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Research
Lysann Schenk
Brandenburg University of Technology Cottbus-Senftenberg
Corresponding Author
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This work is licensed under a CC BY 4.0 License. Read Full License
Background
Lens epithelium derived growth factor splice variant of 75 kDa (LEDGF/p75), is overexpressed in different solid cancers and cancer cell lines and various autoinflammatory diseases. Due to its ability to bind chromatin, it acts as a transcriptional co-activator and promotes anti-apoptotic signalling pathways that lead to increased tumour aggressiveness and resistance to chemotherapy. The role of LEDGF/p75 in DNA-damage repair (DDR) is still not completely elucidated particularly regarding the ubiquitin-dependent regulation and degradation of DDR signalling molecules.
Methods
Different LEDGF model cell lines were generated, a complete knock-out of LEDGF (KO) as well as the re-expression of LEDGF/p75 or LEDGF/p52 using CRISPR/Cas9 technology. Then, various assays were performed to determine their proliferation and migration capacity as well as their chemosensitivity. Moreover, DDR signalling pathways were investigated by western blot and immunofluorescence.
Results
LEDGF-deficient cells exhibited a decreased proliferation (dt (WT) = 21 h, dt (KO) = 26 h) , 60 % decreased migration, as well as an 30-50 % increased sensitivity towards the topoisomerase II inhibitor etoposide. Moreover, LEDGF depleted cells showed a significant reduction by 65 % in the recruitment of downstream DDR-related proteins like replication protein A 32 kDa subunit (RPA32) after exposure to etoposide. Re-expression of LEDGF/p75 rescued all knock-out effects, while re-expression of LEDGF/p52 had no effect.
Surprisingly, untreated LEDGF KO cells showed an increased amount of DNA fragmentation combined with an increased formation of γH2AX and Breast cancer type 1 susceptibility protein (BRCA1). In contrast, the protein levels of ubiquitin-conjugating enzyme UBC13 and nuclear proteasome activator PA28γ were substantially reduced upon LEDGF KO.
Conclusions
This study provides evidence that LEDGF is not only an important player in the DDR after chemotherapeutic treatments but is also involved in the maintenance of the general genome integrity. Moreover, this study provides for the first time an insight into the possible role of LEDGF in the ubiquitin-dependent regulation of DDR signalling molecules and highlights the involvement of LEDGF/p75 in homology-directed DNA repair.
Keywords
LEDGF, CRISPR/Cas9, DNA damage signalling, γH2AX, ubiquitination
Figure 1
Figure 1
Figure 2
Figure 2
Figure 3
Figure 3
Figure 4
Figure 4
Figure 5
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- 201115sup.1.pdf
Verification of LEDGF-modified HEp-2 cells. Workflow generating of LEDGF KO (A.) and EGFP-LEDGF/p75 o/e in HEp-2 cells (B.). C. Verification of LEDGF KO in several single clones by Western Blot with anti C-LEDGF antibody and anti-GAPDH as loading control. Px_E1_GFP+ represents GFP positive bulk cells, whereas px_E1_GFP- represent the sorted fraction without GFP expression. D. Sequencing analysis of a representative LEDGF KO clone showed indel mutation which resulted in a premature stop codon. E. Several predicted off-target site with the highest sequence homology to the E1 sgRNA were analysed by sequencing. No off-target effects for the verified genomic loci were detected.
- 201115sup.1.pdf
Verification of LEDGF-modified HEp-2 cells. Workflow generating of LEDGF KO (A.) and EGFP-LEDGF/p75 o/e in HEp-2 cells (B.). C. Verification of LEDGF KO in several single clones by Western Blot with anti C-LEDGF antibody and anti-GAPDH as loading control. Px_E1_GFP+ represents GFP positive bulk cells, whereas px_E1_GFP- represent the sorted fraction without GFP expression. D. Sequencing analysis of a representative LEDGF KO clone showed indel mutation which resulted in a premature stop codon. E. Several predicted off-target site with the highest sequence homology to the E1 sgRNA were analysed by sequencing. No off-target effects for the verified genomic loci were detected.
- 201115sup.2.pdf
Proliferation rate and Cell cycle analysis of LEDGF-modified cells. A. Proliferation analysis with HEp-2 WT and LEDGF KO cells, amount of cells was determined using SRB assay after 72 h and 96 h, *p < 0.05, **p < 0.01. B. Cell cycle of ethanol-fixed, PI-stained HEp-2 cells was analysed by flow cytometry. Cell number was plotted against PI content leading in a typical cell cycle plot. C. Graphical display of counted cells resulted in 60 % G1-phase, 10-15 % S-phase, 10-15 % G2/M-phase and less than 5 % fragmented and polyploid cells.
- 201115sup.2.pdf
Proliferation rate and Cell cycle analysis of LEDGF-modified cells. A. Proliferation analysis with HEp-2 WT and LEDGF KO cells, amount of cells was determined using SRB assay after 72 h and 96 h, *p < 0.05, **p < 0.01. B. Cell cycle of ethanol-fixed, PI-stained HEp-2 cells was analysed by flow cytometry. Cell number was plotted against PI content leading in a typical cell cycle plot. C. Graphical display of counted cells resulted in 60 % G1-phase, 10-15 % S-phase, 10-15 % G2/M-phase and less than 5 % fragmented and polyploid cells.
- 201115sup.3.pdf
BRCA1 and γH2AX foci formation of LEDGF-modified U2OS cells. A. Generation of LEDGF KO U2OS cells as described in the Method section. The complete knockout was verified on the genomic level by sequencing as shown in the table. B.+C. Immunofluorescence staining of BRCA1 foci (B.) and γH2AX foci (C.) in U2OS WT, LEDGF KO and EGFP-LEDGF/p75 re-expressing cells and analysis by using NucDetect software,, n = 1.
- 201115sup.3.pdf
BRCA1 and γH2AX foci formation of LEDGF-modified U2OS cells. A. Generation of LEDGF KO U2OS cells as described in the Method section. The complete knockout was verified on the genomic level by sequencing as shown in the table. B.+C. Immunofluorescence staining of BRCA1 foci (B.) and γH2AX foci (C.) in U2OS WT, LEDGF KO and EGFP-LEDGF/p75 re-expressing cells and analysis by using NucDetect software,, n = 1.
- 201115sup.4.pdf
C. Different LEDGF expression levels of LEDGF/p75 re-expression clones. Immunoblot shows the level of C-LEDGF and GFP in different LEDGF re-expressing HEp-2 cells, protein amount = 50 µg. As loading control, coomassie was used.
- 201115sup.4.pdf
C. Different LEDGF expression levels of LEDGF/p75 re-expression clones. Immunoblot shows the level of C-LEDGF and GFP in different LEDGF re-expressing HEp-2 cells, protein amount = 50 µg. As loading control, coomassie was used.
- 201115sup.5.pdf
HDR template for EGFP-LEDGF/p75 re-expression in human AAVS1 locus. HDR template was designed with attB1/B2 sites and used for gateway cloning in mammalian expression vector pAAVS1-P-CAG-DEST
- 201115sup.5.pdf
HDR template for EGFP-LEDGF/p75 re-expression in human AAVS1 locus. HDR template was designed with attB1/B2 sites and used for gateway cloning in mammalian expression vector pAAVS1-P-CAG-DEST
- 201115sup.6.pdf
HDR template for mEmarald_LEDGF/p52 re-expression in human AAVS1 locus. HDR template was designed with aatB1/B2 sites and used for gateway cloning in mammalian expression vector pAAVS1-P-CAG-DEST
- 201115sup.6.pdf
HDR template for mEmarald_LEDGF/p52 re-expression in human AAVS1 locus. HDR template was designed with aatB1/B2 sites and used for gateway cloning in mammalian expression vector pAAVS1-P-CAG-DEST
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Lysann Schenk
Brandenburg University of Technology Cottbus-Senftenberg
Corresponding Author
Badges
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This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
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The most recent version of this article is available here.
No community comments so far
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Posted 24 Nov, 2020
No comments provided
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License:
This work is licensed under a CC BY 4.0 License. Read Full License
The most recent version of this article is available here.
Background
Lens epithelium derived growth factor splice variant of 75 kDa (LEDGF/p75), is overexpressed in different solid cancers and cancer cell lines and various autoinflammatory diseases. Due to its ability to bind chromatin, it acts as a transcriptional co-activator and promotes anti-apoptotic signalling pathways that lead to increased tumour aggressiveness and resistance to chemotherapy. The role of LEDGF/p75 in DNA-damage repair (DDR) is still not completely elucidated particularly regarding the ubiquitin-dependent regulation and degradation of DDR signalling molecules.
Methods
Different LEDGF model cell lines were generated, a complete knock-out of LEDGF (KO) as well as the re-expression of LEDGF/p75 or LEDGF/p52 using CRISPR/Cas9 technology. Then, various assays were performed to determine their proliferation and migration capacity as well as their chemosensitivity. Moreover, DDR signalling pathways were investigated by western blot and immunofluorescence.
Results
LEDGF-deficient cells exhibited a decreased proliferation (dt (WT) = 21 h, dt (KO) = 26 h) , 60 % decreased migration, as well as an 30-50 % increased sensitivity towards the topoisomerase II inhibitor etoposide. Moreover, LEDGF depleted cells showed a significant reduction by 65 % in the recruitment of downstream DDR-related proteins like replication protein A 32 kDa subunit (RPA32) after exposure to etoposide. Re-expression of LEDGF/p75 rescued all knock-out effects, while re-expression of LEDGF/p52 had no effect.
Surprisingly, untreated LEDGF KO cells showed an increased amount of DNA fragmentation combined with an increased formation of γH2AX and Breast cancer type 1 susceptibility protein (BRCA1). In contrast, the protein levels of ubiquitin-conjugating enzyme UBC13 and nuclear proteasome activator PA28γ were substantially reduced upon LEDGF KO.
Conclusions
This study provides evidence that LEDGF is not only an important player in the DDR after chemotherapeutic treatments but is also involved in the maintenance of the general genome integrity. Moreover, this study provides for the first time an insight into the possible role of LEDGF in the ubiquitin-dependent regulation of DDR signalling molecules and highlights the involvement of LEDGF/p75 in homology-directed DNA repair.
Figure 1
Figure 1
Figure 2
Figure 2
Figure 3
Figure 3
Figure 4
Figure 4
Figure 5
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- 201115sup.1.pdf
Verification of LEDGF-modified HEp-2 cells. Workflow generating of LEDGF KO (A.) and EGFP-LEDGF/p75 o/e in HEp-2 cells (B.). C. Verification of LEDGF KO in several single clones by Western Blot with anti C-LEDGF antibody and anti-GAPDH as loading control. Px_E1_GFP+ represents GFP positive bulk cells, whereas px_E1_GFP- represent the sorted fraction without GFP expression. D. Sequencing analysis of a representative LEDGF KO clone showed indel mutation which resulted in a premature stop codon. E. Several predicted off-target site with the highest sequence homology to the E1 sgRNA were analysed by sequencing. No off-target effects for the verified genomic loci were detected.
- 201115sup.1.pdf
Verification of LEDGF-modified HEp-2 cells. Workflow generating of LEDGF KO (A.) and EGFP-LEDGF/p75 o/e in HEp-2 cells (B.). C. Verification of LEDGF KO in several single clones by Western Blot with anti C-LEDGF antibody and anti-GAPDH as loading control. Px_E1_GFP+ represents GFP positive bulk cells, whereas px_E1_GFP- represent the sorted fraction without GFP expression. D. Sequencing analysis of a representative LEDGF KO clone showed indel mutation which resulted in a premature stop codon. E. Several predicted off-target site with the highest sequence homology to the E1 sgRNA were analysed by sequencing. No off-target effects for the verified genomic loci were detected.
- 201115sup.2.pdf
Proliferation rate and Cell cycle analysis of LEDGF-modified cells. A. Proliferation analysis with HEp-2 WT and LEDGF KO cells, amount of cells was determined using SRB assay after 72 h and 96 h, *p < 0.05, **p < 0.01. B. Cell cycle of ethanol-fixed, PI-stained HEp-2 cells was analysed by flow cytometry. Cell number was plotted against PI content leading in a typical cell cycle plot. C. Graphical display of counted cells resulted in 60 % G1-phase, 10-15 % S-phase, 10-15 % G2/M-phase and less than 5 % fragmented and polyploid cells.
- 201115sup.2.pdf
Proliferation rate and Cell cycle analysis of LEDGF-modified cells. A. Proliferation analysis with HEp-2 WT and LEDGF KO cells, amount of cells was determined using SRB assay after 72 h and 96 h, *p < 0.05, **p < 0.01. B. Cell cycle of ethanol-fixed, PI-stained HEp-2 cells was analysed by flow cytometry. Cell number was plotted against PI content leading in a typical cell cycle plot. C. Graphical display of counted cells resulted in 60 % G1-phase, 10-15 % S-phase, 10-15 % G2/M-phase and less than 5 % fragmented and polyploid cells.
- 201115sup.3.pdf
BRCA1 and γH2AX foci formation of LEDGF-modified U2OS cells. A. Generation of LEDGF KO U2OS cells as described in the Method section. The complete knockout was verified on the genomic level by sequencing as shown in the table. B.+C. Immunofluorescence staining of BRCA1 foci (B.) and γH2AX foci (C.) in U2OS WT, LEDGF KO and EGFP-LEDGF/p75 re-expressing cells and analysis by using NucDetect software,, n = 1.
- 201115sup.3.pdf
BRCA1 and γH2AX foci formation of LEDGF-modified U2OS cells. A. Generation of LEDGF KO U2OS cells as described in the Method section. The complete knockout was verified on the genomic level by sequencing as shown in the table. B.+C. Immunofluorescence staining of BRCA1 foci (B.) and γH2AX foci (C.) in U2OS WT, LEDGF KO and EGFP-LEDGF/p75 re-expressing cells and analysis by using NucDetect software,, n = 1.
- 201115sup.4.pdf
C. Different LEDGF expression levels of LEDGF/p75 re-expression clones. Immunoblot shows the level of C-LEDGF and GFP in different LEDGF re-expressing HEp-2 cells, protein amount = 50 µg. As loading control, coomassie was used.
- 201115sup.4.pdf
C. Different LEDGF expression levels of LEDGF/p75 re-expression clones. Immunoblot shows the level of C-LEDGF and GFP in different LEDGF re-expressing HEp-2 cells, protein amount = 50 µg. As loading control, coomassie was used.
- 201115sup.5.pdf
HDR template for EGFP-LEDGF/p75 re-expression in human AAVS1 locus. HDR template was designed with attB1/B2 sites and used for gateway cloning in mammalian expression vector pAAVS1-P-CAG-DEST
- 201115sup.5.pdf
HDR template for EGFP-LEDGF/p75 re-expression in human AAVS1 locus. HDR template was designed with attB1/B2 sites and used for gateway cloning in mammalian expression vector pAAVS1-P-CAG-DEST
- 201115sup.6.pdf
HDR template for mEmarald_LEDGF/p52 re-expression in human AAVS1 locus. HDR template was designed with aatB1/B2 sites and used for gateway cloning in mammalian expression vector pAAVS1-P-CAG-DEST
- 201115sup.6.pdf
HDR template for mEmarald_LEDGF/p52 re-expression in human AAVS1 locus. HDR template was designed with aatB1/B2 sites and used for gateway cloning in mammalian expression vector pAAVS1-P-CAG-DEST
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