LEDGF/p75 Is Required for an Efficient DNA Damage Response

Lens epithelium-derived growth factor splice variant of 75 kDa (LEDGF/p75) plays an important role in cancer, but its DNA-damage repair (DDR)-related implications are still not completely understood. Different LEDGF model cell lines were generated: a complete knock-out of LEDGF (KO) and re-expression of LEDGF/p75 or LEDGF/p52 using CRISPR/Cas9 technology. Their proliferation and migration capacity as well as their chemosensitivity were determined, which was followed by investigation of the DDR signaling pathways by Western blot and immunofluorescence. LEDGF-deficient cells exhibited a decreased proliferation and migration as well as an increased sensitivity toward etoposide. Moreover, LEDGF-depleted cells showed a significant reduction in the recruitment of downstream DDR-related proteins such as replication protein A 32 kDa subunit (RPA32) after exposure to etoposide. The re-expression of LEDGF/p75 rescued all knock-out effects. Surprisingly, untreated LEDGF KO cells showed an increased amount of DNA fragmentation combined with an increased formation of γH2AX and BRCA1. In contrast, the protein levels of ubiquitin-conjugating enzyme UBC13 and nuclear proteasome activator PA28γ were substantially reduced upon LEDGF KO. This study provides for the first time an insight that LEDGF is not only involved in the recruitment of CtIP but has also an effect on the ubiquitin-dependent regulation of DDR signaling molecules and highlights the role of LEDGF/p75 in homology-directed DNA repair.


Introduction
Dense fine speckled autoantigen of 70 kDa (DFS70) also known as lens epitheliumderived growth factor (LEDGF), PSIP1 (PC4 and SFRS1 interacting protein 1), or transcriptional co-activator p75 is considered a ubiquitous nuclear transcription co-activator [1]. It is linked to various diseases such as cancer and acquired immunodeficiency syndrome (AIDS), and diverse inflammatory conditions have been described as well [2,3]. LEDGF/p75 shows anti-apoptotic activity by promoting the repair of DNA double-strand breaks (DSBs) via the homology-directed repair pathway (HDR) [4], and its overexpression in different cancer cell lines and solid tumors has been linked to tumor progression, aggressiveness, and chemoresistance [5]. In contrast, the shorter splice variant LEDGF/p52 has been proposed to play a pro-apoptotic role and appears to be involved in RNA-splicing [6,7]. LEDGF/p75 is a multi-functional, chromatin-binding protein upregulated in different solid cancer and cancer cell lines [1], promoting the activation of pathways involved in proliferation, cell survival, and DNA repair [4,8]. The SUMOylation of LEDGF/p75 [9] by sumo-specific protease-1 regulates its binding in promoter regions of stress-related proteins. This protects cells from stress-induced necrosis and enhances the activation of the Akt/ERK signaling

CRISPR/Cas9-Generated LEDGF Cell Models
For complete knock-out of the LEDGF gene PSIP1, the sgRNA was designed within exon 1 of the PSIP1 gene to target all splice variants ( Figure 1). Then, HEp-2 WT and U2OS WT cells were transfected with non-viral px458_DFS70_E1 vector co-expressing EGFP as a marker and Cas9 enzyme and enriched via EGFP-directed FACS sorting ( Figure S1A) following single cell out-growth. The LEDGF KO HEp-2 clones were verified at a protein and genomic level (Figures 1E and S1C,D). Potential genomic off-target loci were checked by sequencing and exhibited all unmodified loci ( Figure S1E). The reconstitution of LEDGF in LEDGF KO was realized by the integration of either EGFP-LEDGF/p75 expression cassette ( Figure 1B) or mEmarald_LEDGF/p52 expression cassette at the human safe harbor locus (AAVS1) (Figures 1E,F and S1B). EGFP-LEDGF/p75 and mEmarald_LEDGF/p52 incorporation and constitutive expression was confirmed by detecting the fluorescent LEDGF fusion protein ( Figure 1C). Both expressed splice variants showed the typical nuclear localization. Additionally, C-terminal LEDGF antibody was used to detect the wild-type LEDGF and EGFP-LEDGF/p75 to show the typical dense fine speckled nuclear staining pattern ( Figure 1D). Note, mEmarald_LEDGF/p52 cannot be detected with this antibody, as p52 is missing the C-terminus. (KO) LEDGF. The Cas9/sgRNA E1 complex induces double-strand breaks, which can be repaired by the cells through non-homologous end joining (NHEJ); however, NHEJ is error-prone, leading to indel mutations, which can cause premature stop codons. (B) LEDGF/p75 and LEDGF/p52 re-expressing cells were created by introducing a DNA DSB at a genomic safe-harbor locus (AAVS1) using an AAVS1-specific sgRNA. After the induction of a DSB, homology-directed repair (HDR) mediates the integration of the donor template containing the EGFP-LEDGF/p75 or a mEmarald_LEDGF/p52 expression cassette at the AAVS1 locus. Generated LEDGF knockout and re-expressing cells were verified by (C) fluorescence analysis with an excitation wavelength of 488 nm (scale bar = 100 µm), (D) indirect immunofluorescence (IF). Anti C-LEDGF antibody appear red due to conjugation to α-rabbit-IgG-Atto 647 secondary antibody, nuclei appear blue due to DAPI incorporation (scale bar = 20 µm). (E) Immunoblot using antibodies against C-terminal LEDGF and vimentin as loading control. (F) Immunoblot with antibodies against N-terminal LEDGF and vimentin as loading control. Verification of CRISPR/Cas9-mediated LEDGF knockout and LEDGF re-expression in HEp-2 cells. (A) Specific sgRNA for Exon 1 of LEDGF-coding gene PSIP1 was designed to knockout (KO) LEDGF. The Cas9/sgRNA E1 complex induces double-strand breaks, which can be repaired by the cells through non-homologous end joining (NHEJ); however, NHEJ is error-prone, leading to indel mutations, which can cause premature stop codons. (B) LEDGF/p75 and LEDGF/p52 re-expressing cells were created by introducing a DNA DSB at a genomic safe-harbor locus (AAVS1) using an AAVS1-specific sgRNA. After the induction of a DSB, homology-directed repair (HDR) mediates the integration of the donor template containing the EGFP-LEDGF/p75 or a mEmarald_LEDGF/p52 expression cassette at the AAVS1 locus. Generated LEDGF knockout and re-expressing cells were verified by (C) fluorescence analysis with an excitation wavelength of 488 nm (scale bar = 100 µm), (D) indirect immunofluorescence (IF). Anti C-LEDGF antibody appear red due to conjugation to α-rabbit-IgG-Atto 647 secondary antibody, nuclei appear blue due to DAPI incorporation (scale bar = 20 µm). (E) Immunoblot using antibodies against C-terminal LEDGF and vimentin as loading control. (F) Immunoblot with antibodies against N-terminal LEDGF and vimentin as loading control.

Depletion of LEDGF Decreases Cellular Migration
LEDGF has been previously shown to affect cell migration. Therefore, the cell migration of HEp-2 and U2OS cells was checked. Indeed, the migratory capacity was significantly reduced upon LEDGF knockout in HEp-2 and U2OS cells ( Figure 2). LEDGF/p52 re-expression failed to restore the migration capacity of the HEp-2 WT (Figure 2A,C). In contrast, LEDGF/p75 re-expression (WT level) reversed the inhibiting effect, and the cell migration ability was further improved with higher LEDGF/p75 levels (oe) in comparison to the unmodified WT cells (Figures 2C and S4). Additionally, EGFP-LEDGF/p75 o/e cells showed a changed morphology toward an elongated, fibroblast-like phenotype in combination ( Figure 2D) with an increased expression of the cytoskeleton subunit α-tubulin ( Figure 2B). Morphological analysis revealed that LEDGF/p75 o/e cells exhibited a significantly increased eccentricity and a significantly decreased round shape by 50% (p < 0.05, Figure 2D). However, LEDGF KO cells showed a reduced expression of α-tubulin but no change in morphology ( Figure 2B).

LEDGF Depletion Sensitizes Cancer Cells toward Etoposide
Already as a result of LEDGF KO, cells showed a significant decrease in cell proliferation in comparison to the WT cells, while no changes in the cell cycle were detectable ( Figures 3A and S2). The re-expression of LEDGF/p75 completely rescued the proliferation rate, implicating that the p75 splice variant is the most relevant for cell growth. For the proliferation analysis, the cell lines were used within 10 passages. During this time, no changes in growth behavior could be detected. As shown in Figure 3A, the lower proliferation rate was reflected by a prolonged doubling time (WT 21 h and KO 26.5 h). In addition, LEDGF KO cells were passaged 30 times without showing any visible increased apoptotic phenotype nor complete death. Upon etoposide exposure, LEDGF KO cells showed a significantly reduced cell survival compared to the WT ( Figure 3B). Furthermore, the recovery rate after etoposide withdrawal was lower in LEDGF KO cells, as confirmed by ED 50 determinations suggesting that LEDGF plays a critical role in chemosensitivity toward etoposide ( Figure 3C). We were able to compensate the reduced survival of LEDGF KO cells (ED 50 = 0.123 ± 0.01) by re-expressing LEDGF (EGFP-LEDGF/p75 o/e, ED 50 = 0.4 ± 0.04), which resulted in an etoposide resistance equal to unmodified WT-cells (ED 50 = 0.317 ± 0.03) ( Figure 3B,C).

LEDGF Depletion Impairs DNA Damage Response via Homology-Directed Repair
LEDGF has been previously implicated to play a crucial role in HDR-mediated damage response involving CtIP-BRCA1-RPA32 signaling [21]. Therefore, we investigated RPA32 foci formation combined with yH2AX foci and DNA fragmentation to detect active HR.
LEDGF knockout in HEp-2 cells caused a significant increase in sensitivity toward etoposide shown by a higher killing rate ( Figure 3B). This phenotype was underlined by phosphorylation of H2AX (γH2AX, Figure 4) and an increased level of DNA fragmentation ( Figure 4B) indicating etoposide-induced DSBs; however, it was similar to wild-type HEp-2 cells. In contrast, the phosphorylation of RPA32 (pRPA upper band in Figure 4A after etoposide treatment) was almost completely abolished in LEDGF KO cells, while wild-type cells showed an elevated phosphorylation level. This was also reflected in significantly reduced RPA32 foci formation ( Figure 4D,E) after etoposide treatment in LEDGF KO cells, indicating an inhibition of CtIP-BRCA1-mediated homology-directed repair. The re-expression of LEDGF/p75 was able to rescue the RPA32 foci formation ( Figure 4D,E).  (B) Immunoblot shows the level of α-tubulin in untreated cells. As loading control, gel was stained with Coomassie brilliant blue to visualize total protein amount (10 µg/lane), and GAPDH antibody was used. (C) The wound-healing capacity of the indicated HEp-2 and U2OS cell clones were analyzed using an ImageJ plugin MRI wound-healing tool after 24 h of the scratch induction and plotted as area/hour. (D) Representative confocal images of HEp-2 WT, LEDGF KO, LEDGF/p75, and LEDGF/p52 overexpressing cells were taken after incubation with Phalloidin-AlexaFluor555 (yellow). Chromatin appears blue due to DAPI incorporation, nuclei of LEDGF/p75 overexpressing cells appear green due to EGFP-tag of LEDGF/p75, scale bar = 20 µm. The images were used to perform a DBSCAN clustering correlating cell roundness and eccentricity in HEp-2 WT, LEDGF K.O., LEDGF/p75 and LEDGF/p52 overexpressing cells (n = 672, whereby only 75 cells are shown per condition). Cells with fibroblast-like morphology are shown in dark gray and cells with roundish morphology are shown in light gray. * p < 0.05, ** p < 0.01, *** p < 0.001. like morphology are shown in dark gray and cells with roundish morphology are shown in light gray. * p < 0.05, ** p < 0.01, *** p < 0.001.

LEDGF Depletion Results in Dysfunctional DNA Damage Response
Interestingly, γH2AX was already increased without DSB-inducing agents in LEDGF KO cells ( Figure 4A). Additionally, pulse-field electrophoresis revealed a significantly increased amount of DNA fragmentation in untreated LEDGF KO cells ( Figure 4B,C). Surprisingly, LEDGF KO cells exhibited not only a significantly higher number of persistent γH2AX foci ( Figure 5 and Figure S3C) but also a significantly increased foci formation (foci/nucleus are shown in Figure S7  Dose-response curves were fitted (95% confidence interval) with multiparametric functions (EXD3: Three-parameter exponential decay model; LL4: Four-parameter log-logistic model, pWT-KO = 0.0004, pWT-p75 = 0.02). The triangles on the x-axis represent the dose of etoposide needed to reach ED 50 . * p < 0.05, ** p < 0.01, *** p < 0.001.

LEDGF Depletion Results in Dysfunctional DNA Damage Response
Interestingly, γH2AX was already increased without DSB-inducing agents in LEDGF KO cells ( Figure 4A). Additionally, pulse-field electrophoresis revealed a significantly increased amount of DNA fragmentation in untreated LEDGF KO cells ( Figure 4B,C). Surprisingly, LEDGF KO cells exhibited not only a significantly higher number of persistent γH2AX foci ( Figures 5 and S3C) but also a significantly increased foci formation (foci/nucleus are shown in Figure S7  Despite the high "basal" γH2AX foci in LEDGF KO cells, indicating permanent DNA damage, the cells were able to be maintained over 20-30 passages without dying even though at a significantly slower growth rate ( Figure 3A and data not shown). Therefore, we were interested in whether the sustained γH2AX foci are related to a dysregulated degradation of the histone. It has been previously stated that ubiquitination plays an important role in the regulation of DNA damage response signaling. For the γH2AX molecule, K63-linked ubiquitination by UBC13 is important in order to activate the BRCA1-A complex, which coordinates the release of γH2AX followed by subsequent degradation of the proteasome (as illustrated in Figure 5G [16]). This happens downstream of LEDGFinduced CtIP-BRCA1-RPA32 DNA damage signaling.
Here, we show for the first time that LEDGF depletion had a significantly reducing effect on the protein expression levels of UBC13 as well as PA28γ also known as REGy (E) Analysis of RPA32 foci (at least 100 cells were counted) was performed using NucDetect software, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001. Despite the high "basal" γH2AX foci in LEDGF KO cells, indicating permanent DNA damage, the cells were able to be maintained over 20-30 passages without dying even though at a significantly slower growth rate ( Figure 3A and data not shown). Therefore, we were interested in whether the sustained γH2AX foci are related to a dysregulated degradation of the histone. It has been previously stated that ubiquitination plays an important role in the regulation of DNA damage response signaling. For the γH2AX molecule, K63-linked ubiquitination by UBC13 is important in order to activate the BRCA1-A complex, which coordinates the release of γH2AX followed by subsequent degradation of the proteasome (as illustrated in Figure 5G [16]). This happens downstream of LEDGFinduced CtIP-BRCA1-RPA32 DNA damage signaling.
Here, we show for the first time that LEDGF depletion had a significantly reducing effect on the protein expression levels of UBC13 as well as PA28γ also known as REGy ( Figure 5E in Hep-2 and 5F in U2OS cells), which was reversed by LEDGF/p75 re-expression but not LEDGF/p52. This indicates a direct or an indirect role of LEDGF/p75 on the expression of UBC13 and PA28γ, which in turn might affect γH2AX and BRCA1 degradation. In Figure 5G, we propose how LEDGF might regulate HDR-mediated DNA repair in addition to the recruitment of CtIP due to the changing UBC13 and PA28γ levels. ( Figure 5E in Hep-2 and 5F in U2OS cells), which was reversed by LEDGF/p75 re-expression but not LEDGF/p52. This indicates a direct or an indirect role of LEDGF/p75 on the expression of UBC13 and PA28γ, which in turn might affect γH2AX and BRCA1 degradation. In Figure 5G, we propose how LEDGF might regulate HDR-mediated DNA repair in addition to the recruitment of CtIP due to the changing UBC13 and PA28γ levels. LEDGFdependent activation of CtlP is interrupted, allowing no complex formation with MRN and BRCA1 and subsequently no activation of RPA32 and later Rad51, resulting in ineffective DNA end resection. Due to UBC13 downregulation, K63-linked ubiquitination to lysine residues (K13/15) of γH2AX molecules are most likely missing. Without these ubiquitination signals, the BRCA1-A complex (containing also Rap80 and Abraxas) is unable to coordinate the release of γH2AX (ubiquitination at K119) from the chromatin. Consequently, degradation by the proteasome (in the nucleus PA28γ) is also impaired, leading to persistent γH2AX foci.

Discussion
LEDGF/p75 has been reported to be overexpressed in different solid tumors and cancer cell lines [10]. Particularly, it is involved in cancer progression by controlling the expression of genes regulating the cell cycle, cell proliferation, and survival [22]. Furthermore, LEDGF/p75 is supposed to enhance HDR by promoting CtIP-BRCA1-dependent DNA endresection after DNA DSB and influences the recruitment of DNA-damage response-related downstream proteins such as RPA32 [21]. Instead of siRNA knockdown with potential residual LEDGF expression, we generated complete PSIP1 knockout using CRISPR/Cas9 technology. We show for the first time that complete LEDGF depletion has an essential influence on DDR signaling.
Firstly, we generated LEDGF KO cell lines. To avoid off-target effects due to plasmid integration and constitutive active Cas9 expression, LEDGF knockout generation was pursued using EGFP reporter expression to enrich cells transiently expressing Cas9 and sgRNA. Thus, effects detectable for EGFP-selected LEDGF KO clones should be based on LEDGF knockout, making the results more reliable. Potential off-target effects due to the sgRNA [23] were checked by sequencing ( Figure S1E). Additionally, different LEDGF KO clones were tested for their uniform behavior using a proliferation assay ( Figure S2A). Moreover, LEDGF recovery experiments showed that the effects induced by LEDGF depletion could be rescued by EGFP-LEDGF/p75 re-expression, indicating a specific knockout.
Increased LEDGF/p75 expression in prostate cancer [24], breast cancer [22], or colon cancer [1] was linked with an aggressive tumor phenotype. Furthermore, the upregulation of LEDGF in prostate and breast cancer cell lines has been shown to play a role in proliferation, migration, and chemoresistance [12,22]. Migration and invasion is an important step in cancer metastasis [25]; however, the involvement of LEDGF in this dynamic process remains unclear. In the presented study, proliferation and migration analysis of LEDGF depleted cells (Figures 2 and 3) showed a significant reduction in cell growth and decreased migration ability, supporting LEDGF's involvement in pro-survival pathways as previously described [12]. This is further underlined by the LEDGF/p75 o/e model in our study where increased expression resulted in enhanced migratory abilities ( Figure 2C). Moreover, these cells showed a change in morphology to a fibroblast-like phenotype, which was accompanied with an increased expression of α-tubulin. Up to now, only acetylation of α-tubulin has been connected to cancer cell migration and invasion [26,27]. However, increased class III ß-tubulin has been associated with a more aggressive tumor phenotype [28]. Taking into account both the increased migration capacity and the changed morphology in consideration, we suggest that LEDGF o/e leads to a more aggressive and invasive cancer phenotype.
The role of LEDGF in DDR signaling should be investigated. Therefore, we induced DNA DSB using the topoisomerase etoposide. Interestingly, LEDGF depletion sensitizes cells toward caspase-dependent cell death on etoposide exposure [12]. In this work, the characterization of LEDGF KO clones confirmed a direct correlation of LEDGF depletion with a reduced proliferation and an increased sensitivity toward topoisomerase II inhibition by etoposide.
As previously shown elsewhere [6,21,29], the re-expression of the shorter splice variant LEDGF/p52 has no enhancing effect on the cell proliferation, migration, or chemosensitivity.
Daugaard et al. [21] demonstrated LEDGF as an important factor in DNA repair via HDR. After induction of a DNA DSB, the ensuing cellular damage response leads either to NHEJ or HDR. In contrast to NHEJ, HDR is only active in late S-and G2-phase [30]. Moreover, LEDGF KO cells exhibit a decreased survival and elevated DNA fragmentation upon etoposide exposure, which might indicate a deficiency to repair DNA DSBs by HDR. Therefore, RPA32 foci formation was investigated, and indeed, in LEDGF knockout clones, less RPA32 was recruited to the DNA damage sites in comparison to the wildtype cells. In fact, LEDGF/p75 is known to bind to methylated histones, supporting the binding of C-terminal binding protein (CtBP) interacting protein (CtIP), which assists DNA damage recognition by the MRN complex [21]. Subsequently, this activates three main damage response-related protein kinases: ATM, ATR, and DNA PKs. RPA32, also active during replication, is activated by ATM and ATR, resulting in phosphorylated RPA32 foci formation, which is necessary for the recruitment of downstream DNA repair proteins [31].
The reduced amount of RPA32 foci in LEDGF-KO clones is consistent with the results of Daugaard et al. [21], which implies that LEDGF is necessary for the recruitment of HDR-related DNA repair proteins and is required for an efficient DNA repair. In addition to the drug-induced DNA damage, elevated γH2AX foci formation was already detected upon LEDGF KO, suggesting that LEDGF plays a role in the maintenance of genome stability. In fact, SETD2 depletion resulted also in an increased γH2AX foci formation [32] SETD2 trimethylates histone-3 lysine-36 (H3K36me3) at sites of active transcription where the histone code reader LEDGF/p75 binds to it [13]. Furthermore, persistent γH2AX is an indicator for "oncogenic stress", DNA damage driving genomic instability or malignant conversion [33]. Chromosomal instability and gene mutations in cancer cells are well described (reviewed in [34]) and are often caused by increased DNA DSBs with dysregulated DNA repair mechanisms. Since LEDGF/p75 is involved in the HDR-mediated DNA repair, its depletion might leave more DNA DSBs unrepaired. In cancer cells, spontaneous DNA damage foci formation (increase in γH2AX foci formation) caused by self-inflicting or endogenous DNA DSBs are often observed. We were able to show that in LEDGF KO cells, those endogenous DNA DSBs are significantly increased ( Figure 4B). Moreover, LEDGF KO cells exhibited elevated γH2AX as well as BRCA1 foci, which are both known as DNA damage sensors [15,35]. Controversially, LEDGF KO cells could be continuously cultured (with decreased proliferation) and are not prone to go directly into complete cell death which suggests that LEDGF interacts with further signaling pathways.
Finally, increased phosphorylation of H2AX can be also caused in response to cell cycle progression during G2/M phase entry [36]. Furthermore, elevated γH2AX and G2/M arrest could also be indicators for senescence [37,38]. Therefore, a potential cell cycle arrest in LEDGF KO cells was investigated, especially because LEDGF knockdown has been previously shown to induce cell cycle arrest in the S/G2 phase and increased apoptosis in prostate cancer cells [10]. Nonetheless, in our study, cell cycle profiles of WT and LEDGF-depleted cells were similar without any sign of cell arrest in LEDGF KO cells ( Figure S2B). As we could not detect a cell cycle arrest but an increased chemosensitivity, we excluded the possibility that the γH2AX foci are senescence related.
Since the persistent DNA fragmentation in LEDGF KO cells still allows proliferation, we investigated whether the persistently high γH2AX could be also related to an insufficient degradation of the γH2AX. An efficient and coordinated degradation of signaling molecules is necessary for an efficient DDR, which is also accompanied by a vast amount of proteasome-independent K63-linked ubiquitin modifications of the signaling molecules. The protein ubiquitylation plays a central role orchestrating the DDR, whereby K63 linkages promote protein recruiting and K48 linkages destabilize the protein (degradation) [39]. At the end of the signal transduction, many DDR-involved molecules e.g., γH2AX, are degraded by the proteasome [16,40,41]. Interestingly, it has been shown that the knockout of the nuclear proteasome activator PA28γ led to persistent γH2AX foci, even after signal termination [20]. These persistent γH2AX foci interfere with new DNA damage signals, which lead to an overall reduced DDR and thus to genetically unstable cells. Consequently, LEDGF-depleted cells were investigated for their PA28γ expression, and indeed, PA28γ protein levels were decreased. As demonstrated for PA28γ knockout cells, LEDGF depletion also resulted in a decreased PA28γ protein level. This might indicate that the sustained γH2AX foci and BRCA1 foci are also a result of the diminished degradation of these molecules. Moreover, the proteasome recycles ubiquitin by degrading proteins, which is required indirectly for the ubiquitin signaling in DDR, as it supplies the DNA repair ligases e.g., UBC13 with ubiquitin [42]. However, an effect of limited ubiquitin is very speculative and has been investigated in this study. Nonetheless, UBC13 has been previously suggested to ubiquitinate γH2AX molecules, which subsequently allows the binding of BRCA1 to initiate the degradation of γH2AX [43]. Therefore, the UBC13 expression was also verified in the LEDGF KO cells and as expected UBC13 protein levels were abrogated ( Figure 5). A possible scenario how the LEDGF KO affects the HR-mediated DDR was illustrated in Figure 5G.
In the past, LEDGF was only known to recruit the DDR protein CtIP; however, here, we show for the first time that LEDGF seems to have an indirect or direct effect on regulatory proteins of the DDR such as UBC13 and PA28γ, which are known to be involved, e.g., in the degradation of γH2AX.

Antibodies
The following antibodies were used in this study: Anti-RPA32 (

Generation of LEDGF-Modified Cell Clones
HEp-2 WT and U2OS WT cells were seeded in 6-well plates (TH. Geyer, Renningen, Germany), incubated for 24 h, and subsequently transfected with px458_sgR_DFS70_E1 using LipofectamineTM 3000 according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA). For the EGFP-LEDGF/p75 o/e, WT and LEDGF KO cells were co-transfected with px458_sgRNA_AAVS1 and pAAVS1_CAG-EGFP-LEDGF/p75. The generation of mEmarald_LEDGF/p52 o/e cells was performed using px458_sgRNA_AAVS1 and pAAVS1_CAG-mEmarald_LEDGF/p52. Transfected cells were enriched by the EGFP selection of biomarkers via FACS using the S3e cell sorter (Bio-Rad). Briefly, cells were resuspended in 1× PBS supplemented with 0.5% FBS, while doublet cells were excluded by bivalent plotting of FSC and SSC, and positively transfected cells were sorted by GFP expression. Per 10 cm 2 cell culture plate, a total of 1 × 10 3 cells were seeded. Outgrown fluorescent, single cell colonies were picked after 7-10 days to establish LEDGF o/e cell lines. Subsequently, the cell clones were analyzed to verify the integration of the expression cassette at the AAVS1 locus was verified by PCR as described in Oceguera-Yanez [45], showing only heterozygous integrations. Subsequently, the expression of LEDGF was verified by Western blot, which exhibited different LEDGF expressions. Therefore, one cell clone expressing WT LEDGF and one LEDGF o/e was selected for the study.

Proliferation Analysis
To determine cell proliferation, cells were seeded at a density of 5 × 10 3 cells/well in a 96-well plate (Th.Geyer, Renningen, Germany), and sulforhodamine B (SRB) assay was performed according to nature protocol [46].
Cell morphology was analyzed via calculation of the roundness and eccentricity of each cell. For that, firstly, each cell with its corresponding nuclei was extracted as a set of points via thresholding (1.96% of maximum possible pixel intensity) and extraction of foreground pixels. Under-segmented areas were detected via the overlap of multiple nuclei with one area. Each region containing multiple nuclei was segmented via a seeded flood fill approach. Thereby, the centers of all corresponding nuclei were used as seed points for segmentation. Cell eccentricity was calculated via the second-order area moments. Roundess R was estimated as the normalized ratio between area A and perimeter P of the cell by the equation R = (4 * π * A)/P.
The morphology of cells was separated into normal and fibroblast-like via Density-Based Spatial Clustering of Applications with Noise (DBSCAN) clustering as implemented in the Python 3.7 package scikit-learn v. 0.23.1 (eps = 0.15, min_samples = 45).

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.