Members of the
DNA transposons have emerged as promising tools in both gene therapy and functional genomics. In particular, the Sleeping Beauty (SB) DNA transposon has advanced into clinical trials due to its ability to stably integrate DNA sequences of choice into eukaryotic genomes. The efficiency of the DNA transposon system depends on the interaction between the transposon DNA and the transposase enzyme that facilitates gene transfer. In this study, we assess the DNA-binding capabilities of variants of the SB transposase and demonstrate that the structural stability of the primary DNA-recognition subdomain, PAI, affects SB DNA-binding affinity and transposition activity. This fundamental understanding of the structure–function relationship of the SB transposase will assist the design of improved transposases for gene therapy applications.
more » « less- PAR ID:
- 10559427
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 53
- Issue:
- 2
- ISSN:
- 0305-1048
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Abstract piggyBac superfamily of DNA transposons are widely distributed in host genomes ranging from insects to mammals. The human genome has retained fivepiggyBac -derived genes as domesticated elements although they are no longer mobile. Here, we have investigated the transposition properties ofpiggyBat fromMyotis lucifugus , the only known active mammalian DNA transposon, and show that its low activity in human cells is due to subterminal inhibitory DNA sequences. Activity can be dramatically improved by their removal, suggesting the existence of a mechanism for the suppression of transposon activity. The cryo-electron microscopy structure of thepiggyBat transposase pre-synaptic complex showed an unexpected mode of DNA binding and recognition using C-terminal domains that are topologically different from those of thepiggyBac transposase. Here we show that structure-based rational re-engineering of the transposase through the removal of putative phosphorylation sites and a changed domain organization - in combination with truncated transposon ends - results in a transposition system that is at least 100-fold more active than wild-typepiggyBat . -
Abstract Transposable elements represent the largest components of many eukaryotic genomes and different genomes harbor different combinations of elements. Here, we discovered a novel DNA transposon in the genome of the clubmoss Selaginella lepidophylla. Further searching for related sequences to the conserved DDE region uncovered the presence of this superfamily of elements in fish, coral, sea anemone, and other animal species. However, this element appears restricted to Bryophytes and Lycophytes in plants. This transposon, named GingerRoot, is associated with a 6 bp (base pair) target site duplication, and 100–150 bp terminal inverted repeats. Analysis of transposase sequences identified the DDE motif, a catalytic domain, which shows similarity to the integrase of Gypsy-like long terminal repeat retrotransposons, the most abundant component in plant genomes. A total of 77 intact and several hundred truncated copies of GingerRoot elements were identified in S. lepidophylla. Like Gypsy retrotransposons, GingerRoots show a lack of insertion preference near genes, which contrasts to the compact genome size of about 100 Mb. Nevertheless, a considerable portion of GingerRoot elements was found to carry gene fragments, suggesting the capacity of duplicating gene sequences is unlikely attributed to the proximity to genes. Elements carrying gene fragments appear to be less methylated, more diverged, and more distal to genes than those without gene fragments, indicating they are preferentially retained in gene-poor regions. This study has identified a broadly dispersed, novel DNA transposon, and the first plant DNA transposon with an integrase-related transposase, suggesting the possibility of de novo formation of Gypsy-like elements in plants.more » « less
-
more » « less
Abstract CRISPR-associated transposases (CASTs) direct DNA integration downstream of target sites using the RNA-guided DNA binding activity of nuclease-deficient CRISPR-Cas systems. Transposition relies on several key protein-protein and protein-DNA interactions, but little is known about the explicit sequence requirements governing efficient transposon DNA integration activity. Here, we exploit pooled library screening and high-throughput sequencing to reveal novel sequence determinants during transposition by the Type I-F Vibrio cholerae CAST system (VchCAST). On the donor DNA, large transposon end libraries revealed binding site nucleotide preferences for the TnsB transposase, as well as an additional conserved region that encoded a consensus binding site for integration host factor (IHF). Remarkably, we found that VchCAST requires IHF for efficient transposition, thus revealing a novel cellular factor involved in CRISPR-associated transpososome assembly. On the target DNA, we uncovered preferred sequence motifs at the integration site that explained previously observed heterogeneity with single-base pair resolution. Finally, we exploited our library data to design modified transposon variants that enable in-frame protein tagging. Collectively, our results provide new clues about the assembly and architecture of the paired-end complex formed between TnsB and the transposon DNA, and inform the design of custom payload sequences for genome engineering applications with CAST systems.
-
more » « less
Abstract Transcription factors (TFs) such as RBPJ in Notch signaling bind to specific DNA sequences to regulate transcription. How TF-DNA binding kinetics and cofactor interactions modulate gene regulation is mostly unknown. We determine the binding kinetics, transcriptional activity, and genome-wide chromatin occupation of RBPJ and mutant variants by live-cell single-molecule tracking, reporter assays, and ChIP-Seq. Importantly, the search time of RBPJ exceeds its residence time, indicating kinetic rather than thermodynamic binding stability. Impaired RBPJ-DNA binding as in Adams-Oliver-Syndrome affect both target site association and dissociation, while impaired cofactor binding mainly alters association and unspecific binding. Moreover, our data point to the possibility that cofactor binding contributes to target site specificity. Findings for other TFs comparable to RBPJ indicate that kinetic rather than thermodynamic DNA binding stability might prevail in vivo. We propose an effective in vivo binding energy landscape of TF-DNA interactions as instructive visualization of binding kinetics and mutation-induced changes.
-
more » « less
Abstract Many eukaryotic transcription factors (TF) form homodimer or heterodimer complexes to regulate gene expression. Dimerization of BASIC LEUCINE ZIPPER (bZIP) TFs are critical for their functions, but the molecular mechanism underlying the DNA binding and functional specificity of homo-
versus heterodimers remains elusive. To address this gap, we present the double DNA Affinity Purification-sequencing (dDAP-seq) technique that maps heterodimer binding sites on endogenous genomic DNA. Using dDAP-seq we profile twenty pairs of C/S1 bZIP heterodimers and S1 homodimers inArabidopsis and show that heterodimerization significantly expands the DNA binding preferences of these TFs. Analysis of dDAP-seq binding sites reveals the function of bZIP9 in abscisic acid response and the role of bZIP53 heterodimer-specific binding in seed maturation. The C/S1 heterodimers show distinct preferences for the ACGT elements recognized by plant bZIPs and motifs resembling the yeast GCN4cis -elements. This study demonstrates the potential of dDAP-seq in deciphering the DNA binding specificities of interacting TFs that are key for combinatorial gene regulation.
