Joint changes in RNA, RNA polymerase II, and promoter activity through the cell cycle identify non-coding RNAs involved in proliferation.

Proper regulation of the cell cycle is necessary for normal growth and development of all organisms. Conversely, altered cell cycle regulation often underlies proliferative diseases such as cancer. Long non-coding RNAs (lncRNAs) are recognized as important regulators of gene expression and are often found dysregulated in diseases, including cancers. However, identifying lncRNAs with cell cycle functions is challenging due to their often low and cell-type specific expression. We present a highly effective method that analyses changes in promoter activity, transcription, and RNA levels for identifying genes enriched for cell cycle functions. Specifically, by combining RNA sequencing with ChIP sequencing through the cell cycle of synchronized human keratinocytes, we identified 1009 genes with cell cycle-dependent expression and correlated changes in RNA polymerase II occupancy or promoter activity as measured by histone 3 lysine 4 trimethylation (H3K4me3). These genes were highly enriched for genes with known cell cycle functions and included 57 lncRNAs. We selected four of these lncRNAs-SNHG26, EMSLR, ZFAS1, and EPB41L4A-AS1-for further experimental validation and found that knockdown of each of the four lncRNAs affected cell cycle phase distributions and reduced proliferation in multiple cell lines. These results show that many genes with cell cycle functions have concomitant cell-cycle dependent changes in promoter activity, transcription, and RNA levels and support that our multi-omics method is well suited for identifying lncRNAs involved in the cell cycle.

Increased p53 signaling impairs neural differentiation in HUWE1-promoted intellectual disabilities.

Essential E3 ubiquitin ligase HUWE1 (HECT, UBA, and WWE domain containing 1) regulates key factors, such as p53. Although mutations in HUWE1 cause heterogenous neurodevelopmental X-linked intellectual disabilities (XLIDs), the disease mechanisms common to these syndromes remain unknown. In this work, we identify p53 signaling as the central process altered in HUWE1-promoted XLID syndromes. By focusing on Juberg-Marsidi syndrome (JMS), one of the severest XLIDs, we show that increased p53 signaling results from p53 accumulation caused by HUWE1 p.G4310R destabilization. This further alters cell-cycle progression and proliferation in JMS cells. Modeling of JMS neurodevelopment reveals majorly impaired neural differentiation accompanied by increased p53 signaling. The neural differentiation defects can be successfully rescued by reducing p53 levels and restoring the expression of p53 target genes, in particular CDKN1A/p21. In summary, our findings suggest that increased p53 signaling underlies HUWE1-promoted syndromes and impairs XLID JMS neural differentiation.

HDACi mediate UNG2 depletion, dysregulated genomic uracil and altered expression of oncoproteins and tumor suppressors in B- and T-cell lines.

BACKGROUND: HDAC inhibitors (HDACi) belong to a new group of chemotherapeutics that are increasingly used in the treatment of lymphocyte-derived malignancies, but their mechanisms of action remain poorly understood. Here we aimed to identify novel protein targets of HDACi in B- and T-lymphoma cell lines and to verify selected candidates across several mammalian cell lines. METHODS: Jurkat T- and SUDHL5 B-lymphocytes were treated with the HDACi SAHA (vorinostat) prior to SILAC-based quantitative proteome analysis. Selected differentially expressed proteins were verified by targeted mass spectrometry, RT-PCR and western analysis in multiple mammalian cell lines. Genomic uracil was quantified by LC-MS/MS, cell cycle distribution analyzed by flow cytometry and class switch recombination monitored by FACS in murine CH12F3 cells. RESULTS: SAHA treatment resulted in differential expression of 125 and 89 proteins in Jurkat and SUDHL5, respectively, of which 19 were commonly affected. Among these were several oncoproteins and tumor suppressors previously not reported to be affected by HDACi. Several key enzymes determining the cellular dUTP/dTTP ratio were downregulated and in both cell lines we found robust depletion of UNG2, the major glycosylase in genomic uracil sanitation. UNG2 depletion was accompanied by hyperacetylation and mediated by increased proteasomal degradation independent of cell cycle stage. UNG2 degradation appeared to be ubiquitous and was observed across several mammalian cell lines of different origin and with several HDACis. Loss of UNG2 was accompanied by 30-40% increase in genomic uracil in freely cycling HEK cells and reduced immunoglobulin class-switch recombination in murine CH12F3 cells. CONCLUSION: We describe several oncoproteins and tumor suppressors previously not reported to be affected by HDACi in previous transcriptome analyses, underscoring the importance of proteome analysis to identify cellular effectors of HDACi treatment. The apparently ubiquitous depletion of UNG2 and PCLAF establishes DNA base excision repair and translesion synthesis as novel pathways affected by HDACi treatment. Dysregulated genomic uracil homeostasis may aid interpretation of HDACi effects in cancer cells and further advance studies on this class of inhibitors in the treatment of APOBEC-expressing tumors, autoimmune disease and HIV-1.

Generation of a Mouse Model Lacking the Non-Homologous End-Joining Factor Mri/Cyren.

Classical non-homologous end joining (NHEJ) is a molecular pathway that detects, processes, and ligates DNA double-strand breaks (DSBs) throughout the cell cycle. Mutations in several NHEJ genes result in neurological abnormalities and immunodeficiency both in humans and mice. The NHEJ pathway is required for V(D)J recombination in developing B and T lymphocytes, and for class switch recombination in mature B cells. The Ku heterodimer formed by Ku70 and Ku80 recognizes DSBs and facilitates the recruitment of accessory factors (e.g., DNA-PKcs, Artemis, Paxx and Mri/Cyren) and downstream core factor subunits X-ray repair cross-complementing group 4 (XRCC4), XRCC4-like factor (XLF), and DNA ligase 4 (Lig4). Accessory factors might be dispensable for the process, depending on the genetic background and DNA lesion type. To determine the physiological role of Mri in DNA repair and development, we introduced a frame-shift mutation in the Mri gene in mice. We then analyzed the development of Mri-deficient mice as well as wild type and immunodeficient controls. Mice lacking Mri possessed reduced levels of class switch recombination in B lymphocytes and slow proliferation of neuronal progenitors when compared to wild type littermates. Human cell lines lacking Mri were as sensitive to DSBs as the wild type controls. Overall, we concluded that Mri/Cyren is largely dispensable for DNA repair and mouse development.

Uracil-DNA glycosylase UNG1 isoform variant supports class switch recombination and repairs nuclear genomic uracil.

UNG is the major uracil-DNA glycosylase in mammalian cells and is involved in both error-free base excision repair of genomic uracil and mutagenic uracil-processing at the antibody genes. However, the regulation of UNG in these different processes is currently not well understood. The UNG gene encodes two isoforms, UNG1 and UNG2, each possessing unique N-termini that mediate translocation to the mitochondria and the nucleus, respectively. A strict subcellular localization of each isoform has been widely accepted despite a lack of models to study them individually. To determine the roles of each isoform, we generated and characterized several UNG isoform-specific mouse and human cell lines. We identified a distinct UNG1 isoform variant that is targeted to the cell nucleus where it supports antibody class switching and repairs genomic uracil. We propose that the nuclear UNG1 variant, which in contrast to UNG2 lacks a PCNA-binding motif, may be specialized to act on ssDNA through its ability to bind RPA. RPA-coated ssDNA regions include both transcribed antibody genes that are targets for deamination by AID and regions in front of the moving replication forks. Our findings provide new insights into the function of UNG isoforms in adaptive immunity and DNA repair.

Targeting the non-canonical roles of PCNA modifies and increases the response to targeted anti-cancer therapy.

Receptor tyrosine kinases (RTKs), such as HER2 and/or EGFR are important therapeutic targets in multiple cancer cells. Low and/or short response to targeted therapies are often due to activation of compensatory signaling pathways, and therefore a combination of kinase inhibitors with other anti-cancer therapies have been proposed as promising strategies. PCNA is recently shown to have non-canonical cytosolic roles, and targeting PCNA with a cell-penetrating peptide containing the PCNA-interacting motif APIM is shown to mediate changes in central signaling pathways such as PI3K/Akt and MAPK, acting downstream of multiple RTKs. In this study, we show how targeting PCNA increased the anti-cancer activity of EGFR/HER2/VEGFR inhibition in vitro as well as in vivo. The combination treatment resulted in reduced tumor load and increased the survival compared to either single agent treatments. The combination treatment affected multiple cellular signaling responses not seen by EGFR/HER2/VEGFR inhibition alone, and changes were seen in pathways determining protein degradation, ER-stress, apoptosis and autophagy. Our results suggest that targeting the non-canonical roles of PCNA in cellular signaling have the potential to improve targeted therapies.

"Two hits - one stone"; increased efficacy of cisplatin-based therapies by targeting PCNA's role in both DNA repair and cellular signaling.

Low response rate and rapid development of resistance against commonly used chemotherapeutic regimes demand new multi-targeting anti-cancer strategies. In this study, we target the stress-related roles of the scaffold protein PCNA with a cell-penetrating peptide containing the PCNA-interacting motif APIM. The APIM-peptide increased the efficacy of cisplatin-based therapies in a muscle-invasive bladder cancer (MIBC) solid tumor model in rat and in bladder cancer (BC) cell lines. By combining multiple omics-levels, from gene expression to proteome/kinome and metabolome, we revealed a unique downregulation of the EGFR/ERBB2 and PI3K/Akt/mTOR pathways in the APIM-peptide-cisplatin combination treated cells. Additionally, the combination treatment reduced the expression of anti-apoptotic proteins and proteins involved in development of resistance to cisplatin. Concurrently, we observed increased levels of DNA breaks in combination treated cells, suggesting that the APIM-peptide impaired PCNA - DNA repair protein interactions and reduced the efficacy of repair. This was also seen in cisplatin-resistant cells, which notably was re-sensitized to cisplatin by the APIM-peptide. Our data indicate that the increased efficacy of cisplatin treatment is mediated both via downregulation of known oncogenic signaling pathways and inhibition of DNA repair/translesion synthesis (TLS), thus the APIM-peptide hits both nuclear and cytosolic functions of PCNA. The novel multi-targeting strategy of the APIM-peptide could potentially improve the efficacy of chemotherapeutic regiments for treatment of MIBC, and likely other solid tumors.

Robust DNA repair in PAXX-deficient mammalian cells.

To ensure genome stability, mammalian cells employ several DNA repair pathways. Nonhomologous DNA end joining (NHEJ) is the DNA repair process that fixes double-strand breaks throughout the cell cycle. NHEJ is involved in the development of B and T lymphocytes through its function in V(D)J recombination and class switch recombination (CSR). NHEJ consists of several core and accessory factors, including Ku70, Ku80, XRCC4, DNA ligase 4, DNA-PKcs, Artemis, and XLF. Paralog of XRCC4 and XLF (PAXX) is the recently described accessory NHEJ factor that structurally resembles XRCC4 and XLF and interacts with Ku70/Ku80. To determine the physiological role of PAXX in mammalian cells, we purchased and characterized a set of custom-generated and commercially available NHEJ-deficient human haploid HAP1 cells, PAXXΔ, XRCC4Δ , and XLFΔ . In our studies, HAP1 PAXXΔ cells demonstrated modest sensitivity to DNA damage, which was comparable to wild-type controls. By contrast, XRCC4Δ and XLFΔ HAP1 cells possessed significant DNA repair defects measured as sensitivity to double-strand break inducing agents and chromosomal breaks. To investigate the role of PAXX in CSR, we generated and characterized Paxx-/- and Aid-/- murine lymphoid CH12F3 cells. CSR to IgA was nearly at wild-type levels in the Paxx-/- cells and completely ablated in the absence of activation-induced cytidine deaminase (AID). In addition, Paxx-/- CH12F3 cells were hypersensitive to zeocin when compared to wild-type controls. We concluded that Paxx-deficient mammalian cells maintain robust NHEJ and CSR.

APIM-peptide targeting PCNA improves the efficacy of docetaxel treatment in the TRAMP mouse model of prostate cancer.

Docetaxel is the chemotherapeutic choice for metastatic hormone-refractory prostate cancer, however, it only marginally improves the survival rate. The purpose of the present study was to examine if a peptide targeting the cellular scaffold protein PCNA could improve docetaxel's efficacy. We found that docetaxel given in combination with a cell penetrating peptide containing the AlkB homolog 2 PCNA interacting motif (APIM-peptide), reduced the prostate volume and limited prostate cancer regrowth in vivo in the immunocompetent transgenic adenocarcinoma model of prostate cancer (TRAMP). In accordance with this, we found that the APIM-peptide enhanced the efficacy of docetaxel in vitro. Gene expression analysis on prostate cancer cell lines indicated that the combination of docetaxel and APIM-peptide alters expression of genes involved in cellular signaling, apoptosis, and prostate cancer development. These changes were not detected in single agent treated cells. Our results suggest that targeting PCNA and thereby affecting multiple cellular pathways simultaneously has the potential to improve docetaxel therapy of advanced prostate cancer.

AID expression in B-cell lymphomas causes accumulation of genomic uracil and a distinct AID mutational signature.

The most common mutations in cancer are C to T transitions, but their origin has remained elusive. Recently, mutational signatures of APOBEC-family cytosine deaminases were identified in many common cancers, suggesting off-target deamination of cytosine to uracil as a common mutagenic mechanism. Here we present evidence from mass spectrometric quantitation of deoxyuridine in DNA that shows significantly higher genomic uracil content in B-cell lymphoma cell lines compared to non-lymphoma cancer cell lines and normal circulating lymphocytes. The genomic uracil levels were highly correlated with AID mRNA and protein expression, but not with expression of other APOBECs. Accordingly, AID knockdown significantly reduced genomic uracil content. B-cells stimulated to express endogenous AID and undergo class switch recombination displayed a several-fold increase in total genomic uracil, indicating that B cells may undergo widespread cytosine deamination after stimulation. In line with this, we found that clustered mutations (kataegis) in lymphoma and chronic lymphocytic leukemia predominantly carry AID-hotspot mutational signatures. Moreover, we observed an inverse correlation of genomic uracil with uracil excision activity and expression of the uracil-DNA glycosylases UNG and SMUG1. In conclusion, AID-induced mutagenic U:G mismatches in DNA may be a fundamental and common cause of mutations in B-cell malignancies.

The UNG2 Arg88Cys variant abrogates RPA-mediated recruitment of UNG2 to single-stranded DNA.

In human cell nuclei, UNG2 is the major uracil-DNA glycosylase initiating DNA base excision repair of uracil. In activated B cells it has an additional role in facilitating mutagenic processing of AID-induced uracil at Ig loci and UNG-deficient patients develop hyper-IgM syndrome characterized by impaired class-switch recombination and disturbed somatic hypermutation. How UNG2 is recruited to either error-free or mutagenic uracil processing remains obscure, but likely involves regulated interactions with other proteins. The UNG2 N-terminal domain contains binding motifs for both proliferating cell nuclear antigen (PCNA) and replication protein A (RPA), but the relative contribution of these interactions to genomic uracil processing is not understood. Interestingly, a heterozygous germline single-nucleotide variant leading to Arg88Cys (R88C) substitution in the RPA-interaction motif of UNG2 has been observed in humans, but with unknown functional relevance. Here we demonstrate that UNG2-R88C protein is expressed from the variant allele in a lymphoblastoid cell line derived from a heterozygous germ line carrier. Enzyme activity as well as localization in replication foci of UNG2-R88C was similar to that of WT. However, binding to RPA was essentially abolished by the R88C substitution, whereas binding to PCNA was unaffected. Moreover, we show that disruption of the PCNA-binding motif impaired recruitment of UNG2 to S-phase replication foci, demonstrating that PCNA is a major factor for recruitment of UNG2 to unperturbed replication forks. Conversely, in cells treated with hydroxyurea, RPA mediated recruitment of UNG2 to stalled replication forks independently of functional PCNA binding. Modulation of PCNA- versus RPA-binding may thus constitute a functional switch for UNG2 in cells subsequent to genotoxic stress and potentially also during the processing of uracil at the immunoglobulin locus in antigen-stimulated B cells.

Overexpression of transcription factor AP-2 stimulates the PA promoter of the human uracil-DNA glycosylase (UNG) gene through a mechanism involving derepression.

The PA promoter in the human uracil-DNA glycosylase gene (UNG) directs expression of the nuclear form (UNG2) of UNG proteins. Using a combination of promoter deletion and mutation analyses, and transient transfection of HeLa cells, we show that repressor and derepressor activities are contained within the region of DNA marked by PA. Footprinting analysis and electrophoretic mobility shift assays of PA and putative AP-2 binding regions with HeLa cell nuclear extract and recombinant AP-2alpha protein indicate that AP-2 transcription factors are central in the regulated expression of UNG2 mRNA. Chromatin immunoprecipitation with AP-2 antibody demonstrated that endogenous AP-2 binds to the PA promoter in vivo. Overexpression of AP-2alpha, -beta or -gamma all stimulated expression from a PA-luciferase reporter gene construct approximately 3- to 4-fold. Interestingly, an N-terminally truncated AP-2alpha, lacking the activation domain but retaining the DNA binding and dimerization domains, stimulated PA to a level approaching that of full-length AP-2, suggesting that AP-2 overexpression stimulates PA activity by a mechanism involving derepression rather than activation, possibly by neutralizing an inhibitory effect of endogenous AP-2 or AP-2-like factors.

Extracts of proliferating and non-proliferating human cells display different base excision pathways and repair fidelity.

Base excision repair (BER) of damaged or inappropriate bases in DNA has been reported to take place by single nucleotide insertion or through incorporation of several nucleotides, termed short-patch and long-patch repair, respectively. We found that extracts from proliferating and non-proliferating cells both had capacity for single- and two-nucleotide insertion BER activity. However, patch size longer than two nucleotides was only detected in extracts from proliferating cells. Relative to extracts from proliferating cells, extracts from non-proliferating cells had approximately two-fold higher concentration of POLbeta, which contributed to most of two-nucleotide insertion BER. In contrast, two-nucleotide insertion in extracts from proliferating cells was not dependent on POLbeta. BER fidelity was two- to three-fold lower in extracts from the non-proliferating compared with extracts of proliferating cells. Furthermore, although one-nucleotide deletion was the predominant type of repair error in both extracts, the pattern of repair errors was somewhat different. These results establish two-nucleotide patch BER as a distinct POLbeta-dependent mechanism in non-proliferating cells and demonstrate that BER fidelity is lower in extracts from non-proliferating as compared with proliferating cells.

Human AlkB homolog 1 is a mitochondrial protein that demethylates 3-methylcytosine in DNA and RNA.

The Escherichia coli AlkB protein and human homologs hABH2 and hABH3 are 2-oxoglutarate (2OG)/Fe(II)-dependent DNA/RNA demethylases that repair 1-methyladenine and 3-methylcytosine residues. Surprisingly, hABH1, which displays the strongest homology to AlkB, failed to show repair activity in two independent studies. Here, we show that hABH1 is a mitochondrial protein, as demonstrated using fluorescent fusion protein expression, immunocytochemistry, and Western blot analysis. A fraction is apparently nuclear and this fraction increases strongly if the fluorescent tag is placed at the N-terminal end of the protein, thus interfering with mitochondrial targeting. Molecular modeling of hABH1 based upon the sequence and known structures of AlkB and hABH3 suggested an active site almost identical to these enzymes. hABH1 decarboxylates 2OG in the absence of a prime substrate, and the activity is stimulated by methylated nucleotides. Employing three different methods we demonstrate that hABH1 demethylates 3-methylcytosine in single-stranded DNA and RNA in vitro. Site-specific mutagenesis confirmed that the putative Fe(II) and 2OG binding residues are essential for activity. In conclusion, hABH1 is a functional mitochondrial AlkB homolog that repairs 3-methylcytosine in single-stranded DNA and RNA.

Repair deficient mice reveal mABH2 as the primary oxidative demethylase for repairing 1meA and 3meC lesions in DNA.

Two human homologs of the Escherichia coli AlkB protein, denoted hABH2 and hABH3, were recently shown to directly reverse 1-methyladenine (1meA) and 3-methylcytosine (3meC) damages in DNA. We demonstrate that mice lacking functional mABH2 or mABH3 genes, or both, are viable and without overt phenotypes. Neither were histopathological changes observed in the gene-targeted mice. However, in the absence of any exogenous exposure to methylating agents, mice lacking mABH2, but not mABH3 defective mice, accumulate significant levels of 1meA in the genome, suggesting the presence of a biologically relevant endogenous source of methylating agent. Furthermore, embryonal fibroblasts from mABH2-deficient mice are unable to remove methyl methane sulfate (MMS)-induced 1meA from genomic DNA and display increased cytotoxicity after MMS exposure. In agreement with these results, we found that in vitro repair of 1meA and 3meC in double-stranded DNA by nuclear extracts depended primarily, if not solely, on mABH2. Our data suggest that mABH2 and mABH3 have different roles in the defense against alkylating agents.

Monoclonal antibodies against TRAIL.

TRAIL (tumor necrosis factor related apoptosis inducing ligand) is a cytokine proposed to be used in cancer therapy, since it kills cancer cells but not normal cells. Also, recent studies report that TRAIL inhibits the development of arthritis. In order to investigate the role of TRAIL in health and disease, monoclonal antibodies against TRAIL have been developed. This chapter gives an overview of different monoclonal antibodies against TRAIL which are published or commercially available. Monoclonal antibodies against TRAIL are useful in different immunological techniques, and this chapter presents an overview of the applications of these antibodies with a focus on immunoassays for detection of soluble TRAIL. In addition, the physiological significance of some results obtained by using monoclonal antibodies against TRAIL are discussed.

Development, characterization and use of monoclonal antibodies against sTRAIL: measurement of sTRAIL by ELISA.

Two monoclonal antibodies against tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), designated VI10E and III6F, have been generated. These antibodies were useful in flow cytometry analysis, immunohistochemistry, immunoprecipitation and in the development of an immunoassay for the detection of soluble TRAIL (sTRAIL)in biological samples. The immunoassay was based on two monoclonal antibodies against TRAIL. VI10E was used as the capture antibody and bound TRAIL was detected with anti-TRAIL from R&D Systems which was digoxigenin (DIG)-labeled. This enzyme-linked immunosorbent assay (ELISA) was specific for TRAIL since a panel of other cytokines did not affect the signal. The immunoassay was suitable for the detection of sTRAIL in human serum and plasma samples, cell culture supernatants and cell lysates. In a preliminary screening, it was found that serum samples from human immunodeficiency virus (HIV)-infected patients contained sTRAIL, and all these positive samples were found in the AIDS group. Using the immunoassay, it was found that phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) to produce significant amounts of sTRAIL, the levels of which increased with exposure time. Thus, the immunoassay for TRAIL presented here represents a useful tool for measuring sTRAIL in various biological samples. It will also permit studies of release mechanisms as well as possible functions of the soluble form of this molecule.