Transposition of a reconstructed Harbinger element in human cells and functional homology with two transposon-derived cellular genes

Sinzelle et al. 10.1073/pnas.0707746105.

Supporting Information

Files in this Data Supplement:

SI Figure 7
SI Table 2
SI Figure 8
SI Figure 9
SI Figure 10
SI Figure 11
SI Figure 12
SI Figure 13
SI Figure 14
SI Figure 15
SI Experimental Procedures
SI Table 2

SI Figure 7

Fig. 7. Phylogenetic trees of the NAIF1 and HARBI1 proteins. The scale for the Jones-Taylor-Thornton (JTT) distances between the protein sequences is indicated as well as bootstrap values >50% at the tree nodes.

SI Figure 8

Fig. 8. Alignment of 23-bp-long integration sites derived from de novo integration events of Harbinger3_DR in human cells. The preferential target site and the alternative target site described in zebrafish are depicted above the alignment of 46 integration sites. The CWG triplet target site located in the middle is boxed in black, the conserved nucleotides compared in the preferential target described in zebrafish are boxed in gray. Of 46 integration events, 44 occurred in a CAG or CTG. For the other two, S14_2_ and S26_1_, integration occurred in CAA and TGG, respectively, which were subsequently duplicated upon insertion.

SI Figure 9

Fig. 9. Distribution of each nucleotide within the 46 integration sites compared to the zebrafish consensus target site. The total number of integration sequences, 46, corresponds to 100%. Each of the 46 integration sites retains at least 12 of the 17 base pairs of the zebrafish target sequence, and three of them (6%) perfectly match the zebrafish consensus.

SI Figure 10

Fig. 10. Transpositional activities of Harbinger3_DR out of donor-flanking DNA of different composition. (A) Sequences of flanking DNA in the donor plasmids. The 12-bp TIRs are boxed in black, and the TSDs are typed in black and boxed in gray. The nucleotide changes within the TSD and/or the flanking target site are typed in white letters with gray background. (B) Transposition assays. Transposition efficiencies were calculated by deriving ratios between the numbers of G418-resistant cell clones obtained in the presence versus in the absence of the transposase and the Myb-like protein. Mean values of relative efficiencies indicated at the bottom are derived from three independent transfections. The error bars show SEM. (C) WebLogo analysis of 23-bp insertion sequences surrounding the TSD for pHarb(SV40-neo)v1, pHarb(SV40-neo)v2, and pHarb(SV40-neo)v4 plasmids. The numbers of integration sites analyzed for each plasmid are indicated. (D) Alignment of consensus target sequences derived from integration events for each of the donor plasmids pHarb(SV40-neo), pHarb(SV40-neo)v1, pHarb(SV40-neo)v2, and pHarb(SV40-neo)v4. The target sequence established from in silico studies in zebrafish is depicted at the top for comparison. The conserved positions surrounding the CWG TSD are boxed in gray.

SI Figure 11

Fig. 11. The predicted HTH motif of the transposase is not required for physical interaction with the Myb-like protein. (A) Alignment of the HTH motif sequence predicted in the transposase (Tnp) and the corresponding disrupted sequence in the transposase mutant (Tnp-HTHm). The HTH motif was disrupted by introducing three amino acid substitutions [Leu®Pro(89), Ala®Pro(90), and Ser®Pro(99)] that are typed against a black background. The secondary structure prediction is depicted below and above the alignment [h, helix (in gray); c, coiled]. (B) Interaction of Tnp-HTHm/HA with Myb-like (80-221)/Myc protein by coimmunoprecipitation. The transposase was precipitated with an anti-HA antibody and the immunoprecipitated proteins (IPs) were analyzed for the presence of the Myb-like (80-221) protein by Western blot (WB) with an anti-Myc antibody. Transfection of JazzSB/HA served as negative control, whereas transfections of Tnp/HA served as positive control. (C) Colocalization assays of Tnp-HTHm/HA and Myb-like/Myc protein by immunofluorescence. Transfections of Tnp/HA with Myb-like/Myc and with Rep78/Myc served as a positive and a negative control, respectively.

SI Figure 12

Fig. 12. Representative fluorescent images of HeLa cells expressing the fusion proteins dsRed/Myb-like and EGFP/Tnp, and the EGFP protein. DAPI-stained nuclei (Left), GFP and dsRedFP fluorescence (Center), and merged images (Right) are shown. (Scale bars, 20 mm.)

SI Figure 13

Fig. 13. Quantification of red fluorescence reflecting the amount of HA-tagged Tnp localized in the nucleus. The percentage of red fluorescence in the nucleus (indicated on the y axis) was measured by using ImageJ sofware. The number of experiments is represented by "n." Numbers of cells analyzed for each experiment are indicated in parentheses.

SI Figure 14

Fig. 14. EMSA of increasing concentrations of MBP/NAIF1 (Left, 1´= 220 nM) and MBP/HARBI1 (Right, 1´ = 220 nM) mixed with a 486-bp Harbinger3_DR transposon probe corresponding to the 5'UTR of the transposase gene, the left TIR, and the flanking consensus target site.

SI Figure 15

Fig. 15. Model of the early steps of Harbinger element transposition. The transposase and the Myb-like protein form a complex in cells. This interaction requires domains located in the N-terminal region of the transposase (amino acids 1-141) and the C-terminal region of the Myb-like protein (amino acids 80-221). The Myb-like protein promotes nuclear import of the complex, and binds to subterminal sequences in the transposon ends through its trihelix domain. The Myb-like protein recruits the transposase to transposon DNA; a synaptic complex is formed and mediates the transposition process.

SI Experimental Procedures

Plasmids. All primer sequences for PCR are provided in SI Table 2. PCR amplifications were carried in a volume of 50 ml with 50 ng of plasmid DNA, 5 pmol of each of the primers, 0.2 mM dNTPs, and 1 unit of PfuUltraII Fusion HS DNA polymerase (Stratagene). The following PCR conditions were used: initial denaturation step at 94°C for 5 min, followed by 28 cycles at 94°C for 30 sec, 56°C for 30 sec, 72°C for 1-3 min depending on the expected size of the amplicons, and a final 5 min at 72°C. The integrity of all coding regions generated by PCR was verified by DNA sequencing.

The transposase and Myb-like protein cDNAs were synthesized by GENEART (Regensburg, Germany), and cloned into the BamHI/XhoI sites of pFV4a. The Harbinger3N_DR element was synthesized by GENEART and cloned into the EcoRI/HindIII sites of pUC19. pHarb(SV40-neo) was constructed by inserting an SV40-neo expression cassette (from pRcCMV-neo, Invitrogen) into the SpeI site of Harbinger3N_DR. The construct series pHarb(SV40-neo)v1, pHarb(SV40-neo)v2, pHarb(SV40-neo)v3, pHarb(SV40-neo)v4, and pHarb(SV40-neo)v5 was generated by PCR with primers 5'EcoRI/Pal/ITRL and 3'HindIII/Pal/ITRR; 5'EcoRI/ITRL and 3'HindIII/Pal/ITRR; 5'EcoRI/ITRL and 3'HindIII/Pal/Perf; 5'EcoRI/TAA/ITRL and 3'HindIII/TAA/ITRR; 5'EcoRI/ITRL and 3'HindIII/TAA/ITRR, respectively. The Harbinger3N_DR element was used as the template. The PCR products were inserted into the EcoRI/HindIII site of pUC19 and an SV40-neo expression cassette was cloned into the SpeI site of Harbinger3N_DR.

The pFV4a(Tnp/HA) plasmid was constructed by generating a PCR fragment encoding two copies of the hemagglutinin (HA) tag (YPYDVPDYA) on the C terminus of the Harbinger3_DR transposase with primers 5'BamHI Tnp and 3'XhoI HA Tnp, followed by cloning into pFV4a. The pFV4a(Tnp/HA) plasmid was used as the template for a series of PCR mutagenesis reactions performed with the QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene). The mutagenic primers 91-92L/A-P and 100-101S/S-P were used to obtain the desired Leu®Pro(89), Ala®Pro(90) and Ser®Pro(99) amino acid substitutions, resulting in the pFV4a(TnpHTHm)/HA plasmid.

pFV4a(Myb-like/Myc) contains two copies of the Myc tag (EQKLISEEDL) on the C terminus of the Myb-like protein, and was made using the primers 5'BamHI Myb-like and 3'XhoI Myc Myb-like, followed by cloning into pFV4a. To obtain the N- and C-terminal regions of the transposase cDNA, residues 1-141 and 136-343, respectively, were PCR-amplified using primers 3'XhoI Tnp HA N-term and 5'BamHI Tnp and primers 5'BamHI Tnp C-term and 3'XhoI HA, followed by cloning into pFV4a. The same PCR-based strategy (primers 5'BamHI Myb-like and 3'XhoI Myb-like Myc N-term and primers 5'BamHI Myb-like C-term and 3'XhoI Myc Myb-like) was followed to obtain pFV4a(Myb-like[1-85]/Myc) and pFV4a(Myb-like[80-221]/Myc) expressing the N-terminal and C-terminal region of the Myb-like cDNA, spanning from residues 1 to 85 and from residues 80 to 221, respectively.

The human HARBI1 coding region was PCR-amplified by using primers 5'NotI HARBI1h and 3'XhoI HARBI1h from the IMAGp998H2211568Q cDNA clone (RZPD). A second PCR amplification was done by using primers 5'NotI HARBI1h and 3'XhoI HA HARBI1h to generate a fragment containing two copies of the HA tag on the C terminus of HARBI1 cDNA. Both PCR products HARBI1_HS and HARBI1_HS/HA were cloned into pFV4a resulting in pFV4a(HARBI1_HS) and pFV4a(HARBI1_HS/HA). The zebrafish HARBI1 coding region from the IRAKp961F12178Q cDNA clone (RZPD) was directly cloned into the SmaI-XhoI restriction sites of pFV4a resulting in pFV4a(HARBI1_DR). The pFV4a(NAIF/Myc) expression vector was constructed by PCR amplification of NAIF1 (also called C9ORF90) cDNA on DKFZp762G199Q cDNA clone (RZPD) by using the primers 5'BamHI C9ORF90 and 3'XhoI Myc tag C9ORF90 containing two copies of the Myc tag and cloning into pFV4a.

To generate MBP fusion proteins, the transposase gene was subcloned into the BamHI-HindIII restriction sites of pMAL-c2 (New Englands Biolabs) resulting in pMAL-c2(Tnp). The coding sequences of Tnp[1-141]/HA, Tnp[136-343]/HA, Myb-like, Myb-like[1-85]/Myc, Myb-like[80-221]/Myc, and NAIF/Myc were cloned into pMAL-c2 following the same strategy. The HARBI1 cDNA was PCR-amplified with primers 5'XbaI HARBI1h and 3'HindIII HARBI1h, and subcloned into pMAL-c2 to create pMAL-c2(HARBI1h).

The pEGFP-C1(Tnp) encoding the Tnp fused to the C terminus of enhanced green fluorescent protein (EGFP) was generated by insertion of the Tnp cDNA (PCR-amplified with primers 5'XhoI Tnp and 3'EcoRI Tnp) into pEGFP-C1 (Clontech). The pDsRed monomer-C1(Myb-like) vector expressing the Myb-like protein fused to the C terminus of a monomeric variant of the red fluorescent protein (RFP) was created by insertion of the Myb-like cDNA (PCR-amplified with primers 5'XhoI Myb-like and 3'EcoRI Myb-like) into pDsRed monomer-C1 (Clontech).

PCR was done on Danio rerio genomic DNA (gDNA) to amplify the 5'-UTR of the transposase gene. The primers 5'-UTR Zf Harbinger locus and 3'-UTR Zf Harbinger locus were designed to amplify the 5'-UTR of a particular copy within the zebrafish genome displaying the highest similarity to the consensus sequence of Harbinger_3DR. PCR amplification was carried out in a volume of 50 ml with 300 ng of Danio rerio gDNA, 5 pmol of each of the primers, 0.2 mM dNTPs, and 1 unit of InviTaq DNA polymerase (Invitek). The cycling procedure was 94°C for 10 min with a hotstart, then 35 amplification cycles (94°C for 45 sec, 56°C for 45 sec, 72°C for 30 sec), followed by 5 min at 72°C. The amplicons were then subcloned into the pGEM-T vector system (Promega). A second PCR was performed on 50 ng of pGEM-T/5'-UTR in a total volume of 100 ml. Primers SalI/left ITR (50 pmol), ITRLuc2 (50 pmol), ITRLuc1 (1 pmol), 0.2 mM dNTPs, 2,5 unit of PfuUltraII Fusion HS DNA polymerase (Stratagene), and 0.1 unit InviTaq DNA polymerase (Invitek). The amplicons were subcloned into the NarI-SalI restriction sites of the reporter construct pCMVtk/Luc (1) to generate p5'-UTR/Luc.

Purification of MBP Fusion Proteins. The MBP fusion proteins were expressed and purified as described (2) with minor modifications. pMAL-c2-based expression plasmids were transformed into E. coli BL21-CodonPlus-RIL competent cells (Stratagene). For each recombinant clone, 800 ml of an overnight culture was transferred to 80 ml of LB medium and grown at 37°C until OD₆₀₀ = 0.5. Protein expression was induced with 0.3 mM IPTG and grown for 2 h at 37°C. Cells were pelleted and resuspended in 5 ml of Column Buffer [20 mM Tris·HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA]. The cell suspensions were sonicated six times for 15 sec each on ice, power 80%, by using a Bandelin Sonopuls sonicator. The lysates were clarified by centrifugation, and 500 ml of the supernatant was mixed with 100 ml of amylose resin (New England Biolabs) for 30 min at 4°C. The resin was washed four times with 1 ml of Column Buffer B [20 mM Tris·HCl (pH 7.4), 500 mM NaCl, 1 mM EDTA], and once with 1 ml of Column Buffer, supplemented with a protease inhibitor mixture. Bound protein was eluted with 100 ml of Column Buffer containing 10 mM maltose. Purified proteins were analyzed by Coomassie staining. Protein purification was also verified by Western blot analysis with an MBP monoclonal antibody. For each MBP fusion protein, the amount of purified protein was determined using the Bradford assay.

Immunofluorescence and Western Blotting. HeLa cells were grown on glass coverslips in 12-well plates and transfected with expression plasmids. Forty-eight hours after transfection, cells were washed three times with PBS supplemented with 1 mM CaCl₂ and 1.5 mM MgCl₂ (Mg/Ca PBS) and fixed in 4% paraformaldehyde in Mg/Ca PBS at room temperature (RT) for 20 min. After three washes with Mg/Ca PBS, cells were permeabilized with 0.5% Triton X-100 in Mg/Ca PBS for 12 min and washed twice again with Mg/Ca PBS. The blocking step was made by using 5% serum in Mg/Ca PBS for 5 min at RT followed by incubation in 0.5% gelatin in Mg/Ca PBS for 5 min at RT. Incubation with the primary antibodies (rabbit polyclonal anti-Myc tag, Abcam, 1:200 diluted, and rat monoclonal anti-HA High affinity, Roche, 1:100 diluted) was done in blocking solution 0.5% gelatin Mg/Ca PBS for 1 h at RT, followed by two washes with Mg/Ca PBS. Blocking was done as described above. The secondary antibodies were a goat polyclonal anti-rat IgG (H+L) coupled to Cyanine 3.5-OSu (Abcam) and a goat anti-rabbit IgG (H+L) coupled to Alexa Fluor 488, 1:100 and 1:500 diluted in blocking solution with 0.5% gelatin, respectively. Coverslips were incubated 30 min at RT with the secondary antibodies and then washed three times for 5 min with Mg/Ca PBS. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Fluorescence images were taken under an Olympus IX81 microscope equipped with CC12 digital camera and the CellˆF software (Soft Imaging System).

Coimmunoprecipitation. Cells were plated into 100-mm plates 1 day before transfection and then transfected with 3 mg each of both plasmid-encoding HA-tagged protein and Myc-tagged protein. After 48 h, cells were lysed in 1 ml of lysis buffer [50 mM Tris·HCl (pH 7.4), 1% Nonidet P-40, 0.25% Na-deoxycholate, 150 mM NaCl ,and 1 mM EDTA] supplemented with protease inhibitor mixture (Roche) and incubated on a shaker at 4°C for 15 min. All of the procedures were done at 4°C. The lysates were clarified by centrifugation at 14,000 ´ g for 15 min. After preclearing with 20 ml of protein G-agarose (Sigma) for 20 min, 200 ml of the lysates were incubated with 300 ng of anti-HA High affinity antibody (Roche) for 2 h. The immunocomplexes were captured by adding 50 ml of protein G-agarose and incubation for 3 h. The beads were washed extensively three times with 800 ml of lysis buffer, resuspended in 200 ml of SDS sample buffer, and boiled for 5 min. After separating by 10% or 15% SDS/PAGE, the proteins were detected by Western blotting by using specific antibodies.

Chromatin Immunoprecipitations. ChIP was performed as described previously (3). Briefly, chromatin preparation was done by using the ChIP-IT kit (Active Motif). Separate aliquots from each chromatin sample were incubated overnight at 4°C with 3 mg of monoclonal ChIP grade anti-HA antibody (Abcam). An aliquot was also retained and used as a loading control for PCR (input control). PCR was performed with total DNA and immunoprecipitated DNA by using the luciferase coding region-specific primers, Luc1 and LucFW. The length of the amplified product was 195 bp. The PCR products were subjected to electrophoresis on 2.5% agarose gel.

Luciferase Reporter Assays. One hundred fifty nanograms of the reporter constructs pTATA/Luc and p5'-UTR/Luc were cotransfected into HeLa cells with 150 ng of the expression plasmid pFV4a(Myb-like) and 100 ng of pCMV-bgal (CLONTECH). Luciferase activity was measured in a Lumat LB 9507 luminometer with a 10-second integration period. Luciferase units were normalized with b-galactosidase readouts for transfection efficiency and with the Bradford assay for total protein.

Bioinformatics and Sequence Analyses. Protein sequences were aligned with T-Coffee (4) and ClustalW (5) by using default parameters. The following sequences were included in the alignment in Fig. 1C. Myb proteins: Zeste (fruit fly), P42POP (human), XP_001134762 (chimpanzee); Harbinger-encoded Myb-like proteins: Harbinger2_DR and Harbinger3_DR (zebrafish), Harbinger-2_XT (frog), Harbinger-1_FR (fugu), Harbinger-N1_OL (medaka), Harbinger-1_CI (sea squirt), Harbinger1_TC (red flour beattle), Harbinger-1_NV (starlet sea anemone); NAIF1 proteins: NAIF1_DR (zebrafish), NAIF1_HS (human), NAIF1_MD (opossum), NAIF1_GG (chicken), NAIF1_XT (frog).

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