Journal: Nucleic Acids Research

Article Title: A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis

doi: 10.1093/nar/gkad247

Figure Lengend Snippet: Effect of TRIM24, TRIM33 and TRIM5α B boxes on L1 retrotransposition in HeLa cells. ( A ) Amino acid sequence alignment of TRIM24, TRIM33, TRIM5α and TRIM28 B boxes (24BB, 33BB, 5αB2 and 28BB, respectively) is performed using Clustal Omega method. Stars indicate the positions of the three amino acids where TRIM28 B box mutations are introduced (shown in Figure ). ( B ) Western blot analysis of the proteins expressed by B box constructs described in A. All indicated B box constructs are FLAG tagged on the C terminus to allow their detection. GAPDH is used as loading control. ( C ) Results of L1 retrotransposition assay in HeLa cells co-transfected with L1Neo-expression plasmid and one of the plasmids containing constructs shown in A. Images of flasks containing Neo R colonies corresponding to L1Neo retrotransposition are shown above the graph. Asterisks (*) denote statistical significance between listed constructs and the control ( n = 3, t -test, **** P < 0.0001, ## P = 0.0016) Dots represent number of Neo R colonies observed in individual experiments. Error bars represent the standard deviation (SD).

Article Snippet: Plasmids containing DNA encoding human and mouse TRIM28 were obtained from Origene (RC201205 and MR210883, respectively) and subcloned into a PCDNA 3.1/Hygro+ vector using NheI and HindIII restriction enzymes.

Techniques: Sequencing, Western Blot, Construct, Control, Transfection, Expressing, Plasmid Preparation, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis

doi: 10.1093/nar/gkad247

Figure Lengend Snippet: Both human TRIM28 (H-TRIM28) and mouse TRIM28 (M-TRIM28) increase L1-Neo retrotransposition. ( A ) Grey bars represent results of L1 retrotransposition assay in HeLa cells co-transfected with a plasmid expressing a Neo-tagged, full-length human wild type L1 and either an empty plasmid (control) or a plasmid expressing either H-TRIM28 or M-TRIM28. Images of flasks containing Neo-resistant (Neo R) colonies corresponding to L1Neo retrotransposition are shown above the graph. Purple bars are results of toxicity assay for which the pIRES2-EGFP vector carrying a constitutive Neo-resistant expression cassette (Neo R) is co-transfected with the control, H-TRIM28, or M-TRIM28 expressing plasmids. ( B ) Western blot analysis of endogenous TRIM28 expression in wild type U2OS cells (U2OS WT) and U2OS TRIM28 knock-out cells (U2OS KO). U2OS KO and U2OS WT cells transfected with a plasmid expressing FLAG tagged human TRIM28 are used as positive control. The lower molecular weight band (indicated by the arrow) corresponds to TRIM28. GAPDH is used as loading control. ( C ) L1 retrotransposition and toxicity assays performed in U2OS WT cells using plasmids and conditions described in (A). ( D ) L1 retrotransposition and toxicity assays performed in U2OS KO cells using plasmids and conditions described in (A). For all experiments asterisks (*) denote statistical significance between indicated experimental data points and the control ( n = 3, t -test, *** P < 0.001, **** P < 0.0001). Dots represent number of Neo R colonies observed in individual experiments. Error bars represent the standard deviation (SD).

Article Snippet: Plasmids containing DNA encoding human and mouse TRIM28 were obtained from Origene (RC201205 and MR210883, respectively) and subcloned into a PCDNA 3.1/Hygro+ vector using NheI and HindIII restriction enzymes.

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Western Blot, Knock-Out, Positive Control, Molecular Weight, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis

doi: 10.1093/nar/gkad247

Figure Lengend Snippet: Human and Mouse TRIM28 specifically interact with human L1 ORF2 protein. ( A1 ) Schematic of a full length L1, containing 5’UTR, two open reading frames (ORF1 and ORF2) and 3’UTR ending with a polyA site and a polyA tail. ( A2 ) Schematic of plasmids used for co-Immunoprecipitation assay in HeLa cells. ORF1p is not tagged. T7 indicates the position of the T7 tag in the ORF2p expressing plasmid. FLAG indicates the position of the FLAG tag in the TRIM28 expressing plasmid. ( B ) Results of co-IP using lysates of HeLa cells transfected with indicated plasmids (+) and anti-FLAG-beads assessed by western blot analysis. ORF1 (the upper bands slightly above GAPDH) is detected using anti-ORF1 antibodies. TRIM28 is detected using anti-FLAG antibodies. GAPDH is used as loading control (the lower bands). Input corresponds to assessment of protein expression in whole cell lysates. CoIP corresponds to the assessment of co-IP results. ( C ) Results of co-IP using lysates of HeLa cells transfected with indicated plasmids (+) and anti-FLAG-beads assessed by western blot analysis. ORF2p is detected using anti-T7 antibodies. TRIM28 is detected using anti-FLAG antibodies. GAPDH is used as loading control. The arrow indicates a non-specific band in the input lysates that masks detection of transfected ORF2p in HeLa cells. The asterisk indicates an ORF2p-specific band.

Article Snippet: Plasmids containing DNA encoding human and mouse TRIM28 were obtained from Origene (RC201205 and MR210883, respectively) and subcloned into a PCDNA 3.1/Hygro+ vector using NheI and HindIII restriction enzymes.

Techniques: Co-Immunoprecipitation Assay, Expressing, Plasmid Preparation, FLAG-tag, Transfection, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis

doi: 10.1093/nar/gkad247

Figure Lengend Snippet: N-terminal B box containing TRIM28 fragments increase L1 retrotransposition. ( A ) Schematic of TRIM28 fragments tested in the L1 retrotransposition assay. All fragments are generated from Human TRIM28, and FLAG-tagged at the C terminus. Names of constructs are reported on the left. The amino acid coordinates corresponding to each fragment are described in materials and method. ( B ) Results of L1 retrotransposition assay in HeLa cells using plasmids depicted in A. The number of Neo R colonies resulting from co-transfection of an empty plasmid with a plasmid expressing Neo-tagged, full-length human wild type L1 is used as control (control). Images of flasks containing Neo R colonies corresponding to L1Neo retrotransposition are shown above the graph. Asterisks (*) denote statistical significance between listed constructs and the control ( n = 3, t -test, **** P < 0.0001). Dots represent number of Neo R colonies observed in individual experiments. Error bars represent the standard deviation (SD).

Article Snippet: Plasmids containing DNA encoding human and mouse TRIM28 were obtained from Origene (RC201205 and MR210883, respectively) and subcloned into a PCDNA 3.1/Hygro+ vector using NheI and HindIII restriction enzymes.

Techniques: Generated, Construct, Cotransfection, Plasmid Preparation, Expressing, Control, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis

doi: 10.1093/nar/gkad247

Figure Lengend Snippet: Amino acids involved in TRIM28 multimerization are required for its ability to increase L1 retrotransposition. ( A ) Schematic of TRIM28 B box variants. BB is B box, WT is wild type, single or triple mutations are indicated using single letter amino acid code and amino acid position in the human wt TRIM28 protein. ( B ) L1 retrotransposition result. HeLa cells are transiently co-transfected with plasmids expressing human neomycin tagged L1 (L1Neo) and indicated TRIM28 BB variants. Images of flasks containing Neo R colonies corresponding to L1Neo retrotransposition are shown above the graph. ( C ) Schematic of full-length wild type TRIM28 (TRIM28 WT) and triple mutant TRIM28 (TRIM28 3M). Amino acid mutations are noted as described in A. ( D ) L1 retrotransposition result. HeLa cells are transiently co-transfected with plasmids expressing human neomycin tagged L1 (L1Neo) and indicated full-length TRIM28 variants. Images of flasks containing Neo R colonies corresponding to L1Neo retrotransposition are shown above the graph. Asterisks (*) denote statistical significance between listed constructs and the control ( n = 3, t-test, **** P < 0.0001). Dots represent number of Neo R colonies observed in individual experiments. Error bars represent the standard deviation (SD).

Article Snippet: Plasmids containing DNA encoding human and mouse TRIM28 were obtained from Origene (RC201205 and MR210883, respectively) and subcloned into a PCDNA 3.1/Hygro+ vector using NheI and HindIII restriction enzymes.

Techniques: Transfection, Expressing, Mutagenesis, Construct, Control, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis

doi: 10.1093/nar/gkad247

Figure Lengend Snippet: Results of analysis of differentially expressed genes in HeLa cells over-expressing control plasmid, TRIM28 WT, or TRIM28 3M. ( A ) The volcano plot shows differentially expressed genes in HeLa cells overexpressing TRIM28 WT versus Control. The horizontal grey line indicates P = 0.05. A fold change cutoff of 1.1 in the graph is shown as the dashed lines running parallel to the y-axis. Multiple DNA repair genes are significantly downregulated in TRIM28 WT, compared to Control. ( B ) The volcano plot shows differentially expressed genes in HeLa cells overexpressing TRIM28 WT versus TRIM28 3M. The horizontal grey line indicates P = 0.05. A fold change cutoff of 1.1 in the graph is shown as the dashed lines running parallel to the y-axis. Multiple DNA repair genes are significantly downregulated in TRIM28 WT, compared to TRIM28 3M. ( C ) The volcano plot shows differentially expressed genes in HeLa cells overexpressing TRIM28 3M versus Control. The horizontal grey line indicates P = 0.05 in Wald test. A fold change cutoff of 1.1 in the graph is shown as the dashed lines running parallel to the y-axis. The indicated DNA repair genes are not differentially expressed in TRIM28 3M compared to Control. ( D ) Heatmap of normalized expression of individual DNA repair genes that are significantly differentially expressed in HeLa cells transfected with TRIM28 WT compared to the control and TRIM28 3M expression plasmids. (Wald test, P < 0.05).

Article Snippet: Plasmids containing DNA encoding human and mouse TRIM28 were obtained from Origene (RC201205 and MR210883, respectively) and subcloned into a PCDNA 3.1/Hygro+ vector using NheI and HindIII restriction enzymes.

Techniques: Expressing, Control, Plasmid Preparation, Transfection

Journal: Nucleic Acids Research

Article Title: A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis

doi: 10.1093/nar/gkad247

Figure Lengend Snippet: Analysis of cDNA products generated by the ORF2p in HeLa cells transfected with wild-type or mutant TRIM28 or TRIM28 B Box. ( A ) Flow chart of the LEAP assay adapted from ( , ). ORF2p-generated cDNA is detected by PCR with a step wise set of ORF2 sequence specific forward primers (O1-O4) and a reverse primer Ro. In parallel, conventional RT-PCR was performed with the same set of step wise ORF2 primers. O1-O4: forward ORF2 specific primers. The expected length of PCR products is shown on the right. ( B , C ) LEAP samples are prepared by harvesting HeLa cells 48h post-transfection with indicated constructs and analyzed with indicated sets of primers. Control is LEAP prep on cells transfected with the empty plasmid (i.e. no ORF2p expression). RNA integrity in LEAP preps is assessed with the same set of ORF2 specific primers shown in A. A PCR product expected to be produced with O4 primer is absent in cells expressing WT full-length H-TRIM28 (TRIM28 WT) or WT B box (BB WT). Mutations of three amino acids responsible for multimerization (TRIM28 3M) eliminate this effect.

Article Snippet: Plasmids containing DNA encoding human and mouse TRIM28 were obtained from Origene (RC201205 and MR210883, respectively) and subcloned into a PCDNA 3.1/Hygro+ vector using NheI and HindIII restriction enzymes.

Techniques: Generated, Transfection, Mutagenesis, Sequencing, Reverse Transcription Polymerase Chain Reaction, Construct, Control, Plasmid Preparation, Expressing, Produced

Journal: Nucleic Acids Research

Article Title: A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis

doi: 10.1093/nar/gkad247

Figure Lengend Snippet: Analysis of L1 insertion in genomic DNA using ruler PCR assays . ( A ) Schematic of the ruler PCR assay adapted from . Three kilobase ruler PCR was performed using primers covering the 3kb target band. The position of primers relative to the L1 vector are shown. ( B ) PCR on genomic DNA sequence of HeLa cells transfected with L1-neo constructs was performed. Forward primer (F) and reverse primer (R) are applied. The L1-neo plasmid gives a band at 3771 bp, while the spliced L1-neo insertion gives a band at 2864 bp. Thirty-two clones were randomly picked and subjected to genomic DNA extraction. Left, number of clones with or without the spliced 3kb L1-Neo insertion in HeLa cells co-expressing L1-Neo construct and the control plasmid (PCDNA 3.1 empty vector). Middle, number of clones with or without the spliced 3kb L1-Neo insertion in HeLa cells co-expressing L1-Neo construct and TRIM28 WT plasmid. Right, number of clones with or without the spliced 3kb L1-Neo insertion in HeLa cells co-expressing L1-Neo construct and TRIM28 3M plasmid.

Article Snippet: Plasmids containing DNA encoding human and mouse TRIM28 were obtained from Origene (RC201205 and MR210883, respectively) and subcloned into a PCDNA 3.1/Hygro+ vector using NheI and HindIII restriction enzymes.

Techniques: Plasmid Preparation, Sequencing, Transfection, Construct, Clone Assay, DNA Extraction, Expressing, Control

Journal: Nucleic Acids Research

Article Title: A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis

doi: 10.1093/nar/gkad247

Figure Lengend Snippet: Analysis of length of tumor specific L1 insertions in WGS data set collected from endometrial, prostate, and ovarian cancer patients. ( A ) Sixteen patients with endometrial cancer are selected and grouped into two groups ( n = 8) according to high or low TRIM28 mRNA expression levels ( t -test, P < 0.0001). ( B ) Length of tumor specific de novo L1 inserts in endometrial cancer patients is significantly shorter in high TRIM28 group ( n = 324) than low TRIM28 group ( n = 491), t -test, P < 0.0001. ( C ) Sixteen patients with prostate cancer are selected and grouped into two groups ( n = 8) according to high or low TRIM28 mRNA expression levels ( t -test, P < 0.0001). ( D ) Length of tumor specific de novo L1 inserts in prostate cancer patients is significantly shorter in high TRIM28 group ( n = 293) than low TRIM28 group ( n = 326), t -test, P = 0.0135. ( E ) Sixteen patients with ovarian cancer are selected and grouped into two groups ( n = 8) according to high or low TRIM28 mRNA expression levels ( t -test, P < 0.0001). ( F ) Length of tumor specific de novo L1 inserts in ovarian cancer patients is significantly shorter in high TRIM28 group ( n = 383) than low TRIM28 group ( n = 386), t -test, P = 0.0319. For each individual figure, error bar represents the standard deviation (SD).

Article Snippet: Plasmids containing DNA encoding human and mouse TRIM28 were obtained from Origene (RC201205 and MR210883, respectively) and subcloned into a PCDNA 3.1/Hygro+ vector using NheI and HindIII restriction enzymes.

Techniques: Expressing, Standard Deviation