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

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

© 2020 The Author(s). 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

Differential effects of pre-mRNA splicing inhibitors on RNA polymerase II transcription

The production of eukaryotic mRNA requires transcription by RNA polymerase (pol) II and co-transcriptional processing, including capping, splicing, and cleavage and polyadenylation (CPA). Pol II can positively affect co-transcriptional processing through interaction of factors with its carboxyl terminal domain (CTD), comprising 52 repeats of the heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Small molecule inhibitors of the splicing factor SF3B1 cause loss of the transcription elongation factor P-TEFb from protein-coding gene templates and major transcription defects, indicating that splicing can, in turn, positively affect transcription. To understand better the relationship between pre-mRNA splicing and pol II transcription, we have investigated the effect of two other splicing inhibitors, Madrasin and Isoginkgetin, on transcription. We found that Madrasin rapidly inhibits pre-mRNA splicing, whereas Isoginkgetin affects transcription before any detectable effect on pre-mRNA splicing. Interestingly, we found that both of these small molecules promote general downregulation of transcription and global transcriptional readthrough, including on intronless and histone genes. Both small molecules affect the association of the mRNA CPA complex with chromatin, likely explaining the transcription termination defect. However, splicing inhibition is not necessarily associated with transcriptional readthrough as small molecule inhibitors of SF3B1 or knockdown of splicing factors do not cause a global transcription termination defect

A genome-wide transcriptional network controlled by the human SETMAR protein

Transposable elements are discrete segments of DNA, which can be mobilized and amplified within a host genome. They are found in almost every living organism where their activities can contribute to gene expression and phenotypic variability. Hsmar1, a DNA transposon from the ITm superfamily, has entered the anthropoid primate lineage 55 to 65 Mya and remained active for ~ 15 My. The human genome contains 8,527 Hsmar1 remnants, which are essentially distributed between Made1 elements, an 80 bp deletion-derivative of the Hsmar1 transposon containing two transposon ends, and solo ITRs, which are deletion-derivative of Made1. Amidst these remnants, a fusion event between a pre-existing histone methyltransferase gene and one copy of the Hsmar1 transposase gave birth to the SETMAR gene. Except for a possible role in non-homologous end joining, the functions of SETMAR in human cells are still poorly understood. Since the DNA-binding domain of SETMAR is under purifying selection and several thousands transposon ends are located within genes, I investigated whether SETMAR binds to the genic transposon ends to regulate gene expression by dimethylating the K36 of histone H3. By using RNA-Seq, I established that a change in SETMAR expression modifies the expression of a third of the 1,523 protein-coding genes containing an ITR, with a bias towards up-regulation. I determined SETMAR binding sites with ChIP-Seq and found that SETMAR binds essentially to genic and intergenic Made1 elements and to a group of putative miRNAs derived from Made1. Even though only 2% of the differentially expressed genes with an ITR are found in the ChIP-Seq data, the genic ChIP peaks are enriched in the genes with an ITR. SETMAR may fine-tune by itself or by interacting with other proteins the expression of the subset of genes, which are differentially expressed and bound by SETMAR. Members of this group of genes encode proteins involved in signalling pathways such as FAM83B and ARSG, which could in turn modify gene expression. SETMAR could also potentially regulate the expression of the miRNAs-like derived from Made1

Transposase subunit architecture and its relationship to genome size and the rate of transposition in prokaryotes and eukaryotes

Cut-and-paste transposons are important tools for mutagenesis, gene-delivery and DNA sequencing applications. At the molecular level, the most thoroughly understood are Tn5 and Tn10 in bacteria, and mariner and hAT elements in eukaryotes. All bacterial cut-and-paste transposases characterized to date are monomeric prior to interacting with the transposon end, while all eukaryotic transposases are multimers. Although there is a limited sample size, we proposed that this defines two pathways for transpososome assembly which distinguishes the mechanism of the bacterial and eukaryotic transposons. We predicted that the respective pathways would dictate how the rate of transposition is related to transposase concentration and genome size. Here, we have tested these predictions by creating a single-chain dimer version of the bacterial Tn5 transposase. We show that artificial dimerization switches the transpososome assembly pathway from the bacterial-style to the eukaryotic-style. Although this had no effect in vitro, where the transposase does not have to search far to locate the transposon ends, it increased the rate of transposition in bacterial and HeLa cell assays. However, in contrast to the mariner elements, the Tn5 single-chain dimer remained unaffected by over-production inhibition, which is an emergent property of the transposase subunit structure in the mariner elements

Thermoresponsive worms for expansion and release of human embryonic stem cells

The development of robust suspension cultures of human embryonic stem cells (hESCs) without the use of cell membrane disrupting enzymes or inhibitors is critical for future clinical applications in regenerative medicine. We have achieved this by using long, flexible, and thermoresponsive polymer worms decorated with a recombinant vitronectin subdomain that bridge hESCs, aiding in hESC's natural ability to form embryoid bodies (EBs) and satisfying their inherent requirement for cell-cell and cell-extracellular matrix contact. When the EBs reached an optimal upper size where cytokine and nutrient penetration becomes limiting, these long and flexible polymer worms facilitated EB breakdown via a temperature shift from 37 to 25 C. The thermoresponsive nature of the worms enabled a cyclical dissociation and propagation of the cells. Repeating the process for three cycles (over eighteen days) provided a >30-fold expansion in cell number while maintaining pluripotency, thereby providing a simple, nondestructive process for the 3D expansion of hESC