Animal, cells, antibodies, and related expression vectors

Adult Chinese amphioxus (~1 year old) B. belcheri were captured by using dense nets from the sea area nearby Zhanjiang city, China. After capturing, the amphioxus were put into a sea water-containing tank and transported to the laboratory. During transportation, the tank was kept at 18–25 °C. The captured amphioxus were cultured in a laboratory incubator under modeled wild conditions. All the experimental protocols for handling of adult Chinese amphioxus were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University, Guangzhou, China. All relevant ethical rules regarding the animals were compliant in this study. The 293T and Hela cell lines from ATCC, were maintained in DMEM (Gibco) supplemented with 10% FBS at 37 °C under 5% CO₂. Transfections were performed using JetPRIME (cat.: 114–15, PolyPlus-transfection Bioparc.) according to the manufacturer‵s instructions. Antibody reagents were purchased from the indicated manufacturers and diluted to appropriate concentrations for blotting analysis. Anti-Flag (1: 5,000; 66008-2-Ig, Proteintech), anti-GAPDH (1:10,000; 60004-1-Ig, Proteintech), anti-GST (1: 5000; 71097-3, Merck), anti-His (1: 5000; 70796-3, Merck), anti-MBP (1: 5,000; 66003-1-Ig, Proteintech or 1: 5,000; M 6295, Sigma)‚ anti-YY1 (1: 5,000; H414; sc-1703, Santa Cruz), anti-YY1 (1: 5000; C20; sc-281, Santa Cruz), goat anti-mouse IgG-HRP (1:10,000; HA1006, HuaBio) and goat anti-rabbit IgG-HRP (1:10,000; HA1001, HuaBio) were used in this study. The pTT5-MBP-bbRAG1L and pTT5-MBP-bbRAG2L vectors^6,7 used for bbRAG1L/2L protein expression were gifts from Prof. David G. Schatz’s lab. The hsYY1 (cat.: EX-F0023-M12), hsUSF1 (cat.: EX-F0238-M12), hsARNTL (cat.: EX-Z1347-M12), hsZBTB33 (cat.: EX-T1082-M12), and hsSTAT6 (cat.: EX-Y2605-M12) genes were cloned and constructed into the pEz-Flag (cat.:EX-NEG-M12) vectors for protein expression. These expression vectors were purchased from iGene Biotechnology Co., Ltd.

Luciferase reporter assays

In all, 293T cells were plated in 12- or 24-well plates, and after 24 h of culture, they were transfected with 500 ng DNA mixture per well. The mixed DNA contained indicated amounts of expression vectors and 30 ng phRL-TK plasmid (Promega). To normalize the transfection efficiency, deficiencies of expression plasmids were filled with empty plasmids. All samples were measured using the luciferase reporter assay system (Promega) according to the manufacturer’s instructions. Values are represented as means of relative simulations for a representative experiment from three independent experiments (n = 3), each performed in triplicate wells.

Electrophoretic mobility shift assays

Biotin-labeled oligomer and unlabeled competitor oligomer were synthesized by Thermo Fisher. These oligomers were annealed to probes in Tris buffer (10 mM Tris (pH = 8.0), 1 mM EDTA, and 50 mM NaCl) by PCR incubation. Briefly, oligo nucleotides were incubated at 95 °C for 5 min and then gradually chilled down to room temperature. Probes were prepared and stored at a concentration of 1 pmol µL⁻¹ and diluted to 10 fmol µL⁻¹ immediately before use. The EMSA probe sequences are listed in Supplementary Table 1.

Nuclear proteins were extracted from the 293T cells according to the NE-PER^® Nuclear extract protocol (cat.: 78835, Thermo Fisher). Volumes of 2–3 μL nuclear extracts or 500 ng–5 μg purified bbYY1 protein per 20 μL binding reaction were used. EMSA was performed according to the Light Shift Chemiluminescent EMSA Kit manual from Thermo Fisher (cat.: 20148). The binding reactions of protein and DNA were resolved on 8% non-denaturing polyacrylamide TBE gel and electrophoretically transferred to nylon membrane. The biotin-labeled DNA was finally detected using the chemiluminescence method.

Characterization of potential TSSs of bbRAG1L and bbRAG2L

Transfection of 293T cells was performed with plasmids containing bbRAG1L and bbRAG2L flanking sequences (pGL-R1-1, pGL-R2-1). At 24 h post-transfection, total RNA was isolated from these cells. Then, cDNA was synthesized from the total RNA using a PrimeScript RT-PCR kit (cat.: RR055B, Takara Bio.) according to the manual. 5′ RACE PCR was further conducted using well-designed primers for the identification of potential transcription start sites (TSSs). The RACE primers are listed in Supplementary Table 1.

Characterization of bbYY1

For bbYY1 gene cloning, the total RNA was then isolated from adult lancelet tissues using TRIzol (cat.: 11667165001, Roche). In total, three male and three female adult amphioxus were randomly selected, quickly frozen, and euthanized by liquid nitrogen in one round of the experiment. cDNA was synthesized from the total RNA using a PrimeScript RT-PCR kit (cat.: RR055B, Takara Bio.) according to the manual. Specific primers targeting the bbYY1 gene were designed and synthesized according to the prediction of the lancelet genome database. 5′ RACE and 3′ RACE PCR were then conducted for amplification of the bbYY1 gene from the newly synthesized cDNA of lancelet. The RACE PCR products were recovered from the DNA gel and inserted into the pGEM-T-easy vector for further sequencing. The identified bbYY1 gene fragments were finally assembled into a complete bbYY1 gene. The bbYY1 gene has been deposited in the NCBI under the accession numbers MF966513 [https://www.ncbi.nlm.nih.gov/nuccore/MF966513] and MF966514 [https://www.ncbi.nlm.nih.gov/nuccore/MF966514].

The bbYY1 homologous genes were searched in the NCBI database using the BLASTN program. Certain homologs from representative species were then downloaded from the GenBank database for alignment using CLUSTALW⁵³. The alignment results were finally colored using ESPript for display⁵⁴.

For analysis of the bbYY1 expression profile, qPCR was performed according to the ReverTra Ace qPCR RT Master Mix with gDNA Remover kit manual (cat.: FSQ-301, TOYOBO). The expression level of bbYY1 was calculated using the comparative 2^−ΔΔCt method. Values are represented as means of three independent experiments, with each performed in triplicate reactions. The RACE primers and qPCR primers are listed in Supplementary Table 1.

For analysis of the bbYY1 cellular sublocation, expression vectors of GFP-tagged bbYY1 or bbYY1 truncations were transfected into HeLa cells. At 24 h after transfection, cells were fixed in a 4% formaldehyde solution, washed, stained for 5 min with DAPI, and then washed and imaged using a confocal microscope SP5 (LEICA).

HsYY1 was knocked down by siRNA or shRNA in 293T cells

Synthetic siRNA oligos targeting hsYY1 and non-targeting control oligos were obtained from Guangzhou Ribo Bio Co. Ltd. The transfection of 293 T cells was performed with the indicated siRNA using Lipofectamine^® RNAiMAX according to the manual (cat.: 11668-019, Thermo Fisher Scientific). Cells were harvested at 48 h post-transfection. The hsYY1 knockdown efficiency was analyzed by western blotting (using anti-YY1 (H414; sc-1703) or anti-YY1 (C20; sc-281) antibodies, Santa Cruz). Signals were normalized to GAPDH.

For 293T^shYY1 cell construction, three pairs of hsYY1 shRNA oligomers were synthesized and annealed according to the manual’s instructions (Thermo Fisher Scientific). The lentiviral pLKO.1-shYY1 plasmids were first constructed. Viral particles were then prepared by transfecting psPAX2 and pMD2.G with pLKO.1-shYY1 plasmids into 293T cells. A more detailed description of the lentiviral package can be obtained from the instructions for Addgene Plasmid 10878. Finally, the constructed 293T^shYY1 cells were screened out via puromycin resistance and maintained as usual with puromycin (2 µg mL⁻¹) addition. The hsYY1 knockdown efficiency was confirmed by western blotting. Three pairs of hsYY1 shRNA oligomers are listed in Supplementary Table 1.

Flow cytometry assays

The 293T or 293T^shYY1 cells in 12 or 24-well plates were cultured and transfected with 100 ng substrate vectors and 200 ng protein expression vectors per well according to the requirements (including bbRAG1L/2 L protein expression vectors or the bbYY1 expression vector). After 48 h of culture, the cells were centrifuged, washed, and resuspended in PBS. The GFP signals were then analyzed using a Beckman CytoFLEX. For quantification of mCherry- and GFP-positive cells, the Beckman MoFlo Astrios EQs was used. CytExpert 1.2 and FlowJo 10 software were used for data analysis. All samples in each independent experiment were performed in triplicate. Values are represented as means of relative simulations for a representative experiment from three separate experiments (n = 3).

Bacterial colony assays

Bacterial colony assays with pTIR104 were conducted according to the following protocol. First, the pTIR104 (200 ng) vector together with the bbRAG1L (200 ng) and bbRAG2L (200 ng) expression vectors were co-transfected into 293T or 293T^shYY1 cells on a six-well culture plate. After the transfection, the cells were continually cultured for a further 48 h. Subsequently, the recombinant plasmid was recovered from the transfected cells through the alkaline lysis method and transformed into the E. coli DH5α. Kanamycin (100 µg mL⁻¹) and chloramphenicol (50 µg mL⁻¹) containing LB plates were used to select the positive clones. These positive clones were sent to sequencing for further analysis.

For the detection of HDJs from pdTIR, two iterations of PCR were performed. Briefly, after transfection of bbRAG1L/2L expression vectors, pdTIR together with hsYY1 siRNA were transfected into 293T cells for 48 h, and the recombinant pdTIR plasmids were isolated. The primer pair P1 and P2 (indicated in Supplementary Fig. 5C) was used to amplify HDJs after TIR-dependent recombination. The newly amplified HDJ products were then recovered and inserted into pGEM-T-easy vector followed by their transformation into E. coli. DH5α cells. The positive bacterial colonies were then selected and subjected to a second PCR using primer pairs P3 and P4 (indicated in Supplementary Fig. 5C). This second series of PCR products were then resolved on 2% agarose gels for electrophoresis band analysis. Moreover, the selected positive clones were sent for further sequencing analysis. The PCR primers used are listed in Supplementary Table 1.

Analysis of the 5′ and 3′ TIREs by LM-PCR and sequencing

Briefly, bbRAG1L/2L expression vectors (2 µg) together with pTIR104 (2 µg) substrate were co-transfected into 293T or 293T^shYY1 cells according to the JETPRIME protocol (cat.: 114-15, polyplus-transfection Bioparc.). After 48 h of incubation, recombinant DNA was recovered from these cells using the alkaline-SDS lysis method (see the E.Z.N.A.® Plasmid DNA Mini Kit I protocol). For LM-PCR, adapters were prepared by annealing FM1 with FM2 on the PCR instrument; 200 ng of the recovered DNA was ligated to the well-prepared adapters and then used as a PCR template for the reactions. Primer pairs P5/P6 and P6/P7 were used for PCR amplification of the 3′ and 5′ TIREs, respectively (adapters and PCR primers are listed in Supplementary Table 1). The two different rTaq Mix (30 cycles, cat.: RR901A, Takara) and KOD-plus-neo (45 cycles, cat.: KOD-401, TOYOBO) DNA polymerases and corresponding PCR procedures according to the manuals were used for each sample test. The PCR products of TIREs were resolved by electrophoresis of 1.5–2% agarose gels. After gel electrophoresis, target bands (TIREs) were then recovered through gel extraction kits and subjected to TA cloning for further sequencing analysis.

Analysis of DNA and protein sequences

The TIRE sequencing data were filtered using SeqMan 7.1.0 (44.1) software (Gene Star). The HDJs were analyzed using ApE v2.0.49.10 software with manual adjustment of the alignment parameter settings. The CREB, ATF, and PRC protein sequences were downloaded from the public NCBI database and aligned against the Chinese lancelet genome database⁵⁵ [http://genome.bucm.edu.cn/lancelet/] for homolog searching via the BLASTP program with the default parameter settings. The highest scoring homologs from the lancelet genome database were then downloaded. The online SMART server⁵⁶ [http://smart.embl-heidelberg.de/] was used for the prediction of the conservative domain architectures of these homologs. The MUSCLE in MEGA 5.2 or T-Coffee server⁵⁷ [http://www.tcoffee.org/] was used for evaluating and aligning DNA or protein sequences. Genedoc 2.7, BioEdit 7.0.5.2 software and ESPript 3.0 web server⁵⁴ [http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi] were used for the refinement or shading of the sequence alignments.

A co-crystal structure of the human YY1 zinc-finger domain bound to the adeno-associated virus P5 initiator was downloaded from the PDB database⁵⁸ (PDB: 1UBD [https://doi.org/10.2210/pdb1UBD/pdb]) and used for remodeling the core ZNF (cZNF) domain of bbYY1 (bbYY1_cZNF). The I-TASSER program⁵⁹ [https://zhanglab.ccmb.med.umich.edu/I-TASSER/] was used for the prediction of the bbYY1 structure. A model structure of the bbRAGL-3′ TIR synaptic complex with nicked DNA was downloaded from the PDB database⁷ (PDB: 6B40 [https://doi.org/10.2210/pdb6B40/pdb]). BbRAG2L and bbYY1_cZNF were displayed using PyMOL (TM) 1.7.4.5 Edu software as required.

Protein expression and purification

For His-tagged bbYY1 expression and purification, the pET28a-bbYY1 plasmid was transformed into E. coli. BL21(DE3) bacteria. The transformed bacteria were cultured to an OD = 0.6–0.8, chilled to 18 °C and induced with 0.5 mM IPTG overnight. After induction, the bacterial cells were collected and sonicated for lysis. The cell lysis supernatant was purified through Ni-NTA resin (cat.: 30210, Qiagen) with binding buffer A (25 mM Tris (pH = 8.0), 500 mM NaCl, 40 mM imidazole, 1 mM PMSF, and 1 mM DTT). The resin was slowly washed with increasing imidazole concentrations to ~160 mM. The eluate was collected and concentrated in vials and dialyzed with dialysis buffer (25 mM Tris, pH = 7.5, 150 mM KCl, 10% glycerol, and 2 mM DTT) through ultra-filtration.

For GST-tagged bbYY1 (or GST) expression and purification, the pGEX-6p-1-bbYY1 (or pGEX-6p-1)-containing plasmid was transformed into E. coli. BL21 bacteria. After culturing and inducing (the conditions were similar to those used for His-tagged bbYY1), the cultured bacterial cells were homogenized with lysis buffer B (25 mM Tris, pH = 7.5, 1 M KCl, and 1 mM DTT). The supernatant of the lysate was then purified through Glutathione Sepharose 4B beads (cat.: 17-5113-01, GE). The beads were washed with 10 column volumes of lysis buffer B and then eluted to obtain protein using 10 mM GSH-containing buffer (25 mM Tris, pH 7.5, 0.5 M KCl, 1 mM DTT, and 10 mM GSH). After elution, the target protein was collected, concentrated, and dialyzed using the same conditions described above.

All purified proteins were divided into small aliquots and stored at −80 °C before use. Protein purity was determined by SDS-PAGE followed by Coomassie Brilliant Blue (CBB) staining. The protein concentration was determined using the Bradford method.

Co-IP assays

The 293T cells were plated on six-well dishes (5 × 10⁶ cells per well) and transfected with 3 μg DNA plasmid (1.5 μg for each expression vector; if the target expression vector was insufficient, parallel empty plasmid was added). At 48 h post-transfection, the cells were lysed using western and IP lysis buffer (cat.: P0013, Beyotime Biotechnology). The lysate was incubated with primary antibodies (0.5∼1 μg Flag antibody (cat.:66008-2-Ig, Proteintech)) at 4 °C overnight and then incubated with Protein G Sepharose (cat.: 17061802, GE) for an additional 4 h at 4 °C. Analysis was conducted using SDS-PAGE electrophoresis followed by western blotting. The results were visualized using the ECL method (cat.: #C900376 and #C900377, Sangon Biotech (Shanghai) Co. Ltd).

GST-pulldown assays

Fragments of bbYY1 or hsYY1 were inserted into the pGEX-6P-1 vector for GST-fused protein constructions. The constructs were then transformed into E. coli. BL21 bacteria. The transformed bacteria were cultured at 37 °C to an OD = 0.6–0.8, chilled to 18 °C and induced with 0.5 mM IPTG. After 12 h of induction, the cells were collected, resuspended, and sonicated with lysis buffer (TBST with protease inhibitor cocktail). The lysate supernatant was incubated with 40 μL of Glutathione-Sepharose slurry (cat.: 17-5113-01, GE) for 1 h at 4 °C to pull down GST-fused protein.

The MBP-bbRAG1L/2L expression vectors were co-transfected into 293 T cells for MBP fusion protein preparation. The transfected cells were continually cultured 60 h after vector transfection and lysed using western and IP lysis buffer (cat.: P0013, Beyotime Biotechnology). The lysate supernatant was rotated with 40 μL of the above well-prepared slurry (containing GST or GST fusion protein) in TBST buffer (with protease inhibitor cocktail addition) at 4 °C overnight and washed four times with TBST. Finally, the slurry binding with proteins was eluted in Laemmli buffer (supplemented with 200 mM 2-mercaptoethanol) and resolved on 10% SDS-PAGE gels for further western blotting analysis. The ECL chemiluminescence method was used for the western blot analysis.

DNA pulldown assays

The method used was referenced from a previous description³³. First, for protein preparation, the MBP-bbRAG1L/2L and Flag-tagged bbYY1 expression constructs were co-transfected into 293T cells for protein expression. After 48 h of culture, the associated proteins were extracted from 293T cell lysates using western and IP lysis buffer (cat.: P0013, Beyotime Biotechnology). Second, for biotin-labeled probe preparation, the single-stranded biotinylated oligonucleotide and an equal quantity of antisense oligonucleotide were dissolved in deionized sterile water and annealed on the PCR instrument as described above for the EMSA method. Third, for DNA pulldown, 20 pmol biotin-labeled probes and appropriate competitor DNA (poly dA:dT or unlabeled 5′TIR) were added to 500 μL of the cell lysates. The ratios of competitor DNA and biotin-labeled probe were set to ranges of 1.25:1, 2.5:1, 5:1, 10:1, and 20:1. After mixing of the DNA and cell lysates, the mixtures were then set on a rotator for 30 min of pre-incubation at 4 °C. After pre-incubation, 30 μL streptavidin-agarose (cat.: S1638, Sigma or cat.: 20359, Thermo Scientific) was added to each mixture for an additional 1 h of incubation. Each sample was then washed with TBS (pH = 8.0, with protease inhibitor cocktails added (cat.: 5892791001, Roche)) by centrifugation at 1000 × g for 2 min three times. The centrifuged agarose pellets were then collected, resuspended in 50 μL Laemmli sample buffer and resolved on 8% SDS-PAGE gels for western blotting analysis. Images were obtained using ECL chemiluminescence.

Intermolecular transposition assays

The ex vivo plasmid-to-plasmid transposition assay was performed as recently described⁷. As follows, each 4 μg pTT5-MBP-bbRAG1L and pTT5-MBP-bbRAG2L expression vector, together with 6 μg donor plasmid (pTet-dTIR), 10 μg target plasmid (pEGFP-N1), and 4 μg YY1 expression vector (or empty vector), was co-transfected into 293T or 293T^shYY1 cells using polyethyleneimine. The medium was changed after transfection of cells for 24 h. Cells were continuously cultured for another 36 h and collected. Plasmid DNA was extracted from cell lysates using the alkaline-SDS lysis method (see the E.Z.N.A.® Plasmid DNA Mini Kit II protocol). Each 300 ng DNA was then transformed into MC1061 bacterial cells, which were plated onto the kanamycin-tetracycline-streptomycin (KTS) or kanamycin plates. The transposition efficiency was determined by calculating the ratio of KTS resistant to kanamycin resistant clones. All the KTS resistant clones were sent for sequencing for further transposition site analysis.

Statistical analysis

The results are expressed as means (±s.d.). The unpaired Student’s t test was used for comparisons. Values of P < 0.05 were considered statistically significant (two tailed), indicated by “*”; P < 0.01, indicated by “**”; P < 0.001, indicated by “***”; and P < 0.0001, indicated by “****”. P > 0.05 was not significant and is indicated by “ns”. All samples in each independent experiment were conducted in triplicate. Relative simulations for a representative experiment were from three separate experiments (n = 3) unless otherwise noted. The exact number of replicates and exact P values are indicated in figures or figure legends. Statistical analysis was performed using GraphPad Prism 8 and Microsoft Excel 2013.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.