Figures and data
DNA hypomethylation results in activation of L1TD1 expression and loss of L1TD1 affects cell viability in HAP cells.
(A) Quantification of DNA methylation levels at the L1TD1 promoter in HAP1 wildtype (WT), DNMT1 KO and DNMT1/L1TD1 DKO cells using the MethyLight assay. DNA methylation is shown as percentage of methylation ratio (PMR). (B) qRT-PCR analysis of L1TD1 mRNA expression in HAP1 wildtype (WT), DNMT1 KO and DNMT1/L1TD1 DKO cells. GAPDH was used as a normalization control and relative L1TD1 mRNA levels in DNMT1 KO cells were set to 1. All data in the figure are shown as a mean of ± SD of 3 biological replicates. (C) Western blot analysis of L1TD1 levels in HAP1 WT, DNMT1 KO and DNMT1/L1TD1 DKO cells. LAMIN B was used as a loading control. (D) Western blot analysis of L1TD1 protein expression in HAP1 WT and DNMT1 KO cells and OV-90 cells. β-actin was used as a loading control. (E) Indirect immunofluorescent staining of L1TD1 in HAP1 DNMT1 KO and DNMT1/L1TD1 DKO cells and OV-90 cells. Nuclear DNA was stained with DAPI (blue). (F) Cell viability analysis of HAP1 WT, DNMT1 KO and DNMT1/L1TD1 DKO cells using the CellTiter-Glo assay measured over 72h. (G) Bar graph representing the percentage of apoptotic cells of cultured cell lines quantified by flow cytometry analysis of cleaved caspase 3. (H) Western blot analysis of ɣH2AX levels in HAP1 WT, DNMT1 KO and DNMT1/L1TD1 DKO cells in nuclear extracts. Antibodies specific for histone H3 C-terminus and LAMIN B were used as loading controls. (A-B, F-G) Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparison correction. * p≤0.05, ** p ≤0.01, ** p ≤0.01, *** p ≤0.001, ns = not significant.
RIP-seq identifies a set of RNAs and transposon transcripts associated with L1TD1.
(A) Schematic representation of the RNA immunoprecipitation sequencing (RIP-seq) method (Figure created with BioRender.com). L1TD1-RNA complexes were isolated from HAP1 KO cell extracts with an L1TD1-specific antibody, RNA was isolated from complexes and input and cDNA libraries were prepared using the Smart-seq3 protocol. Sequencing data was analyzed by DEseq2 and TEtranscript software, separately. (B) Volcano plot showing L1TD1-associated transcripts as a result of DESeq2 analysis (cut-off log2FC > 2 and adj. p-value < 0.05). Selected hits are highlighted in green. (C) RIP-qPCR analysis confirms the interaction of L1TD1 to the transcripts L1TD1, ARMC1, YY2. The bar graphs represent the fold enrichments of the transcripts in the IP samples normalized to input samples in the indicated cells. GAPDH was used as a negative control for the top hit transcripts. (D) Volcano plot showing L1TD1-associated transposon transcripts as result of the TEtranscript analysis with a log2FC > 1 and adj. p-value < 0.05. (E) RIP-qPCR analysis confirms the association of L1TD1 with LINE-1 transcripts. Statistical significance was determined using paired two-tailed t-test. All data in the figure are shown as a mean of ± SD of 3 biological replicates. * p ≤0.05, ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001, ns = not significant.
© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.
L1TD1 cross-talk with its ancestor L1 ORF1p.
(A) Proteome analysis: Volcano plot displaying proteomics of HAP1 DNMT1 KO and DNMT1/L1TD1 DKO cells determined by mass spectrometry. Differentially abundant proteins were plotted as DNMT1/L1TD1 DKO over DNMT1 KO (log2FC ≥ 1, adj. p-value < 0.05 (red) and log2FC ≤ -1, adj. p-value < 0.05 (blue)). (B) Transcriptome analysis: Volcano plot illustrating DESeq2 analysis of RNA-seq performed in HAP1 DNMT1 KO and DNMT1/L1TD1 DKO cells. Differentially expressed genes are plotted as DNMT1/L1TD1 DKO over DNMT1 KO (log2FC ≥ 1, adj. p-value < 0.05 (red) and log2FC ≤ -1, adj. p-value < 0.05 (blue)). (C) Western blot analysis illustrating protein levels of L1TD1 and L1 ORF1p in HAP1 WT, DNMT1 KO and DNMT1/L1TD1 DKO cells. β-actin was used as loading control. (D) Physical interaction of L1 ORF1p and L1TD1. L1 ORF1p was immunoprecipitated with an L1 ORF1p-specific antibody from whole cell extracts prepared from DNMT1 KO and DNMT1/L1TD1 DKO cells and precipitated L1 RNP complexes were analyzed on a Western blot. IgG was used as a negative IP control and β-actin was used as loading control. (E) Confocal microscopy images of indirect immunofluorescence co-stainings using mouse ORF1p (green) and rabbit L1TD1 (red) antibodies in DNMT1 KO cells. Nuclear DNA was stained with DAPI.
L1TD1 promotes L1 retrotransposition.
(A) Schematic representation of plasmids used for retrotransposition (Figure modified from (Kopera et al., 2016)) (Figure created with BioRender.com). The pJJ101/L1.3 construct contains the full length human L1.3 element with a blasticidin deaminase gene (mblast) inserted in antisense within the 3’UTR. The mblast gene is disrupted with an intron and mblast expression occurs only when L1 transcript is expressed, reverse transcribed and inserted into the genome. The pJJ105/L1.3 plasmid contains a mutation in the reverse transcriptase (RT) resulting in defective retrotransposition. The backbone plasmid pCEP4 was used as additional negative control. The blasticidin deaminase gene containing plasmid pLenti6.2 was used as transfection/selection control. (B) Workflow of retrotransposition assay. DNMT1 KO and DNMT1/L1TD1 DKO cells were separately transfected with pJJ101 and control plasmids. Equal number of cells were seeded for each condition. Blasticidin selection (10µg/ml) was started at day 4 and resistant colonies were counted on day 13. (C) Bar graph showing the average number of retrotransposition events per 106 cells seeded in three independent experiments. Blasticidin resistant colonies in pLenti6.2 transfected cells were used for normalization. Statistical significance was determined using unpaired t-test. All data in the figure are shown as a mean of ± SD of three independent experiments, **** p ≤0.0001. (D) Representative pictures of bromophenol blue stainings of resistant colonies for each genotype and each transfection.
© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.
L1TD1 interacts with a specific set of mRNAs and transposon transcripts.
(A) Volcano plot shows the L1TD1-associated transcripts by L1TD1 enriched in DNMT1 KO IP over DNMT1/L1TD1 DKO IP with a log2FC ≥ 2 and adj. p-val < 0.05. The transcripts of L1TD1, SYNJ2BP, ARMC1 are highlighted in green. (B) and (C) DAVID Gene Ontology enrichment of 228 common transcripts identified as L1TD1 targets in both DESeq2 comparisons (DNMT1 KO versus input and DNMT1 KO versus DNMT1/L1TD1 DKO) in terms of biological processes (B) and sequence features (C).
GSEA analysis of deregulated proteins in HAP1 DNMT1/L1TD1 DKO versus DNMT1 KO cells.
(A) Gene ontology enrichment analysis of differentially expressed proteins upon loss of L1TD1. (B) Venn plot of the overlap of mRNAs identified in the RIP-seq analysis and differentially expressed proteins (up and down) identified in the mass spectrometry analysis. The single common mRNA is L1TD1.
Ablation of L1TD1 leads to changes in the transcriptome of HAP1 cells.
(A and B) qRT-PCR analyses of selected L1TD1-associated transcripts. Statistical significance was determined using unpaired two-tailed t-test. All data in the figure are shown as mean of ± SD of 3 biological replicates. ** p ≤0.01, ns not significant. (C) Volcano plot illustrating TEtranscript analysis of RNA-seq performed in HAP1 DNMT1 KO and DNMT1/L1TD1 DKO cells.
RNA-independent interaction of L1TD1 and ORF1p in HAP1 and OV-90 cells.
(A) Western blot analysis of ORF1p IPs with and without RNase A/T1 digestion in DNMT1 KO HAP1 cells using antibodies specific for ORF1p and L1TD1. DNMT1/L1TD1 DKO HAP1 cells were used as a negative IP control and β-actin was used as Western blot loading control. (B) Western blot analysis of ORF1p IPs with and without RNase A/T1 digestion in OV-90 cells using antibodies specific for ORF1p and L1TD1 in OV-90 cells. Mouse IgG was used as a negative control for the IP and β-actin was used as Western blot loading control. (C) Indirect immunofluorescence in OV-90 cells marks ORF1p (green) and L1TD1 (red) in cytoplasmic granules.
L1TD1 enhances LINE-1 retrotransposition.
(A) The bar graphs separately show the number of retrotransposition events per 106 cells seeded for three independent biological replicates. Blasticidin resistant colonies in pLenti6.2 transfected cells were used for normalization. Statistical significance was determined using unpaired t-test. All data in the figure are shown as a mean of ± SD of 3 technical replicates. ** p ≤0.01**** p ≤0.0001. (B) Representative pictures of bromophenol blue stainings of resistant colonies for transfection with the backbone plasmid pCEP4 and the blasticidin deaminase gene containing plasmid pLenti6.2 used as transfection/selection control.
