 to synergistically activate the promoter for the P450scc gene by forming a ternary complex of TReP-132, CBP/p300 and SF-1 (Morohashi et al. 1993; Takayama et al. 1994; Watanabe et al. 1994; Monte et al. 1998).

In this study, we demonstrated the direct binding between TdIF1 and TReP-132, co-localization of TdIF1 and TReP-132 or TdT and TReP-132 in COS7 cells and DNA binding region in TReP-132. We further clarified the negative effects of TReP-132 on TdT activity. The reason why TReP-132 reduces TdT activity will also be discussed.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Isolation of cDNA clones binding to TdIF1

We isolated cDNA clones with products that directly bind to TdIF1 in yeast cells using the two-hybrid system. The full-length human cDNA coding for TdIF1 was fused to a plasmid encoding a GAL4 DNA-binding domain, and the human thymus cDNA library was screened. Sixteen candidate clones were isolated by screening 1.64 x 106 clones on Leu-, His- and Trp-dropout plates. All the plasmids constructed were transformed in E. coli (HB101) and their DNA sequences were determined. One (TS32) of the 16 clones showed a strong signal indicating its interaction with a bait protein (Fig. 1). When the DNA sequence of TS32 was compared with the NCBI sequences obtained using BLASTN and BLASTP algorithms (Altschul et al. 1997), TS32 was found to lack the N-terminal of TReP-132 variant 3 (Fig. 2A). TReP-132 has three transcriptional variants that are produced by the alternative splicing of its mRNA (Fig. 2). One variant (variant 2) lacks a proline-rich region, and another (variant 3) has, in addition to the loss of the proline-rich region, 12 extra amino acids inserted in the zinc finger motif at the C-terminal. We isolated the gene coding for variant 3 by yeast two-hybrid screening. In comparison between the DNA sequence of the gene for TReP-132 and that in the NCBI database, T's at positions 1511 and 2064 in the NCBI database were found to be converted to C's. The conversion from T to C at position 2064 indicates a change from serine to proline. The gene coding for TReP-132 was located at 6p21.2 on chromosome 6 and consisted of 17 exons (Fig. 2B), which were clarified by BLAST for the human genome.



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  		Figure 1  Interaction between TdIF1 and TS32 in yeast cell. Protein-protein interactions were detected on the basis of , ß-galactosidase activity by colony-lift filter assay. pACT2-TdIF1 encoded GAL4-AD fused with human TdIF1; pAS2-1-TdT, GAL4 DNA-BD fused with human TdT; pAS2-1-TS32, GAL4 DNA-BD fused with TS32. pACT2-TdIF1 and pAS2-1-TdT were co-transfected into yeast cells as a positive control and pACT2-TdIF1 was transfected into yeast cells as a negative control.

 


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  		Figure 2  Schematic representation of isoforms and genomic organization of TReP-132. (A) Three isoforms of human TReP-132 and TS32. TReP-132 has three transcript variants: variant 1, variant 2 (RAPA-1), and variant 3 (RAPA-2). In variant 3, 12 extra amino acids were inserted in the C2H2 zinc finger motif, located at amino acids 1015–1037 in variant 1. TS32 is absent in the N-terminal region in variant 3, which was isolated from human thymus cDNA library. LXXLL represents the nuclear-receptor-binding motif; GI, glutamine-rich region; C2H2, C2H2 zinc finger motif; P, proline-rich region; E, ELM2 domain; S, SANT domain; GA, glutamate-rich region. (B) Genomic organization variant 3. Boxes show the exons of TReP-132. Numbers in the boxes correspond to each other. Variant 3 consists of 17 exons in chromosome 6p21.1-p12.1.

 
Binding between TdIF1 and TReP-132 in vitro

To confirm the direct binding between TdIF1 and TReP-132 in vitro, we constructed GST-fusion vectors that express GST-TReP-132 (TS32) fusion proteins in E. coli. The fusion proteins expressed in E. coli were coupled with glutathione Sepharose 4B beads and the E. coli cell lysates expressing His-TdIF1 were reacted with GST-TReP-132 or GST bound to beads. After washing the beads thoroughly, we determined whether TdIF1 binds to TReP-132 bound to beads by Western blotting using a polyclonal rabbit antibody against TdIF1. As shown in Fig. 3A, TdIF1 was found to bind to GST-TReP-132 bound to beads (lane 3), but not to GST bound to beads (lane 2). These data support the results of the direct binding between TdIF1 and TReP-132 from the two-hybrid screening based on protein-protein interactions in yeast cells.



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  		Figure 3  Assay of binding between TReP-132 and TdIF1 or TdT in vitro. (A) Binding activity between deletion mutant TReP-132 del1 (TS32) and TdIF1 was analyzed by GST pull-down assay. Glutathione Sepharose beads were mixed and precipitated with E. coli lysates expressing GST and His-TdIF1 (lane 2), and GST-TReP-132 del1 and His-TdIF1 (lane 3). Lane 1 is a positive control. The bound proteins were analyzed by Western blotting. TReP-132 and TdIF1 were detected with rabbit anti-TReP-132 and anti-TdIF1 antibodies, respectively. (B) Binding activities between deletion mutants of TReP-132 (del1-8) and TdIF1 or TdT. These binding activities were analyzed by GST pull-down assay. Crude extracts from E. coli expressing GST-fused TReP-132 del1-8 were incubated with His-TdIF1 or His-TdT coupled to glutathione Sepharose 4B. Bound proteins were analyzed by Western blotting. B.A., binding activities; (+) positive interaction; (–) no interaction; (+/–) weak interaction.

 
TdIF1 binding region in TReP-132

Next, to identify the TdIF1 binding region in TReP-132, we constructed TReP-132 deletion mutants del2-del8 (Fig. 3B), and the binding between TdIF1 and TReP-132 deletion mutants was investigated by pull-down assay (column TdIF1 B.A. in Fig. 3B). TReP-132 contains two LXXLL motifs in the N- and C-terminal regions, which are protein-protein interaction motifs found in proteins bound to members of the nuclear receptor superfamily. We initially expected that TReP-132 would bind to TdIF1 through the C-terminal LXXLL motif, because the cDNA coding for TReP-132 containing only the C-terminal LXXLL motif had been isolated by yeast two-hybrid screening. However, as shown in Fig. 3B, TdIF1 unexpectedly bound to truncated TReP-132 residues ranging from 387 to 444 without the LXXLL motif and did not bind to del7, suggesting that TdIF1 binds to a confined region from 387 to 407 of TReP-132. Recently, TReP-132 has been reported to bind to SF-1 only through its N-terminal LXXLL, not through its C-terminal LXXLL (Gizard et al. 2002b). Thus, we then examined the possibility of binding through the N-terminal LXXLL. As shown in Fig. 3B (del8), no binding through the N-terminal LXXLL was observed. We therefore conclude that TdIF1 mainly binds to the novel confined region between residues 387 and 407 in TReP-132.

TReP-132 associates with TdIF1 in vivo

To confirm the binding between TReP-132 and TdIF1 in vivo, we performed immunoprecipitation assay. We initially examined the tissue for TReP-132 expression using swine organs, namely, the thymus, spleen, kidneys, testis and liver, which were homogenized in a lysis buffer, Western blotting was then performed. As shown in Fig. 4A, TReP-132 was detected only in the thymus, although mRNA coding for TReP-132 has been reported to be expressed in the kidneys, testis and thymus (Gizard et al. 2002a; Duguay et al. 2003). Since TdIF1 was also expressed in the thymus (Fig. 4B), an immunoprecipitation assay was performed using extracts from the thymus with a specific antibody for TReP-132. Figure 5A shows that TdIF1 was co-precipitated with TReP-132, indicating that TdIF1 associates with TReP-132 in vivo.



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  		Figure 4  Expression of swine TReP-132 and TdIF1. Swine tissue (0.15 g) was sonicated briefly with 135 µL of buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Triton X-100, 1 mM PMSF, and 10 µM pepstatin A) and centrifuged at 10 000 g for 20 min. The supernatant was examined by SDS-PAGE and Western blotting was performed with an (A) anti-TReP-132 or (B) anti-TdIF1 antibody. The tissues applied were thymus (lane 1), spleen (lane 2), kidney (lane 3), testis (lane 4) and liver (lane 5). V1, V2 and V3 represent TReP-132 variants 1, 2 and 3, respectively.

 


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  		Figure 5  Assays of binding between TReP-132 and TdIF1 or TdT in vivo and in vitro. (A) TReP-132 associates with TdIF1 and TdT in vivo. Lysate was prepared from swine thymus with a buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Triton X-100, 1 mM PMSF, and 10 nM pepstatin A). Immunoprecipitation was carried out with rabbit preimmune serum (lanes 2, 5 and 8) and a rabbit anti-TReP-132 antibody (lanes 3, 6 and 9), and the precipitate obtained was examined by SDS-PAGE. TReP-132, TdIF1 and TdT were detected with rabbit anti-TReP-132 (lanes 1–3), anti-TdIF1 (lanes 4–6) and anti-TdT (lanes 7–9) antibodies, respectively. One hundred micrograms of lysate was applied to lanes 1, 4 and 7 as a control. (B) Binding activity between TReP-132 and TdT in vitro. GST pull-down assay: His-TdT was incubated with GST-fused deletion mutants of TReP-132 (del1) or GST coupled to glutathione Sepharose 4B. The bound proteins were analyzed by Western blotting using rabbit anti-TReP-132 (lanes 1–3), anti-TdT (lanes 4–6) and mouse anti-GST (lanes 7–9) antibodies. Glutathione Sepharose beads were mixed with TdT and E. coli lysate expressing GST (lanes 2, 5 and 8) and TdT and E. coli lysate expressing GST-TReP-132 del1 (lanes 3, 6 and 9). Lanes 1, 4 and 7 are positive controls. (C) Competitive binding assay: His-TdT and His-TdIF1 were incubated with GST-fused TReP-132 del 5 coupled to glutathione Sepharose 4B. Five hundred micrograms of E. coli lysate expressing TdT was mixed with 0, 50, 500 and 5000 µg of E. coli lysates expressing TdIF1 (lanes 1, 2, 3 and 4, respectively). Five hundred micrograms of E. coli lysate expressing TdIF1 was mixed with 0, 50, 500 and 5000 µg of E. coli lysates expressing TdT (lanes 5, 6, 7 and 8, respectively). The bound proteins were analyzed by Western blotting using the rabbit anti-TdT (lanes 1–4) or anti-TdIF1 (lanes 5–8) antibody.

 
TReP-132 directly binds to TdT in vitro

TdIF1 directly binds to TdT and TReP-132. We therefore suspected that TdIF1, TdT and TReP-132 form a ternary complex. In such a case, TdT should be contained in the immunoprecipitant with TReP-132 as a bait. We checked for the presence of TdT by Western blotting. As shown in Fig. 5A, TdT was detected together with TdIF1, suggesting that TReP-132, TdIF1 and TdT form a ternary complex. However, if TdT and TReP-132 form a protein complex independently of the TdT/TdIF1 complex, TdIF1, TdT and TReP-132 may outwardly form a ternary complex obtained by immunoprecipitation assay, as shown in Fig. 5A. We then examined whether TdT directly binds to TReP-132 in vitro. As shown in Fig. 5B, surprisingly, TdT directly bound to TReP-132. To date, 16 DNA polymerases have been found in eukaryotic cells, and none of them have been reported to directly bind to transcriptional coactivators. Next, we attempted to determine the binding region in TReP-132 by pull-down assay. As shown in Fig. 3B (column TdT B.A.), the region corresponding to residues 407–457 in TReP-132 bound to TdT, indicating that the binding region of TReP-132 to TdT is present between residues 407–444. We first suspected that TdT binds to TReP-132 through the BRCT domain in its N-terminal region, which is considered to function as a protein-protein interaction domain. Thus, we constructed plasmids expressing the N- and C-terminal regions of TdT, expressed them in E. coli and performed pull-down assay. However, the C-terminal region bound to TReP-132, while the N-terminal region containing the BRCT domain did not (data not shown). As shown in Fig. 3B, the binding regions between TReP-132 and TdT or between TReP-132 and TdIF1 are confined in the contiguous region in TReP-132. We then determined whether both TdT and TdIF1 can simultaneously bind to TReP-132. As shown in Fig. 5C, the binding between TdT and TReP-132 decreased with increasing amount of TdIF1, strongly suggesting that TdT and TdIF1 cannot simultaneously bind to TReP-132.

Subcellular localization in a cell

We have clarified that TdIF1 binds to TReP-132 both in vitro and in vivo. Next, to examine whether TdIF1 can bind to TReP-132 in a cell, we constructed the EGFP-fusion plasmids pEGFP-TReP-132 and pDsRed1-TdIF1, and co-transfected them into COS7 cells, which were observed by immunofluorescence microscopy. As shown in Fig. 6A, when only EGFP-TReP-132 was expressed in the cells, immunofluorescence was observed within the entire nucleus and, in many cells, a few broad areas strongly fluoresced. Several signals of TdIF1 were observed as bright spots within the nucleus. A strong fluorescence from TdT was observed within the entire nucleus and many small spots were also detected. However, as shown in Fig. 6B, when EGFP-TReP-132 and DsRed1-TdIF1 or EGFP-TdT and DsRed1-TReP-132 were co-expressed, EGFP-TReP-132 and DsRed1-TdIF1 or EGFP-TdT and DsRed1-TReP-132 co-localized as distinctive foci within the nuclei of the COS7 cells, respectively, strongly supporting the speculation of the association between TReP-132 and TdIF1 or between TReP-132 and TdT in vivo. We also observed the co-localization of TdT and TdIF1 as many small spots within a nucleus.



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  		Figure 6  Subcellular localizations of TReP-132, TdIF1 and TdT in cell. (A) EGFP-TReP-132, TdIF1 and TdT fusion proteins were expressed in COS7 cells; these fusion proteins were observed by fluorescence microscopy. EGFP fusion proteins were localized within the nucleus. DNA was counterstained with 4,6-diamino-2-phenyl indole. EGFP was expressed in COS7 as a control. (B) Subcellular co-localization of TReP-132, TdIF1 and TdT. (1) EGFP-TReP-132 and RFP-TdIF1 were co-expressed in COS7 cells. Green and red represent the expressions of EGFP-TReP-132 and RFP-TdIF1 within the nucleus, respectively. (2) RFP-TReP-132 and EGFP-TdT were co-expressed in COS7 cells. Red and green represent RFP-TReP-132 and EGFP-TdT expressed within the nucleus, respectively. (3) EGFP-TdT and RFP-TdIF1 were co-expressed in COS7 cells. Green and red represent the expressions of EGFP-TdT and RFP-TdIF1 within the nucleus, respectively. Yellow represent the merged fluorescence image of EGFP and RFP.

 
TReP-132 binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA)

TReP-132 variant 3 has three C2H2 DNA-binding domains (Fig. 2A). To investigate whether these domains really function as DNA-binding domains, we analyzed the DNA-binding activity in five deletion mutants (del2–del6), each containing DNA-binding domains (Fig. 3B). GST-fused TReP-132 del2 was applied to ssDNA- and dsDNA-cellulose columns (Fig. 7A,H). TReP-132 del2 was eluted from ssDNA and dsDNA columns at 0.5 M and 0.7 M NaCl, respectively. The deletion mutants of TReP-132 del3, del4, del5 and del6 were eluted at 0.4, 0.3, and 0.2 M NaCl, respectively. Especially, when almost all of the ELM2 motif was deleted, the DNA binding ability was reduced to the level of del5, which contains a C2H2 motif, suggesting that C2H2 and ELM2 motifs are critical to the binding to DNA. On the other hand, the TReP-132 del7 mutant N-terminal region showed no DNA-binding activity for ssDNA cellulose (data not shown). Unexpectedly, both mutants with two C2H2 domains (residues 684-968) and without a C2H2 domain in which 12 amino acids (residues 824–968) are inserted showed no ssDNA-binding activity (Fig. 7F,G).



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  		Figure 7  DNA-cellulose column chromatography. GST-tagged TReP-132 deletion mutants (del2–6, 9 and 10) were applied to single-stranded or double-stranded calf thymus DNA-cellulose (Sigma-Aldrich) columns. After washing with buffer, the proteins bound to the column were eluted with a 0–2.0 M NaCl gradient. The eluate was analyzed by Western blotting. GST-tagged TReP-132 del2 (A), TReP-132 del3 (B), TReP-132 del4 (C), TReP-132 del5 (D), TReP-132 del6 (E), TReP-132 del9 (F) and TReP-132 del10 (G) were loaded on to ssDNA cellulose columns and detected with a rabbit anti-TReP-132 antibody. TReP-132 del9 and 10 correspond to the regions of the amino acid 684–968 and 824–903 of TReP-132 V3, respectively. (H) GST-tagged TReP-132 del2 was loaded on to a dsDNA-cellulose column and detected with a rabbit anti-TReP-132 antibody. The upper figures show the elution patterns of the column chromatography. Closed circles and triangles indicate salt concentration and elution pattern, respectively, when E. coli lysates containing GST-tagged TReP-132 deletion mutants (del2–6 and del9–10) were loaded on to the columns. Lower figures are the Western blotting profiles of the eluates. P, F and W1–W3 denote the positive controls, flow-through fraction and wash fractions with buffer, respectively; lanes 1–11 correspond to the fractional numbers 1–11, respectively.

 
TReP-132 and TdIF1 reduce TdT activity in vitro

First, we examined the utility efficiency of several types of ssDNA and dsDNA as template primers for TdT. When four types of ssDNA were used, TdT activity was dependent on the DNA sequences of the primers, as shown in Fig. 8(1–4). In particular, when a primer containing many dTs (S-R) was used, TdT expressed the highest nucleotidyltransferase activity. When we assessed the effect of TReP-132 or TdIF1 on TdT activity, TReP-132 and TdIF1 reduced TdT activity to 5% or 32% of its maximum value, respectively (Fig. 8(4)). Next, when dsDNA with a 3' protrusion, which mimics the DNA structure as a primer for TdT during V(D)J recombination, was used as a primer, TdT also efficiently utilized the primer and showed high activities. TdT activity was reduced to 2.5% and 24% of its maximum value in the presence of TReP-132 and TdIF1 (Fig. 8(5) and (6)), respectively. When a blunt-ended dsDNA was used as a primer, a low TdT activity was detected compared with that obtained with the use of dsDNA with a 3' protrusion. In this case, TReP-132 and TdIF1 also reduced TdT activity (Fig. 8(7)).



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  		Figure 8  Reduction of TdT activity by TReP-132 and TdIF1. TdT activities in the presence of TReP-132, TdIF1 or TReP-132 and TdIF1 were determined under standard assay conditions in vitro. Four types of ssDNA (S-A, S-C, S-F and S-R) and three types of dsDNA ("A" protrusion, "C" protrusion and blunt end) were used as primers. "A" and "C" protrusions have the 3' protrudent sequence with "AAAA" and ‘CCCC,’ respectively. Purified His-tagged TReP-132 del1, TdIF1 and TdT were used for each assay. TReP + TdT and TdIF1 +TdT show that TReP-132 and TdT, TdIF1 and TdT were mixed prior to the reaction with the primer, respectively. TdIF1 + (TReP + TdT) represents that TdIF1 and primers were incubated and then the mixture was added to the reaction mixture with TReP-132 and TdT. Bovine serum albumin (BSA) was used as a control for TdT activity. The data shown are the average of triplicate determinations.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
We isolated TReP-132 as a protein that binds to TdIF1, which directly binds to TdT, by yeast two-hybrid screening and clarified that TReP-132 binds to both TdIF1 and TdT, strongly suggesting that TReP-132 is involved in N region synthesis together with TdIF1 and TdT during V(D)J recombination in thymocytes. We initially expected that TReP-132 increases TdT activity because TReP-132 has been reported to function as a transcriptional activator (Gizard et al. 2001, 2002a, 2002b, 2004). However, TReP-132 unexpectedly reduced TdT activity. We suspect that this reduction in TdT activity by TReP-132 is caused by a unique mode of TdT action, by which TdT polymerizes a deoxynucleotide at the 3' end of ssDNA in a distributive manner. That is, TdT should be released from the catalyzing site after the addition of a nucleotide to the 3' end of DNA in order to add another nucleotide. However, if TReP-132 preliminarily binds to the 3' primer end, TdT could be captured by TReP-132 through protein-protein interaction; therefore, TdT cannot be easily released from the protein complex. As in the case of the use of the TdT/TReP-132 complex instead of TdT alone, a similar reduction in TdT activity was observed, which could be caused by the strong binding of TReP-132 to ssDNA. This could be the reason TdT activity is reduced by TReP-132. As shown in Fig. 8, TdIF1 as well as TReP-132 reduced TdT activity. TReP-132 and TdIF1 might reduce TdT activity by the same mode of action. Next, we examined whether TdIF1 influences the inhibitory effect of TReP-132 on TdT activity. Consequently, the level of reduction by TReP-132 was not influenced by the preliminary binding of TdIF1 to a primer, indicating the absence of the synergistic effect of TReP-132 and TdIF1 on TdT activity. This situation, in which a DNA-polymerase-binding protein keeps DNA polymerase on a DNA to reduce its polymerization activity, is very similar to the association between polymerase {lambda} (pol {lambda}) and PCNA. Pol {lambda} polymerizes nucleotides in a distributive manner, whereas PCNA is an auxiliary protein of DNA polymerase {lambda} that markedly increases the activity of this DNA polymerase. However, PCNA exhibits an inhibitory function against pol {lambda}. PCNA is considered to keep pol {lambda} at the DNA primer end and consequently reduces pol {lambda} activity to 24% of its maximum value (Shimazaki et al. 2002, 2005). TdT can synthesize several hundreds nucleotides at the 3' end of ssDNA in vitro, whereas N regions synthesized during V(D)J recombination consists of a few nucleotides in genes coding for Ig or TcR (Bollum 1974). Therefore, a reduced TdT activity by TReP-132 or TdIF1 might be sufficient for N nucleotide synthesis. Yamashita et al. (2001) reported that TdIF1 enhances TdT activity using oligo (dT)16 as primer. In their experiment, bovine TdT with {alpha}- and ß-subunits was used because of its high activity. On the other hand, in this study, we used purified full-length human TdT for experimental consistency. We suspect that the difference between our results and those of Yamashita et al. (2001) is caused by the difference between the enzymes used in our study and theirs, namely, full-length human TdT and bovine TdT with two subunits, respectively.

TReP-132 contains three C2H2 zinc fingers in V1 (Fig. 2A). However, no DNA-binding activity was detected in its C-terminal region containing the second and third zinc fingers using ssDNA-cellulose columns. The two C2H2 zinc fingers in the C-terminal of V1 are indispensable for P450scc gene expression (Gizard et al. 2002a). Twelve amino acids are inserted at the center of the consensus sequence of the second zinc finger in V3. The second zinc finger may originally have a DNA-binding ability in V1 and therefore be indispensable for functional expression, whereas it may be unnecessary in V3. Namely, we are tempted to speculate that there is no need for the second and third zinc fingers in V3 to have an ssDNA-binding ability and that only the region containing the first zinc finger is sufficient for V3 to perform N region synthesis.

This is the first report that TReP-132, which is a coactivator of the nuclear receptor, binds to TdT and affects the activity of deoxynucleotidyltransferase. Since TdT cross-talks with many proteins to synthesize N nucleotides of Ig or TcR genes in the thymus, further studies of the factors involved in V(D)J recombination are needed to clarify the TReP-132 function.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Yeast two-hybrid screening

Target clones were isolated with the Matchmaker GAL4 two-hybrid system (BD Clontech, USA). Full-length cDNA coding for human TdIF1 was subcloned into pAS2-1. Protein-protein interactions were analyzed from the expressions of the two reporter genes HIS3 and lacZ in yeast strain Y190 cells. The expressions of HIS3 and lacZ activity were detected on the basis of histidine prototrophy and ß-galactosidase activity, respectively. The DNA sequences of the isolated plasmids were determined using a fluorescent-dye-labeled primer cycle sequencing kit (Amersham Biosciences Corp., USA) and compared with NCBI sequences obtained using BLAST algorithms (National Center for Biotechnology Information (NCBI)) to search for homologous genes (Altschul et al. 1997).

Plasmids

cDNA for the N terminus of TReP-132 was subcloned into the pGEX4T-1 vector (Amersham Biosciences) by RT-PCR with SuperScript II (Invitrogen Corp., USA) from mRNA in MOLT4 cells. TdIF1 and TdT cDNAs were subcloned into the pET28 (EMD Biosciences, Inc., USA) vector to express His-fusion proteins in E. coli, as well as into pEGFP-C2 or pDsRed1-N1 (BD Clontech) to determine the localization of gene products. To determine the region of TReP-132 binding to TdIF1 and TdT, TReP-132 deletion mutants (del1-8) were subcloned into the pET28 or pGEX4T vector. The plasmids constructed are shown in Fig. 3. Full-length cDNA coding for TReP-132 was subcloned into pEGFP-C2 and pDsRed-N1 to elucidate the localization of gene products in the cells.

Antibodies

Rabbit polyclonal anti-TdIF1 and anti-TReP-132 antibodies were raised against human TdIF1 and TReP-132, respectively. A goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (HRP) and a horse anti-mouse IgG antibody conjugated with HRP were purchased from New England Biolabs.

GST pull-down assay

Glutathione S-transferase (GST) fusion proteins with TReP-132, TdIF1 and TdT were expressed in E. coli (BL21 (DE3) strain) using the expression vector pGEX4T-1. E. coli cells expressing fusion proteins were lyzed with a buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, and 10% glycerol). GST-fused TReP-132 (500 µg) was mixed with glutathione Sepharose beads (Sepharose 4B; Amersham Biosciences). The resulting mixture was incubated for 1 h at 4 °C, sufficiently washed with the buffer, mixed with 500 µg of His-fused TdIF1 or TdT, and incubated again for 1 h at 4 °C. In competitive binding assay, 0, 50, 500 and 5000 µg of E. coli lysates expressing His-fused TdIF1 or His-fused TdT were added to the reaction mixture containing His-fused TdT and GST-fused TReP-132 or His-fused TdIF1 and GST-fused TReP-132, respectively. The protein complexes obtained were extensively washed with the buffer and analyzed by Western blotting using the ECL Plus Western Blotting Detection system (Amersham Biosciences).

Tissue expression and immunoprecipitation

To determine whether TReP-132 is expressed in tissues, extracts from swine thymus, spleen, kidney, testis and liver were examined by SDS-PAGE and Western blotting. Swine thymus was lyzed with a buffer (40 mM Tris-HCl (pH 7.4), 40 mM KCl, 5 mM MgCl2, 0.1% Nonidet P-40, 1 µM pepstatin A, 1 mM PMSF, and 10% glycerol). Protein A/G bound Sepharose beads were mixed with 1 mg of tissue protein and 1 µg of normal rabbit IgG. The mixture was rotated for 1 h at 4 °C and centrifuged. The supernatant was collected, mixed with 2 µg of antibody for TReP-132, incubated for 12 h at 4 °C, mixed with ProteinA/G and incubated again for 12 h at 4 °C. The protein complex was immunoprecipitated with anti-TReP-132 and extensively washed with the buffer. All proteins complexed with TReP-132 were separated by SDS-PAGE. Immunoblotting was performed with the anti-TReP-132, anti-TdIF1 and anti-TdT antibodies.

Co-localization of TReP-132 and TdIF1 or TdT

TReP-132, TdIF1 and TdT were ligated with a fluorescent protein expression vector, pEGFP-C or pDsRed-N1. pEGFP-C2 and pDsRed-N1 into which TReP-132, TdIF1 or TdT was subcloned were transfected into COS7 cells with Lipofectin Reagent (Invitrogen) and observed by fluorescence microscopy 36 h after transfection. COS7 was cultured at 37 °C in DMEM supplemented with 10% (v/v) fetal bovine serum.

DNA-cellulose column chromatography

GST-fused TReP-132 was loaded on to DNA-cellulose columns. Single- or double-stranded calf thymus DNA-cellulose (Sigma-Aldrich, USA) column chromatography was performed to examine the DNA-binding activity of TReP-132. 250 µg each of GST-tagged TReP-132 del2-6, 9, 10 and GST protein was loaded on to an ssDNA- or dsDNA-cellulose column. The column was washed with 10 column volumes of a buffer (50 mM Tris-HCl (pH 7.5), 40 mM NaCl, 1 mM DTT and 10% glycerol) and eluted with a 0–2 M NaCl gradient. The fractionalized eluates were analyzed by Western blotting and protein bands were analyzed to determine protein concentration using an NIH image (NCBI).

TdT activity assay in vitro

The effect of TReP-132 on TdT activity was examined by standard assay for TdT activity using ssDNA or dsDNA as a primer. The reaction mixture used contained 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol, 1 mM MnCl2, 200 µg/mL BSA, 0.12 µM primer, 40 µM dTTP contains 0.5 µCi [3H]dTTP with a specific activity of 0.55 Ci/mmol, and proteins. The proteins used in the reactions were 0.12 µM each of His-tagged TReP-132 del 1 and His-tagged TdT, and 0.24 µM His-tagged TdIF1. The total volume of each reaction mixture was 25 µL. After preincubation of TReP-132 and TdT at 37 °C for 30 min, the mixture was added to the reaction mixture containing the ssDNA or dsDNA primer (Fig. 8) and TdIF1, which was preincubated at 37 °C for 30 min. Finally, dTTP was added to the resulting mixture and incubated at 37 °C for 60 min. Another procedure was also performed to assay the TdT activity. After preincubation of TdT and TReP-132 or TdIF1 at 37 °C for 30 min, the mixture was added to the reaction mixture containing the DNA primer and finally TdT was added to the mixture containing the ssDNA or dsDNA primer. The reaction mixture was spotted on a Whatman DE-81 disc, which was then washed five times with 5% disodium hydrogen phosphate and twice with water, and then dehydrated with ethanol. A dried disc was used for counting radioactivity with a scintillation counter (PerkinElmer Inc., USA).


   Footnotes
 
Communicated by: Kozo Kaibuchi

* Correspondence: E-mail: koiwai{at}rs.noda.sut.ac.jp


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 Introduction
 Results
 Discussion
 Experimental procedures
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Received: 13 September 2005
Accepted: 3 October 2005




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T. Kubota, S. Maezawa, K. Koiwai, T. Hayano, and O. Koiwai
Identification of functional domains in TdIF1 and its inhibitory mechanism for TdT activity
Genes Cells, August 1, 2007; 12(8): 941 - 959.
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