Lens Epithelium-derived Growth Factor Research Articles

LEDGF interacts with the NID domain of MeCP2 and modulates MeCP2 condensates.

Methyl-CpG-binding protein 2 (MeCP2) is a ubiquitously expressed nuclear protein involved in transcriptional regulation and chromatin remodeling. MeCP2 exists in two isoforms, MeCP2 E1 and E2, which share the same functional domains. Loss-of-function mutations in the MeCP2 gene are the main cause of Rett syndrome (RTT). Previous studies identified a complex formation between MeCP2 and lens epithelium derived growth factor (LEDGF), a transcriptional regulator that exists in two isoforms, LEDGF/p75 and LEDGF/p52. Here, we characterized the molecular and functional interaction between MeCP2 and LEDGF. The NCoR interaction domain (NID) domain in MeCP2 is essential for the direct binding to the PWWP-CR1 region of LEDGF. Introduction of R306C, an RTT mutation in the NID of MeCP2, reduced the interaction with LEDGF. Our data reveal mutual inhibition of MeCP2 and LEDGF multimerization due to overlapping binding sites. Aligning with this observation, LEDGF depletion resulted in larger MeCP2 and DNA foci in NIH3T3 cells, suggesting a role for the MeCP2-LEDGF complex in chromatin organization.

Rational fragment-based design of compounds targeting the PWWP domain of the HRP family

Lens epithelium-derived growth factor p75 (LEDGF/p75), member of the hepatoma-derived growth-factor-related protein (HRP) family, is a transcriptional co-activator and involved in several pathologies including HIV infection and malignancies such as MLL-rearranged leukemia. LEDGF/p75 acts by tethering proteins to the chromatin through its integrase binding domain. This chromatin interaction occurs between the PWWP domain of LEDGF/p75 and nucleosomes carrying a di- or trimethylation mark on histone H3 Lys36 (H3K36me2/3). Our aim is to rationally devise small molecule drugs capable of inhibiting such interaction. To bootstrap this development, we resorted to X-ray crystallography-based fragment screening (FBS-X). Given that the LEDGF PWWP domain crystals were not suitable for FBS-X, we employed crystals of the closely related PWWP domain of paralog HRP-2. As a result, as many as 68 diverse fragment hits were identified, providing a detailed sampling of the H3K36me2/3 pocket pharmacophore. Subsequent structure-guided fragment expansion in three directions yielded multiple compound series binding to the pocket, as verified through X-ray crystallography, nuclear magnetic resonance and differential scanning fluorimetry. Our best compounds have double-digit micromolar affinity and optimally sample the interactions available in the pocket, judging by the Kd-based ligand efficiency exceeding 0.5 kcal/mol per non-hydrogen atom. Beyond π-stacking within the aromatic cage of the pocket and hydrogen bonding, the best compounds engage in a σ-hole interaction between a halogen atom and a conserved water buried deep in the pocket. Notably, the binding pocket in LEDGF PWWP is considerably smaller compared to the related PWWP1 domains of NSD2 and NSD3 which feature an additional subpocket and for which nanomolar affinity compounds have been developed recently. The absence of this subpocket in LEDGF PWWP limits the attainable affinity. Additionally, these structural differences in the H3K36me2/3 pocket across the PWWP domain family translate into a distinct selectivity of the compounds we developed. Our top ranked compounds are interacting with both homologous LEDGF and HRP-2 PWWP domains, yet they showed no affinity for the NSD2 PWWP1 and BRPF2 PWWP domains which belong to other PWWP domain subfamilies. Nevertheless, our developed compound series provide a strong foundation for future drug discovery targeting the LEDGF PWWP domain as they can further be explored through combinatorial chemistry. Given that the affinity of H3K36me2/3 nucleosomes to LEDGF/p75 is driven by interactions within the pocket as well as with the DNA-binding residues, we suggest that future compound development should target the latter region as well. Beyond drug discovery, our compounds can be employed to devise tool compounds to investigate the mechanism of LEDGF/p75 in epigenetic regulation.

Abstract C096: The GR-LEDGF/p75 transcriptional network as a promising target to reduce therapy cross-resistance in prostate cancer

Abstract Prostate cancer (PCa) is the second-leading cause of cancer mortality in U.S. men, with African American (AA) men showing higher incidence and mortality compared to European American men. In addition, AA men are more likely to be diagnosed with more aggressive PCa resulting in reduction of treatment options, especially as tumors develop therapy resistance. Thus, understanding the mechanisms underlying PCa therapy resistance is crucial for developing new therapeutic options. The upregulation of the Lens Epithelium Derived Growth Factor p75 (LEDGF/p75) by glucocorticoid receptor (GR) is a novel mechanism of PCa therapy resistance recently identified by our group. GR upregulation in PCa bypasses the blockade of androgen receptor by inhibitors such as enzalutamide and abiraterone. LEDGF/p75 is an oncogenic transcription co-activator that promotes DNA damage repair and cancer cell survival against environmental stressors including chemotherapeutic drugs such as docetaxel (DTX). An emerging concern in PCa treatment is therapy cross-resistance, in which the molecular mechanisms of resistance to a particular drug confer resistance to another drug. Our recent studies showed that GR upregulates and interacts with LEDGF/p75 forming a transcriptional complex that includes other oncoproteins such as Menin, JPO2, c-Myc and HRP2, and promotes DTX resistance (DTX-R) in PCa cells. In addition, we reported that both GR and LEDGF/p75 are upregulated in enzalutamide resistant (ENZ-R) PCa cells. We hypothesized that the GR- LEDGF/p75 transcriptional network plays a role in PCa therapy cross-resistance to both ENZ and DTX and is a promising therapeutic target. Cell viability assays revealed that ENZ-R in PCa cells confers cross-resistance to DTX. Western blot analysis revealed upregulation of members of the GR-LEDGF/p75 network such as GR, LEDGF/p75, Menin and c-Myc in ENZ-R PCa cells, consistent with similar observations in DTX-R PCa cells. Upon knockdown of GR and LEDGF/p75 in DTX-R PCa cells, we identified by RNA-seq analysis overlapping DEGs associated with both ENZ-R and DTX-R, suggesting that the GR-LEDGF/p75 transcriptional network may contribute to therapy cross-resistance. Consistent with this, mining of publicly available RNA-seq databases identified 46 overlapping differentially expressed genes (DEGs) between ENZ-R and DTX-R PCa cells. Pathway analysis of DEGs regulated by both GR and LEDGF/p75 revealed significant enrichment of Apoptosis, p53, and DNA repair pathways, which are associated with therapy resistance. Ongoing studies are evaluating the anti-cancer properties and synergistic/additive potential of various inhibitors of the GR-LEDGF/p75 transcriptional network in cellular models of ENZ-R and DTX-R. These studies provide novel mechanistic insights into PCa therapy cross-resistance and may lead to the development of novel therapeutic strategies to reduce PCa mortality disparities. Citation Format: Pedro T. Ochoa, Evelyn S. Sanchez-Hernandez, Kai Wen Cheng, Isaac Kremsky, Charles Wang, Carlos A. Casiano. The GR-LEDGF/p75 transcriptional network as a promising target to reduce therapy cross-resistance in prostate cancer [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C096.

N-acetylgalactosaminyltransferase GALNT6 is a potential therapeutic target of clear cell renal cell carcinoma progression.

High expression of truncated O-glycans Tn antigen predicts adverse clinical outcome in patients with clear cell renal cell carcinoma (ccRCC). To understand the biosynthetic underpinnings of Tn antigen changes in ccRCC, we focused on N-acetylgalactosaminyltransferases (GALNTs, also known as GalNAcTs) known to be involved in Tn antigen synthesis. Data from GSE15641 profile and local cohort showed that GALNT6 was significantly upregulated in ccRCC tissues. The current study aimed to determine the role of GALNT6 in ccRCC, and whether GALNT6-mediated O-glycosylation aggravates malignant behaviors. Gain- and loss-of-function experiments showed that overexpression of GALNT6 accelerated ccRCC cell proliferation, migration, and invasion, as well as promoted ccRCC-derived xenograft tumor growth and lung metastasis. In line with this, silencing of GALNT6 yielded the opposite results. Mechanically, high expression of GALNT6 led to the accumulation of Tn antigen in ccRCC cells. By undertaking immunoprecipitation coupled with liquid chromatography/mass spectrometry, vicia villosa agglutinin blot, and site-directed mutagenesis assays, we found that O-glycosylation of prohibitin 2 (PHB2) at Ser161 was required for the GALNT6-induced ccRCC cell proliferation, migration, and invasion. Additionally, we identified lens epithelium-derived growth factor (LEDGF) as a key regulator of GALNT6 transcriptional induction in ccRCC growth and an upstream contributor to ccRCC aggressive behavior. Collectively, our findings indicate that GALNT6-mediated abnormal O-glycosylation promotes ccRCC progression, which provides a potential therapeutic target in ccRCC development.

Design and synthesis of novel and potent allosteric HIV-1 integrase inhibitors with a spirocyclic moiety

We report herein the design and discovery of novel allosteric HIV-1 integrase inhibitors. Our design concept utilized the spirocyclic moiety to restrain the flexibility of the conformation of the lipophilic part of the inhibitor. Compound 5 showed antiviral activity by binding to the nuclear lens epithelium-derived growth factor (LEDGF/p75) binding site of HIV-1 integrase (IN). The introduction of a lipophilic amide substituent into the central benzene ring resulted in a significant increase in antiviral activity against HIV-1 WT X-ray crystallography of compound 15 in complex with the integrase revealed the presence of a hydrogen bond between the oxygen atom of the amide of compound 15 and the hydroxyl group of the T125 side chain. Chiral compound 17 showed high antiviral activity, good bioavailability, and low clearance in rats.

DNA methylation as an epigenetic mechanism in the regulation of LEDGF expression and biological response in aging and oxidative stress

The physiological quantum of stress-inducible transcriptional protein, Lens Epithelium-Derived Growth Factor (LEDGF), is vital for the maintenance of cellular physiology. Erratic epigenetic reprogramming in response to oxidative stress or with advancing age is found to be a major cause in the gene silencing, leading to pathobiologies. Using aging human (h) eye lens/lens epithelial cells (LECs) coupled with redox-active Peroxiredoxin 6 (Prdx6)-deficient (Prdx6−/−) mLECs as model systems, herein, we showed that in aging/oxidative stress, the human LEDGF gene was regulated by unique methylation patterns of CGs nucleotides within and around the Sp1 binding site(s) of CpG island of the LEDGF promoter (−170 to −27nts). The process caused the repression of LEDGF and its target, Hsp27, resulting in reactive oxygen species (ROS) amplification and cellular insults. This phenomenon was opposed to the unmethylated promoter in LECs. Clinically, we observed that the loss of LEDGF in the Prdx6−/− mLECs or aging lenses/LECs, correlating with increased expression of DNMT1, DNMT3a, and DNMT3b along with the methyl CpG binding protein 2 (MeCP2). Upon oxidative stress, the expression of these molecules was increased with the dramatic reduction in LEDGF expression. While demethylating agent, 5-Aza deoxycytidine (5-AzaC) transposed the aberrant methylation status, and revived LEDGF and Hsp27 expression. Mechanistically, the chloramphenicol acetyltransferase (CAT) reporter gene driven by the LEDGF promoter (−170/ + 35) and ChIP assays uncovered that 5-AzaC acted on GC/Sp1 sites to release LEDGF transcription. The data argued, for the first time, that de novo methylation of CGs around and within Sp1 sites of the CpG island directly disrupted Sp1 activity, which ensued in LEDGF repression and its biological functions. The findings should improve our understanding of cellular insults-associated with aberrant DNMTs-mediated LEDGF’s activity, and can offer strategies for therapeutic intervention to halt aging/oxidative stress-induced abnormalities.

Abstract 5834: The GR-LEDGF/p75 transcriptional network promotes therapy cross-resistance in prostate cancer cells

Abstract Prostate cancer (PCa) is the second leading cause of cancer mortality for men in the U.S. Patients who develop advanced PCa ultimately acquire therapy resistance and succumb to the disease. An emerging challenge in the treatment of advanced PCa is the development of therapy cross-resistance, in which molecular pathways promoting resistance to a particular drug or treatment also promote resistance to subsequent treatments. Although the mechanisms driving PCa therapy cross-resistance are not clearly understood, recent studies from our group and others implicate the glucocorticoid receptor (GR), a transcription factor, in resistance to both androgen receptor signaling inhibitors such enzalutamide (ENZ), and taxane drugs such as docetaxel (DTX). Our studies also showed that GR upregulates and interacts with the lens epithelium derived growth factor of 75 kD (LEDGF/p75) in a large panel of PCa cell lines. LEDGF/p75 is a stress transcription co-activator and oncoprotein that promotes cancer cell resistance to DTX and DNA-damaging drugs. GR and LEDGF/p75 are part of a large transcriptional network that include other oncogenic transcription factors linked to cancer chemoresistance such as β-catenin, Menin, MLL, JPO2, and c-Myc. We hypothesized that this transcriptional network plays a role in PCa therapy cross-resistance and could be a promising therapeutic target. We observed by Western blotting (WB) that both GR and LEDGF/p75 proteins are upregulated in ENZ-resistant and DTX-resistant PCa cell lines, and that GR silencing in both contexts abrogated LEDGF/p75 expression. MTT viability assays showed that ENZ-resistant PCa cells also exhibited cross-resistance to DTX. Bioinformatics studies, using publicly available RNA-seq datasets, identified overlapping differentially expressed genes (DEGs) between ENZ-resistant and DTX-resistant PCa cells. Individual silencing of GR and LEDGF/p75 in two DTX-resistant PCa cell lines followed by RNA-seq analysis also identified an overlap between DEGs regulated by GR and LEDGF/p75 with DEGs associated with both ENZ and DTX resistance in PCa. GSEA analysis of DEGs targeted by both GR and LEDGF/p75 in DTX-resistant PCa cells revealed enriched pathways associated with stress responses, androgen responses, and regulation of cell death and cell cycle. Further, we initiated studies to evaluate the efficacy of small molecule inhibitors targeting multiple components of the GR-LEDGF/p75 transcriptional network in sensitizing therapy-resistant PCa cells to DTX and ENZ. In conclusion, our results implicate the GR-LEDGF/p75 axis in promoting resistance to both ENZ and DTX in PCa cells. Further mining of RNA-seq data will identify potential molecular targets associated with PCa therapy cross-resistance, leading to the design of novel therapeutic strategies to attenuate this resistance. Citation Format: Pedro T. Ochoa, Evelyn S. Sanchez-Hernandez, Kai Wen Cheng, Isaac Kremsky, Charles Wang, Carlos A. Casiano. The GR-LEDGF/p75 transcriptional network promotes therapy cross-resistance in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5834.

The Impact of Lens Epithelium-Derived Growth Factor p75 Dimerization on Its Tethering Function.

The transcriptional co-activator lens epithelium-derived growth factor/p75 (LEDGF/p75) plays an important role in the biology of the cell and in several human diseases, including MLL-rearranged acute leukemia, autoimmunity, and HIV-1 infection. In both health and disease, LEDGF/p75 functions as a chromatin tether that interacts with proteins such as MLL1 and HIV-1 integrase via its integrase-binding domain (IBD) and with chromatin through its N-terminal PWWP domain. Recently, dimerization of LEDGF/p75 was shown, mediated by a network of electrostatic contacts between amino acids from the IBD and the C-terminal α6-helix. Here, we investigated the functional impact of LEDGF/p75 variants on the dimerization using biochemical and cellular interaction assays. The data demonstrate that the C-terminal α6-helix folds back in cis on the IBD of monomeric LEDGF/p75. We discovered that the presence of DNA stimulates LEDGF/p75 dimerization. LEDGF/p75 dimerization enhances binding to MLL1 but not to HIV-1 integrase, a finding that was observed in vitro and validated in cell culture. Whereas HIV-1 replication was not dependent on LEDGF/p75 dimerization, colony formation of MLLr-dependent human leukemic THP-1 cells was. In conclusion, our data indicate that intricate changes in the quaternary structure of LEDGF/p75 modulate its tethering function.

Dense fine speckled nuclear immunofluorescence: A mildly reassuring antinuclear antibody pattern meriting consideration.

Antinuclear antibodies (ANAs) are regarded as a hallmark of connective tissue diseases (CTDs) and play a key role in their diagnosis, but the value of some particular antibodies in management of patients and the disease prognosis is controversial. The mechanism underlying the production of ANAs in CTDs, other chronic inflammatory conditions and even in healthy people, is not completely elucidated. Anti-DFS70 antibodies connected with the dense fine speckled autoantigen of 70 kD, known as the lens epithelium-derived growth factor p75, are a subgroup of ANAs. Their presence and coexistence with other antibodies and their clinical significance are the matter of debate. Based on literature data, the authors focused on current knowledge explaining the role of anti-DFS70 antibodies in selected CTDs. However, the literature data is ambiguous and does not fully support the validity of the anti-DFS70 assay for a specific CTD diagnosis. Most researchers claim that the presence of anti-DFS70 as the only one usually exclude the diagnosis of CTD. Nevertheless, its coexistence with other ANAs is not an excluding factor but has predictive value due to more favorable course of CTD. Such situations may also suggest an enhanced risk of the development of a CTD in the future. Although more studies are needed in this field, it seems reasonable to ascertain the presence of anti-DFS70 in routine clinical practice.

Glucocorticoid Receptor Regulates and Interacts with LEDGF/p75 to Promote Docetaxel Resistance in Prostate Cancer Cells.

Patients with advanced prostate cancer (PCa) invariably develop resistance to anti-androgen therapy and taxane-based chemotherapy. Glucocorticoid receptor (GR) has been implicated in PCa therapy resistance; however, the mechanisms underlying GR-mediated chemoresistance remain unclear. Lens epithelium-derived growth factor p75 (LEDGF/p75, also known as PSIP1 and DFS70) is a glucocorticoid-induced transcription co-activator implicated in cancer chemoresistance. We investigated the contribution of the GR-LEDGF/p75 axis to docetaxel (DTX)-resistance in PCa cells. GR silencing in DTX-sensitive and -resistant PCa cells decreased LEDGF/p75 expression, and GR upregulation in enzalutamide-resistant cells correlated with increased LEDGF/p75 expression. ChIP-sequencing revealed GR binding sites in the LEDGF/p75 promoter. STRING protein-protein interaction analysis indicated that GR and LEDGF/p75 belong to the same transcriptional network, and immunochemical studies demonstrated their co-immunoprecipitation and co-localization in DTX-resistant cells. The GR modulators exicorilant and relacorilant increased the sensitivity of chemoresistant PCa cells to DTX-induced cell death, and this effect was more pronounced upon LEDGF/p75 silencing. RNA-sequencing of DTX-resistant cells with GR or LEDGF/p75 knockdown revealed a transcriptomic overlap targeting signaling pathways associated with cell survival and proliferation, cancer, and therapy resistance. These studies implicate the GR-LEDGF/p75 axis in PCa therapy resistance and provide a pre-clinical rationale for developing novel therapeutic strategies for advanced PCa.

The SETD2 Methyltransferase Supports Productive HPV31 Replication through the LEDGF/CtIP/Rad51 Pathway.

The human papillomavirus (HPV) life cycle takes place in the stratified epithelium, with the productive phase being activated by epithelial differentiation. The HPV genome is histone-associated, and the life cycle is epigenetically regulated, in part, by histone tail modifications that facilitate the recruitment of DNA repair factors that are required for viral replication. We previously showed that the SETD2 methyltransferase facilitates the productive replication of HPV31 through the trimethylation of H3K36 on viral chromatin. SETD2 regulates numerous cellular processes, including DNA repair via homologous recombination (HR) and alternative splicing, through the recruitment of various effectors to histone H3 lysine 36 trimethylation (H3K36me3). We previously demonstrated that the HR factor Rad51 is recruited to HPV31 genomes and is required for productive replication; however, the mechanism of Rad51 recruitment has not been defined. SET domain containing 2 (SETD2) promotes the HR repair of double-strand breaks (DSBs) in actively transcribed genes through the recruitment of carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) to lens epithelium-derived growth factor (LEDGF)-bound H3K36me3, which promotes DNA end resection and thereby allows for the recruitment of Rad51 to damaged sites. In this study, we found that reducing H3K36me3 through the depletion of SETD2 or the overexpression of an H3.3K36M mutant leads to an increase in γH2AX, which is a marker of damage, on viral DNA upon epithelial differentiation. This is coincident with decreased Rad51 binding. Additionally, LEDGF and CtIP are bound to HPV DNA in a SETD2-dependent and H3K36me3-dependent manner, and they are required for productive replication. Furthermore, CtIP depletion increases DNA damage on viral DNA and blocks Rad51 recruitment upon differentiation. Overall, these studies indicate that H3K36me3 enrichment on transcriptionally active viral genes promotes the rapid repair of viral DNA upon differentiation through the LEDGF-CtIP-Rad51 axis. IMPORTANCE The productive phase of the HPV life cycle is restricted to the differentiating cells of the stratified epithelium. The HPV genome is histone-associated and subject to epigenetic regulation, though the manner in which epigenetic modifications contribute to productive replication is largely undefined. In this study, we demonstrate that SETD2-mediated H3K36me3 on HPV31 chromatin promotes productive replication through the repair of damaged DNA. We show that SETD2 facilitates the recruitment of the homologous recombination repair factors CtIP and Rad51 to viral DNA through LEDGF binding to H3K36me3. CtIP is recruited to damaged viral DNA upon differentiation, and, in turn, recruits Rad51. This likely occurs through the end resection of double-strand breaks. SETD2 trimethylates H3K36me3 during transcription, and active transcription is necessary for Rad51 recruitment to viral DNA. We propose that the enrichment of SETD2-mediated H3K36me3 on transcriptionally active viral genes upon differentiation facilitates the repair of damaged viral DNA during the productive phase of the viral life cycle.

Discovery of arylsulfonamides as a novel class of allosteric integrase inhibitors with antiviral activity

Lens epithelial-derived growth factor (LEDGF) increases the efficiency of proviral DNA integration into the host genome by interacting with HIV integrase (IN) and directing it to a chromatin environment that favors viral transcription. Allosteric integrase inhibitors (ALLINIs), such as known 2-(tert-butoxy)acetic acid (1), bind to the LEDGF pocket on the catalytic core domain (CCD) of IN, but exert more potent antiviral activities by inhibition of late-stage HIV-1 replication events than through disruption of proviral integration at an earlier phase. A high-throughput screen (HTS) for compounds that disrupt IN-LEDGF interaction led to the identification of a novel arylsulfonamide series, as exemplified by 2, possessing ALLINI-like properties. Further SAR studies led to more potent compound 21 and provided key chemical biology probes revealing that arylsulfonamides are a novel class of ALLINIs with a distinct binding mode than that of 2-(tert-butoxy)acetic acids.

Over-Expression of LEDGF/p75 in HEp-2 Cells Enhances Autoimmune IgG Response in Patients with Benign Prostatic Hyperplasia—A Novel Diagnostic Approach with Therapeutic Consequence?

Lens epithelium-derived growth factor splice variant of 75 kDa (LEDGF/p75) is an autoantigen over-expressed in solid tumors and acts as a stress-related transcriptional co-activator. Participation of autoimmune responses in the pathophysiology of benign prostatic hyperplasia (PBH) and a corresponding immunosuppressive therapy by TNFalpha antagonists has been recently suggested. Thus, autoAb testing could aid in the diagnosis of BPH patients profiting from such therapy. We generated CRISPR/Cas9 modified HEp-2 LEDGF knock-out (KO) and HEp-2 LEDGF/p75 over-expressing (OE) cells and examined IgG autoantibody reactivity to LEDGF/p75 in patients with prostate cancer (PCa, n = 89), bladder cancer (BCa, n = 116), benign prostatic hyperplasia (BPH, n = 103), and blood donors (BD, n = 60) by indirect immunofluorescence assay (IFA). Surprisingly, we could not detect elevated binding of autoAbs against LEDGF/p75 in cancer patients, but autoAb reactivity to LEDGF/p75 OE cells in about 50% of patients with BPH was unexpectedly significantly increased. Furthermore, a line immunoassay enabling the detection of 18 different autoAbs revealed a significantly increased occurrence of anti-dsDNA autoAbs in 34% of BPH patients in contrast to tumor patients and BD. This finding was confirmed by anti-mitochondrial (mDNA) autoAb detection with the Crithidia luciliae immunofluorescence test, which also showed a significantly higher prevalence (34%) of anti-mDNA autoAbs in BPH. In summary, our study provided further evidence for the occurrence of autoimmune responses in BPH. Furthermore, LEDGF/p75 over-expression renders HEp-2 cells more autoantigenic and an ideal target for autoAb analysis in BPH with a potential therapy consequence.

Nucleosome chaperone activity of LEDGF and HDGF2 characterized with single molecule force spectroscopy.

The nucleosome, two turns of DNA wrapped around a histone octamer, is the fundamental structure of DNA organization in eukaryotic cells. Lens epithelium-derived growth factor (LEDGF) and hepatoma-derived growth factor 2 (HDGF2) are recently discovered proteins that facilitate transcription without ATP hydrolysis, suggesting roles as nucleosome chaperones during the integration of lentiviral DNA, including HIV-1 DNA, into infected cell genomes. Both proteins have methyl-lysine reading PWWP domains that can recognize H3K36me2/3 marks. To better understand these activities, we used optical tweezers to stretch reconstituted DNA nucleosome arrays containing twelve Widom 601 positioning sequences with unmodified (WT) and methylated histones (mimicking the H3K36me2/3 modifications), in the presence of LEDGF and HDGF2. To test the function of these nucleosome chaperone proteins during HIV-1 DNA integration, we exposed the modified nucleosomes to HIV-1 integrase (IN) together with equal concentrations of LEDGF and HDGF2. Analysis of force-extension data showed that while some destabilization of the inner wrapping of nucleosomes occurs due to histone methylation, exposure to chaperones results in further destabilization. Notably, the outer half turn unwrapping of nucleosomes that typically occurs at lower forces disappears with chaperone binding. Kinetic analysis of histone-DNA interactions in the presence of these chaperones via survival probability and confocal fluorescence measurements indicates that LEDGF not only facilitates nucleosome unwrapping but also moderately promotes nucleosome reassembly. These results offer insight into the functions of LEDGF and HDGF2 as nucleosome chaperone proteins and their effect on IN binding to the nucleosome array.

Identification of the H3K36me3 reader LEDGF/p75 in the pancancer landscape and functional exploration in clear cell renal cell carcinoma

Lens epithelium-derived growth factor (LEDGF/p75) is a reader of epigenetic marks and a potential target for therapeutic intervention. Its involvement in human immunodeficiency virus (HIV) integration and the development of leukemia driven by MLL (also known as KMT2A) gene fusion make it an attractive candidate for drug development. However, exploration of LEDGF/p75 as an epigenetic reader of H3K36me3 in tumors is limited. Here, for the first time, we analyze the role of LEDGF/p75 in multiple cancers via multiple online databases and in vitro experiments. We used pancancer bulk sequencing data and online tools to analyze correlations of LEDGF/p75 with prognosis, genomic instability, DNA damage repair, prognostic alternative splicing, protein interactions, and tumor immunity. In summary, the present study identified that LEDGF/p75 may serve as a prognostic predictor for tumors such as adrenocortical carcinoma, kidney chromophobe, liver hepatocellular carcinoma, pancreatic adenocarcinoma, skin cutaneous melanoma, and clear cell renal cell carcinoma (ccRCC). In addition, in vitro experiments and gene microarray sequencing were performed to explore the function of LEDGF/p75 in ccRCC, providing new insights into the pathogenesis of the nonmutated SETD2 ccRCC subtype.

G-Quadruplex DNA and Other Non-Canonical B-Form DNA Motifs Influence Productive and Latent HIV-1 Integration and Reactivation Potential.

The integration of the HIV-1 genome into the host genome is an essential step in the life cycle of the virus and it plays a critical role in the expression, long-term persistence, and reactivation of HIV expression. To better understand the local genomic environment surrounding HIV-1 proviruses, we assessed the influence of non-canonical B-form DNA (non-B DNA) on the HIV-1 integration site selection. We showed that productively and latently infected cells exhibit different integration site biases towards non-B DNA motifs. We identified a correlation between the integration sites of the latent proviruses and non-B DNA features known to potently influence gene expression (e.g., cruciform, guanine-quadruplex (G4), triplex, and Z-DNA). The reactivation potential of latent proviruses with latency reversal agents also correlated with their proximity to specific non-B DNA motifs. The perturbation of G4 structures in vitro using G4 structure-destabilizing or -stabilizing ligands resulted in a significant reduction in integration within 100 base pairs of G4 motifs. The stabilization of G4 structures increased the integration within 300-500 base pairs from G4 motifs, increased integration near transcription start sites, and increased the proportion of latently infected cells. Moreover, we showed that host lens epithelium-derived growth factor (LEDGF)/p75 and cleavage and polyadenylation specificity factor 6 (CPSF6) influenced the distribution of integration sites near several non-B DNA motifs, especially G4 DNA. Our findings identify non-B DNA motifs as important factors that influence productive and latent HIV-1 integration and the reactivation potential of latent proviruses.

The choice of anti-LEDGF/DFS70 assay matters: a comparative study of six assays

Lens-epithelial derived growth factor (LEDGF/DFS70) autoantibodies result in the commonly observed dense fine speckled (DFS) pattern by anti-nuclear antibody (ANA) assay. However, there is no consensus approach for confirmation of this autoantibody specificity. To evaluate current approaches, we examined inter-assay agreement between six anti-LEDGF/DFS70 assays. A total of 395 consecutive sera samples from routine ANA diagnostics were obtained, tested by routine ANA, anti-ENA line immunoblot assay (LIA) and anti-dsDNA assay and with six anti-DFS/LEDGF assays: the EuroLine-LIA (Euro-LIA), Medical and Biological Laboratories ELISA (MBL-ELISA), Phadia-EliA (EliA), QUANTA Flash CLIA, EuroImmun ELISA (Euro-ELISA) and Immco-Diagnostics HEp-2 ELITE/DFS-Knockout (HEp-2KO). Of 395 sera, 108 tested positive by at least one assay. Despite general good concordance between all assays across the cohort (Gwet's AC1=0.89), within the target DFS-ANA pattern group inter-assay agreement was poor (AC1=0.59). Euro-LIA, CLIA and MBL-ELISA assays were most concordant, but CLIA and Euro-LIA were also most likely to identify discordant positive results. EliA and Euro-ELISA had poorer agreement, which could be attributable to ill-matched cut-offs between assays. HEp-2KO was frequently discordant with all other assays tested. Euro-LIA, CLIA and MBL-ELISA were most concordant at manufacturer's specifications and are suited for use in clinical laboratories. Modified assay thresholds are required to ensure comparative results for Euro-ELISA and EliA. HEp-2KO assay is frequently discordant with all other assays, making it less suited for routine diagnostics. The study highlights the importance of considering inter-assay variability when developing a diagnostic strategy for anti-LEDGF/DFS70 autoantibodies in clinical laboratories.

Allosteric Integrase Inhibitor Influences on HIV-1 Integration and Roles of LEDGF/p75 and HDGFL2 Host Factors.

Allosteric integrase (IN) inhibitors (ALLINIs), which are promising preclinical compounds that engage the lens epithelium-derived growth factor (LEDGF)/p75 binding site on IN, can inhibit different aspects of human immunodeficiency virus 1 (HIV-1) replication. During the late phase of replication, ALLINIs induce aberrant IN hyper-multimerization, the consequences of which disrupt IN binding to genomic RNA and virus particle morphogenesis. During the early phase of infection, ALLINIs can suppress HIV-1 integration into host genes, which is also observed in LEDGF/p75-depelted cells. Despite this similarity, the roles of LEDGF/p75 and its paralog hepatoma-derived growth factor like 2 (HDGFL2) in ALLINI-mediated integration retargeting are untested. Herein, we mapped integration sites in cells knocked out for LEDGF/p75, HDGFL2, or both factors, which revealed that these two proteins in large part account for ALLINI-mediated integration retargeting during the early phase of infection. We also determined that ALLINI-treated viruses are defective during the subsequent round of infection for integration into genes associated with speckle-associated domains, which are naturally highly targeted for HIV-1 integration. Class II IN mutant viruses with alterations distal from the LEDGF/p75 binding site moreover shared this integration retargeting phenotype. Altogether, our findings help to inform the molecular bases and consequences of ALLINI action.

LEDGF is a new growth factor in fetal serum

Fetal serum supports the immortal growth of mammalian cell lines in culture while adult serum leads to the terminal differentiation and death of cells in culture. Many of the proteins in fetal serum that support the indefinite division and growth of cancerous cell lines remain obscure. The peptides and proteins of fetal versus adult serum were analyzed by liquid chromatography, nano electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS). Three batches of fetal serum contained the Alpha Fetoprotein marker while adult serum batches did not. Insulin (INS), and insulin-like growth factor (ILGF), fibroblast growth factor (FGF), epidermal growth factor (EGF) and platelet derived growth factor (PDGF) were increased in fetal serum. New fetal growth factors including MEGF, HDGFRP and PSIP1 and soluble growth receptors such as TNFR, EGFR, NTRK2 and THRA were discovered. Addition of insulin or the homeotic transcription factor PSIP1, also referred to as Lens Epithelium Derived Growth Factor (LEDGF), partially restored the rounded phenotype of rapidly dividing cells but was not as effective as fetal serum. Thus, a new growth factor in fetal serum, LEDGF/PSIP1, was directly observed by tandem mass spectrometry and confirmed by add back experiments to cell culture media alongside insulin.

Expansion microscopy allows high resolution single cell analysis of epigenetic readers.

Interactions between epigenetic readers and histone modifications play a pivotal role in gene expression regulation and aberrations can enact etiopathogenic roles in both developmental and acquired disorders like cancer. Typically, epigenetic interactions are studied by mass spectrometry or chromatin immunoprecipitation sequencing. However, in these methods, spatial information is completely lost. Here, we devise an expansion microscopy based method, termed Expansion Microscopy for Epigenetics or ExEpi, to preserve spatial information and improve resolution. We calculated relative co-localization ratios for two epigenetic readers, lens epithelium derived growth factor (LEDGF) and bromodomain containing protein 4 (BRD4), with marks for heterochromatin (H3K9me3 and H3K27me3) and euchromatin (H3K36me2, H3K36me3 and H3K9/14ac). ExEpi confirmed their preferred epigenetic interactions, showing co-localization for LEDGF with H3K36me3/me2 and for BRD4 with H3K9/14ac. Moreover addition of JQ1, a known BET-inhibitor, abolished BRD4 interaction with H3K9/14ac with an IC50 of 137 nM, indicating ExEpi could serve as a platform for epigenetic drug discovery. Since ExEpi retains spatial information, the nuclear localization of marks and readers was determined, which is one of the main advantages of ExEpi. The heterochromatin mark, H3K9me3, is located in the nuclear rim whereas LEDGF co-localization with H3K36me3 and BRD4 co-localization with H3K9/14ac occur further inside the nucleus.