HIV-1 integrase inhibitor raltegravir promotes DNA damage-induced apoptosis in multiple myeloma.

Raltegravir, the first integrase inhibitor approved for the treatment of HIV infection, has been implicated as a promising potential in cancer treatment. Therefore, the present study aimed to investigate the repurposing of raltegravir as an anticancer agent and its mechanism of action in multiple myeloma (MM). Human MM cell lines (RPMI-8226, NCI H929, and U266) and normal peripheral blood mononuclear cells (PBMCs) were cultured with different concentrations of raltegravir for 48 and 72 h. Cell viability and apoptosis were then measured by MTT and Annexin V/PI assays, respectively. Protein levels of cleaved PARP, Bcl-2, Beclin-1, and phosphorylation of histone H2AX were detected by Western blotting. In addition, the mRNA levels of V(D)J recombination and DNA repair genes were analyzed using qPCR. Raltegravir treatment for 72 h significantly decreased cell viability, increased apoptosis, and DNA damage in MM cells while having minimum toxicity on cell viability of normal PBMCs approximately from 200 nM (0.2 µM; p < .01 for U66 and p < .0001 for NCI H929 and RPMI 8226 cells). Furthermore, raltegravir treatment altered the mRNA levels of V(D)J recombination and DNA repair genes. We report for the first time that treatment with raltegravir is associated with decreased cell viability, apoptosis induction, DNA damage accumulation, and alteration of mRNA expression of genes involved in V(D)J recombination and DNA repair in MM cell lines, all of which show its potential for anti-myeloma effects. Hence, raltegravir may significantly impact the treatment of MM, and further studies are required to confirm its efficacy and mechanism of action in more detail in patient-derived myeloma cells and in-vivo models.

SETMAR, a case of primate co-opted genes: towards new perspectives.

BACKGROUND: We carry out a review of the history and biological activities of one domesticated gene in higher primates, SETMAR, by discussing current controversies. Our purpose is to open a new outlook that will serve as a framework for future work about SETMAR, possibly in the field of cognition development. MAIN BODY: What is newly important about SETMAR can be summarized as follows: (1) the whole protein sequence is under strong purifying pressure; (2) its role is to strengthen existing biological functions rather than to provide new ones; (3) it displays a tissue-specific pattern of expression, at least for the alternative-splicing it undergoes. Studies reported here demonstrate that SETMAR protein(s) may be involved in essential networks regulating replication, transcription and translation. Moreover, during embryogenesis, SETMAR appears to contribute to brain development. SHORT CONCLUSION: Our review underlines for the first time that SETMAR directly interacts with genes involved in brain functions related to vocalization and vocal learning. These findings pave the way for future works regarding SETMAR and the development of cognitive abilities in higher primates.

Structural and genome-wide analyses suggest that transposon-derived protein SETMAR alters transcription and splicing.

Extensive portions of the human genome have unknown function, including those derived from transposable elements. One such element, the DNA transposon Hsmar1, entered the primate lineage approximately 50 million years ago leaving behind terminal inverted repeat (TIR) sequences and a single intact copy of the Hsmar1 transposase, which retains its ancestral TIR-DNA-binding activity, and is fused with a lysine methyltransferase SET domain to constitute the chimeric SETMAR gene. Here, we provide a structural basis for recognition of TIRs by SETMAR and investigate the function of SETMAR through genome-wide approaches. As elucidated in our 2.37 Å crystal structure, SETMAR forms a dimeric complex with each DNA-binding domain bound specifically to TIR-DNA through the formation of 32 hydrogen bonds. We found that SETMAR recognizes primarily TIR sequences (∼5000 sites) within the human genome as assessed by chromatin immunoprecipitation sequencing analysis. In two SETMAR KO cell lines, we identified 163 shared differentially expressed genes and 233 shared alternative splicing events. Among these genes are several pre-mRNA-splicing factors, transcription factors, and genes associated with neuronal function, and one alternatively spliced primate-specific gene, TMEM14B, which has been identified as a marker for neocortex expansion associated with brain evolution. Taken together, our results suggest a model in which SETMAR impacts differential expression and alternative splicing of genes associated with transcription and neuronal function, potentially through both its TIR-specific DNA-binding and lysine methyltransferase activities, consistent with a role for SETMAR in simian primate development.

Structure, Activity, and Function of SETMAR Protein Lysine Methyltransferase.

SETMAR is a protein lysine methyltransferase that is involved in several DNA processes, including DNA repair via the non-homologous end joining (NHEJ) pathway, regulation of gene expression, illegitimate DNA integration, and DNA decatenation. However, SETMAR is an atypical protein lysine methyltransferase since in anthropoid primates, the SET domain is fused to an inactive DNA transposase. The presence of the DNA transposase domain confers to SETMAR a DNA binding activity towards the remnants of its transposable element, which has resulted in the emergence of a gene regulatory function. Both the SET and the DNA transposase domains are involved in the different cellular roles of SETMAR, indicating the presence of novel and specific functions in anthropoid primates. In addition, SETMAR is dysregulated in different types of cancer, indicating a potential pathological role. While some light has been shed on SETMAR functions, more research and new tools are needed to better understand the cellular activities of SETMAR and to investigate the therapeutic potential of SETMAR.

Genome-wide mapping of binding sites of the transposase-derived SETMAR protein in the human genome.

Throughout evolution, DNA transposons provide a recurrent supply of genetic information to give rise to novel gene functions by fusion of their transposase domain to various domains of host-encoded proteins. One of these "domesticated", transposase-derived factors is SETMAR/Metnase which is a naturally occurring fusion protein that consists of a histone-lysine methyltransferase domain and an HsMar1 transposase. To elucidate the biological role of SETMAR, it is crucial to identify genomic targets to which SETMAR specifically binds and link these sites to the regulation of gene expression. Herein, we mapped the genomic landscape of SETMAR binding in a near-haploid human leukemia cell line (HAP1) in order to identify on-target and off-target binding sites at high resolution and to elucidate their role in terms of gene expression. Our analysis revealed a perfect correlation between SETMAR and inverted terminal repeats (ITRs) of HsMar1 transposon remnants, which are considered as natural target sites for SETMAR binding. However, we did not detect any untargeted events at non-ITR sequences, calling into question previously proposed off-target binding sites. We identified sequence fidelity of the ITR motif as a key factor for determining the binding affinity of SETMAR for chromosomes, as higher conservation of ITR sequences resulted in increased affinity for chromatin and stronger repression of SETMAR-bound gene loci. These associations highlight how SETMAR's chromatin binding fine-tune gene regulatory networks in human tumour cells.

SETMAR Shorter Isoform: A New Prognostic Factor in Glioblastoma.

Recent evidence suggests that the chimeric protein SETMAR is a factor of interest in cancer, especially in glioblastoma. However, little is known about the expression of this protein in glioblastoma tissues, and no study has been done to assess if SETMAR could be a prognostic and/or diagnostic marker of glioblastoma. We analyzed protein extracts of 47 glioblastoma samples coming from a local and a national cohort of patients. From the local cohort, we obtained localized biopsies from the central necrosis area, the tumor, and the perilesional brain. From the French Glioblastoma Biobank (FGB), we obtained three types of samples: from the same tumors before and after treatment, from long survivors, and from very short survivors. We studied the correlations between SETMAR amounts, clinical profiles of patients and other associated proteins (PTN, snRNP70 and OLIG2). In glioblastoma tissues, the shorter isoform of SETMAR (S-SETMAR) was predominant over the full-length isoform (FL-SETMAR), and the expression of both SETMAR variants was higher in the tumor compared to the perilesional tissues. Data from the FGB showed that SETMAR amounts were not different between the initial tumors and tumor relapses after treatment. These data also showed a trend toward higher amounts of S-SETMAR in long survivors. In localized biopsies, we found a positive correlation between good prognosis and large amounts of S-SETMAR in the perilesional area. This is the main result presented here: survival in Glioblastoma is correlated with amounts of S-SETMAR in perilesional brain, which should be considered as a new relevant prognosis marker.

NONO Inhibits Lymphatic Metastasis of Bladder Cancer via Alternative Splicing of SETMAR.

Bladder cancer patients with lymph node (LN) metastasis have an extremely poor prognosis and no effective treatment. The alternative splicing of precursor (pre-)mRNA participates in the progression of various tumors. However, the precise mechanisms of splicing factors and cancer-related variants in LN metastasis of bladder cancer remain largely unknown. The present study identified a splicing factor, non-POU domain-containing octamer-binding protein (NONO), that was significantly downregulated in bladder cancer tissues and correlated with LN metastasis status, tumor stage, and prognosis. Functionally, NONO markedly inhibited bladder cancer cell migration and invasion in vitro and LN metastasis in vivo. Mechanistically, NONO regulated the exon skipping of SETMAR by binding to its motif, mainly through the RRM2 domain. NONO directly interacted with splicing factor proline/glutamine rich (SFPQ) to regulate the splicing of SETMAR, and it induced metastasis suppression of bladder cancer cells. SETMAR-L overexpression significantly reversed the metastasis of NONO-knockdown bladder cancer cells, both in vitro and in vivo. The further analysis revealed that NONO-mediated SETMAR-L can induce H3K27me3 at the promotor of metastatic oncogenes and inhibit their transcription, ultimately resulting in metastasis suppression. Therefore, the present findings uncover the molecular mechanism of lymphatic metastasis in bladder cancer, which may provide novel clinical markers and therapeutic strategies for LN-metastatic bladder cancer.

Inhibition of SETMAR-H3K36me2-NHEJ repair axis in residual disease cells prevents glioblastoma recurrence.

BACKGROUND: Residual disease of glioblastoma (GBM) causes recurrence. However, targeting residual cells has failed, due to their inaccessibility and our lack of understanding of their survival mechanisms to radiation therapy. Here we deciphered a residual cell-specific survival mechanism essential for GBM relapse. METHODS: Therapy resistant residual (RR) cells were captured from primary patient samples and cell line models mimicking clinical scenario of radiation resistance. Molecular signaling of resistance in RR cells was identified using RNA sequencing, genetic and pharmacological perturbations, overexpression systems, and molecular and biochemical assays. Findings were validated in patient samples and an orthotopic mouse model. RESULTS: RR cells form more aggressive tumors than the parental cells in an orthotopic mouse model. Upon radiation-induced damage, RR cells preferentially activated a nonhomologous end joining (NHEJ) repair pathway, upregulating Ku80 and Artemis while downregulating meiotic recombination 11 (Mre11) at protein but not RNA levels. Mechanistically, RR cells upregulate the Su(var)3-9/enhancer-of-zeste/trithorax (SET) domain and mariner transposase fusion gene (SETMAR), mediating high levels of H3K36me2 and global euchromatization. High H3K36me2 leads to efficiently recruiting NHEJ proteins. Conditional knockdown of SETMAR in RR cells induced irreversible senescence partly mediated by reduced H3K36me2. RR cells expressing mutant H3K36A could not retain Ku80 at double-strand breaks, thus compromising NHEJ repair, leading to apoptosis and abrogation of tumorigenicity in vitro and in vivo. Pharmacological inhibition of the NHEJ pathway phenocopied H3K36 mutation effect, confirming dependency of RR cells on the NHEJ pathway for their survival. CONCLUSIONS: We demonstrate that the SETMAR-NHEJ regulatory axis is essential for the survival of clinically relevant radiation RR cells, abrogation of which prevents recurrence in GBM.

Compensating for over-production inhibition of the Hsmar1 transposon in Escherichia coli using a series of constitutive promoters.

BACKGROUND: Transposable elements (TEs) are a diverse group of self-mobilizing DNA elements. Transposition has been exploited as a powerful tool for molecular biology and genomics. However, transposition is sometimes limited because of auto-regulatory mechanisms that presumably allow them to cohabit within their hosts without causing excessive genomic damage. The papillation assay provides a powerful visual screen for hyperactive transposases. Transposition is revealed by the activation of a promoter-less lacZ gene when the transposon integrates into a non-essential gene on the host chromosome. Transposition events are detected as small blue speckles, or papillae, on the white background of the main Escherichia coli colony. RESULTS: We analysed the parameters of the papillation assay including the strength of the transposase transcriptional and translational signals. To overcome certain limitations of inducible promoters, we constructed a set of vectors based on constitutive promoters of different strengths to widen the range of transposase expression. We characterized and validated our expression vectors with Hsmar1, a member of the mariner transposon family. The highest rate of transposition was observed with the weakest promoters. We then took advantage of our approach to investigate how the level of transposition responds to selected point mutations and the effect of joining the transposase monomers into a single-chain dimer. CONCLUSIONS: We generated a set of vectors to provide a wide range of transposase expression which will be useful for screening libraries of transposase mutants. The use of weak promoters should allow screening for truly hyperactive transposases rather than those that are simply resistant to auto-regulatory mechanisms, such as overproduction inhibition (OPI). We also found that mutations in the Hsmar1 dimer interface provide resistance to OPI in bacteria, which could be valuable for improving bacterial transposon mutagenesis techniques.

The roles of the human SETMAR (Metnase) protein in illegitimate DNA recombination and non-homologous end joining repair.

SETMAR is a fusion between a SET-domain methyltransferase gene and a mariner-family transposase gene, which is specific to anthropoid primates. However, the ancestral SET gene is present in all other mammals and birds. SETMAR is reported to be involved in transcriptional regulation and a diverse set of reactions related to DNA repair. Since the transcriptional effects of SETMAR depend on site-specific DNA binding, and are perturbed by inactivating the methyltransferase, we wondered whether we could differentiate the effects of the SET and MAR domains in DNA repair assays. We therefore generated several stable U2OS cell lines expressing either wild type SETMAR or truncation or point mutant variants. We tested these cell lines with in vivo plasmid-based assays to determine the relevance of the different domains and activities of SETMAR in DNA repair. Contrary to previous reports, we found that wild type SETMAR had little to no effect on the rate of cell division, DNA integration into the genome or non-homologous end joining. Also contrary to previous reports, we failed to detect any effect of a strong active-site mutation that should have knocked out the putative nuclease activity of SETMAR.

SETMAR isoforms in glioblastoma: A matter of protein stability.

Glioblastomas (GBMs) are the most frequent and the most aggressive brain tumors, known for their chemo- and radio-resistance, making them often incurable. We also know that SETMAR is a protein involved in chromatin dynamics and genome plasticity, of which overexpression confers chemo- and radio-resistance to some tumors. The relationships between SETMAR and GBM have never been explored. To fill this gap, we define the SETMAR status of 44 resected tumors and of GBM derived cells, at both the mRNA and the protein levels. We identify a new, small SETMAR protein (so called SETMAR-1200), enriched in GBMs and GBM stem cells as compared to the regular enzyme (SETMAR-2100). We show that SETMAR-1200 is able to increase DNA repair by non-homologous end-joining, albeit with a lower efficiency than the regular SETMAR protein. Interestingly, the regular/small ratio of SETMAR in GBM cells changes depending on cell type, providing evidence that SETMAR expression is regulated by alternative splicing. We also demonstrate that SETMAR expression can be regulated by the use of an alternative ATG. In conclusion, various SETMAR proteins can be synthesized in human GBM that may each have specific biophysical and/or biochemical properties and characteristics. Among them, the small SETMAR may play a role in GBMs biogenesis. On this basis, we would like to consider SETMAR-1200 as a new potential therapeutic target to investigate, in addition to the regular SETMAR protein already considered by others.

Crystallization of and selenomethionine phasing strategy for a SETMAR-DNA complex.

Transposable elements have played a critical role in the creation of new genes in all higher eukaryotes, including humans. Although the chimeric fusion protein SETMAR is no longer active as a transposase, it contains both the DNA-binding domain (DBD) and catalytic domain of the Hsmar1 transposase. The amino-acid sequence of the DBD has been virtually unchanged in 50 million years and, as a consequence, SETMAR retains its sequence-specific binding to the ancestral Hsmar1 terminal inverted repeat (TIR) sequence. Thus, the DNA-binding activity of SETMAR is likely to have an important biological function. To determine the structural basis for the recognition of TIR DNA by SETMAR, the design of TIR-containing oligonucleotides and SETMAR DBD variants, crystallization of DBD-DNA complexes, phasing strategies and initial phasing experiments are reported here. An unexpected finding was that oligonucleotides containing two BrdUs in place of thymidines produced better quality crystals in complex with SETMAR than their natural counterparts.

SOX11 and HIG-2 are cross-regulated and affect growth in mantle cell lymphoma.

The transcriptional factor SOX11 is a disease-defining antigen in mantle cell lymphoma (MCL) and absent in most non-malignant tissues. To explore the role of SOX11-related cell signaling, and potentially take benefit from these for targeted therapy, associated networks and proteins need to be defined. In this study, we used an inducible SOX11 knock-down system followed by gene expression analysis to identify co-regulated genes and associated signaling pathways. A limited number (n = 27) of significantly co-regulated genes were identified, including SETMAR, HIG-2, and CD24. Further analysis confirmed co-regulation of SOX11 with HIG-2 and CD24 at the protein level. Of major interest, knock-down of HIG-2 reduced SOX11 levels and increased proliferation, the proteins are thus cross-regulated. HIG-2 was localized at the plasma cell membrane in both cell lines and primary MCL cells, and could potentially be of interest for targeted therapy.