scispace - formally typeset

Posted ContentDOI

Development of a papillation assay using constitutive promoters to find hyperactive transposases

21 Sep 2018-bioRxiv (Cold Spring Harbor Laboratory)-pp 423012

TL;DR: An improvement of the well-known papillation assay is presented where in place of an inducible promoter, a set of constitutive promoters cloned into a one or five copies vector in presence or absence of a ribosome binding site is designed.
Abstract: Background Transposable elements is an extremely diverse group of genetic elements encoding their own mobility. This ability has been exploited as a powerful tool for molecular biology and genomics techniques. However, transposition activity is regulated by cis and/or trans mechanisms because of the need to co-exist with their host. This represents a limitation to their usage as biotechnological tools. The development of screening assays and the improvement of current ones is therefore needed to find hyperactive transposases. Results We present in this study an improvement of the well-known papillation assay where in place of an inducible promoter, we designed a set of constitutive promoters cloned into a one or five copies vector in presence or absence of a ribosome binding site. This set of vectors provides a wide range of transposase expression and offers a more uniform expression of the transposase across cells compared to inducible promoters. These constructs can therefore be used to screen for hyperactive transposases or for transposases resistant to overproduction inhibition, a mechanism affecting DNA transposases such as Hsmar1, which decreases the transposition rate when the transposase concentration increases. We characterized and validated our set of vectors with the Hsmar1 transposase and took advantage of our approach to investigate the effects on the transposition rate of inserting mutations in the Hsmar1 dimer interface or of covalently binding two Hsmar1 monomer. Conclusions This improved papillation assay should be applicable to a wide variety of DNA transposases. It also provides a straightforward approach to screen transposase mutant libraries with a specific expression level to find hypoactive, hyperactive or overproduction inhibition resistant transposases. Our approach could also be useful for synthetic biology as a combination of the wild type or covalently bound Hsmar1 transposase with a library of weak promoters offers the possibility to find promoters expressing on average one or two proteins per cell.
Topics: Transposase (60%), Transposable element (56%)

Content maybe subject to copyright    Report

1
A series of constitutive expression vectors to accurately measure the rate of DNA
1
transposition and correct for auto-inhibition
2
3
Michael Tellier* and Ronald Chalmers*
4
School of Life Sciences, University of Nottingham, Queen’s Medical Centre, Nottingham,
5
NG7 2UH, UK
6
* Correspondence: michael.tellier@path.ox.ac.uk; ronald.chalmers@nottingham.ac.uk
7
Present Address: Michael Tellier, Sir William Dunn School of Pathology, University of
8
Oxford, Oxford, OX1 3RF, UK
9
10
11
12
13
14
15
16
17
18
19
20
21
.CC-BY-NC-ND 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted October 18, 2019. ; https://doi.org/10.1101/423012doi: bioRxiv preprint

2
Abstract
22
Background
23
Transposable elements (TEs) form a diverse group of DNA sequences encoding functions
24
for their own mobility. This ability has been exploited as a powerful tool for molecular biology
25
and genomics techniques. However, their use is sometimes limited because their activity is
26
auto-regulated to allow them to cohabit within their hosts without causing excessive genomic
27
damage. To overcome these limitations, it is important to develop efficient and simple
28
screening assays for hyperactive transposases.
29
Results
30
To widen the range of transposase expression normally accessible with inducible promoters,
31
we have constructed a set of vectors based on constitutive promoters of different strengths.
32
We characterized and validated our expression vectors with Hsmar1, a member of the
33
mariner transposon family. We observed the highest rate of transposition with the weakest
34
promoters. We went on to investigate the effects of mutations in the Hsmar1 transposase
35
dimer interface and of covalently linking two transposase monomers in a single-chain dimer.
36
We also tested the severity of mutations in the lineage leading to the human SETMAR gene,
37
in which one copy of the Hsmar1 transposase has contributed a domain.
38
Conclusions
39
We generated a set of vectors to provide a wide range of transposase expression which will
40
be useful for screening libraries of transposase mutants. We also found that mutations in the
41
Hsmar1 dimer interface provides resistance to overproduction inhibition in bacteria, which
42
could be valuable for improving bacterial transposon mutagenesis techniques.
43
44
45
.CC-BY-NC-ND 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted October 18, 2019. ; https://doi.org/10.1101/423012doi: bioRxiv preprint

3
Keywords
46
Papillation assay, Hsmar1, overproduction inhibition, SETMAR, transposase, transposable
47
elements.
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
.CC-BY-NC-ND 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted October 18, 2019. ; https://doi.org/10.1101/423012doi: bioRxiv preprint

4
Background
66
Transposable elements (TEs) are DNA sequences encoding their own ability to move in a
67
genome from one place to another. They are found in virtually all organisms and are
68
particularly present in eukaryotes where they can represent a high percentage of the
69
genome (1-3). Originally described as selfish elements since they were considered parasites
70
which use the host for propagation but do not provide any particular advantage, TEs have
71
now been shown to be important drivers of genome evolution (4, 5). Indeed, TEs can provide
72
novel transcription factor binding sites, promoters, exons or poly(A) sites and can also be co-
73
opted as microRNAs or long intergenic RNAs (6-8). TEs are a diverse group of DNA
74
sequences using a wide range of mechanisms to transpose within their hosts. One particular
75
mechanism prevalent in eukaryotes, and used by the mariner family, is known as “cut-and-
76
paste” transposition (9). Over the past several years, our group and others have described
77
the mechanisms regulating the transposition rate of different mariner transposons, such as
78
Himar1, Hsmar1 or Mos1 (10-15). In Hsmar1, a regulatory mechanism was first recognized
79
because of the phenomenon of overproduction inhibition (OPI) (16). The mechanism of OPI
80
was eventually explained by the realization that double occupancy of the transposon ends
81
with transposase dimers blocks assembly of the transpososome (12). Thus, OPI curbs
82
Hsmar1 transposition rate to avoid damaging the host genome by excessive transposition
83
(12).
84
However, OPI represents a limitation in the development of hyperactive transposases, which
85
would facilitate transposon mutagenesis. Several approaches such as modifying the binding
86
kinetics of the transposase to the inverted terminal repeat (ITR) or the monomer-dimer
87
equilibrium can be used to overcome OPI. Indeed, we and others previously showed that
88
most mutations in the conserved motif, WVPHEL, in Himar1 and Hsmar1, located at the
89
subunit interface, result in hyperactive transposases but at the cost of producing non-
90
productive DNA double-strand breaks and therefore DNA damage (17, 18).
91
.CC-BY-NC-ND 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted October 18, 2019. ; https://doi.org/10.1101/423012doi: bioRxiv preprint

5
To facilitate the isolation of suitable transposase mutants, the papillation assay was
92
developed as an efficient screening procedure (Supplementary Figure 1) (19, 20). This
93
assay is based on a promoter-less lacZ gene flanked by transposon ends. This reporter is
94
integrated in a silent region of the genome of Escherichia coli. The transposase gene is
95
provided in trans on a plasmid to simplify mutagenesis and library handling. Transposition
96
events into an expressed ORF give rise to lacZ gene fusion proteins. When this happens
97
within a colony growing on an X-gal indicator plate, it converts the cell to a lac+ phenotype,
98
which allows the outgrowth of blue microcolonies (papillae) on a background of white cells.
99
The transposition rate is estimated by the number of papillae per colony and by the rate of
100
their appearance.
101
A limitation of the papillation assay is that it generally employs a transposase gene whose
102
expression is under the control of an inducible promoter which cannot be finely regulated.
103
We have constructed a set of vectors maintained in single copy or as five copies per cell
104
which carry various constitutive promoters in the absence or presence of a ribosome binding
105
site (RBS). This set of vectors allows transposase expression across a wide range of
106
expression levels facilitating the screening of hyperactive and/or OPI-resistant transposases.
107
We used this set of vectors to compare an Hsmar1 transposase monomer to a single-chain
108
dimer and to test for hyperactivity and OPI-resistance several Hsmar1 transposase mutants.
109
We found that one Hsmar1 mutant in the dimer interface, R141L, is resistant to OPI in E. coli.
110
111
Results and Discussion
112
Characterization of the papillation assay using a strong inducible promoter
113
The papillation assay provides a visual assessment of the transposition rate, which can be
114
determined from the rate of papillae appearance and their number per colony (19). The
115
transposition rate is dependent on the concentration and activity of the transposase (12). We
116
defined the transposition rate as the average number of papillae per colony after five days of
117
.CC-BY-NC-ND 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted October 18, 2019. ; https://doi.org/10.1101/423012doi: bioRxiv preprint

Citations
More filters

Journal Article
01 Jan 2003-Molecular Cell
TL;DR: The mechanism by which mariner, a eukaryotic transposable element, performs DNA cleavage is examined and it is shown that the nontransferred strand is cleaved initially, unlike prokaryotictransposons which cleave the transferred strand first.
Abstract: It has been proposed that the modern immune system has evolved from a transposon in an ancient vertebrate. While much is known about the mechanism by which bacterial transposable elements catalyze double-strand breaks at their ends, less is known about how eukaryotic transposable elements carry out these reactions. We have examined the mechanism by which mariner, a eukaryotic transposable element, performs DNA cleavage. We show that the nontransferred strand is cleaved initially, unlike prokaryotic transposons which cleave the transferred strand first. First strand cleavage is not tightly coupled to second strand cleavage and can occur independently of synapsis, as happens in V(D)J recombination but not in transposition of prokaryotic transposons. Unlike V(D)J recombination, however, second strand cleavage of mariner does not occur via a hairpin intermediate.

80 citations


Journal ArticleDOI
Shivam Bhatt1, Ronald Chalmers1Institutions (1)
TL;DR: An in vitro mechanistic study that demonstrates efficient Cas9 targeting of the mariner transposon Hsmar1 and shows that the transposase moiety behaved normally and was proficient for integration in vitro and in Escherichia coli.
Abstract: Homology-directed genome engineering is limited by transgene size. Although DNA transposons are more efficient with large transgenes, random integrations are potentially mutagenic. Here we present an in vitro mechanistic study that demonstrates efficient Cas9 targeting of the mariner transposon Hsmar1. Integrations were unidirectional and tightly constrained to one side of the sgRNA binding site. Further analysis of the nucleoprotein intermediates demonstrated that the transposase and Cas9 moieties can bind their respective substrates independently or in concert. Kinetic analysis of the reaction in the presence of the Cas9 target-DNA revealed a delay between first and second strand cleavage at the transposon end. This step involves a significant conformational change that may be hindered by the properties of the interdomainal linker. Otherwise, the transposase moiety behaved normally and was proficient for integration in vitro and in Escherichia coli. Specific integration into the lacZ gene in E. coli was obscured by a high background of random integrations. Nevertheless, Cas9 is an attractive candidate for transposon-targeting because it has a high affinity and long dwell-time at its target site. This will facilitate a future optogenetic strategy for the temporal control of integration, which will increase the ratio of targeted to untargeted events.

17 citations


Journal ArticleDOI
Michael Tellier1, Ronald Chalmers1Institutions (1)
01 Aug 2019-DNA Repair
TL;DR: Full length SETMAR expression has no effect on DNA repair and integration in vivo and SETMAR putative nuclease activity is not required in vivo.
Abstract: 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.

7 citations


Posted ContentDOI
Shivam Bhatt1, Ronald Chalmers1Institutions (1)
08 Mar 2019-bioRxiv
Abstract: Transposases are attractive tools for the integration of therapeutic transgenes into the chromosome for gene therapy applications. Typically, transgenes can be flanked with inverted-terminal repeat sequences, which are recognised by the transposase and integrated at random sites. Minimising detrimental insertions of transgenes is a key goal in the development of gene delivery vectors for gene therapy. We fused the Hsmar1 transposase to a catalytically inactive Cas9. Our aim was to bias transposon insertions into the vicinity of the target site bound by a guide RNA-dCas9 ribonucleoprotein complex. Although we could not detect any targeted transposition events in vivo, we achieved a 15-fold enrichment of transposon insertions into a 600-bp target site in an in vitro plasmid-to-plasmid assay. Additionally, we show that among those integrations that were successfully targeted, the location is tightly constrained to a site immediately to one side of the guide RNA target site. We present an in vitro proof-of-concept study demonstrating that the transposase insertion profile can be biased using a catalytically inactive Cas9 variant as a programmable DNA-binding module. One factor that limits the utility of this approach is that the transposon continues to integrate randomly. Although the dCas9 domain can be targeted to chromosomal lacZ, as evidenced by transcriptional repression, we were unable to detect any targeted insertions in the vicinity of the target site. Any targeted insertions that did occur were masked be a much larger number of random insertions. It is therefore necessary to develop a method for the temporal control of the transposase to allow Cas9 time to locate its target site.

3 citations


Posted ContentDOI
Shivam Bhatt1, Ronald Chalmers1Institutions (1)
08 Mar 2019-bioRxiv
TL;DR: An in vitro proof-of-concept study demonstrating that the transposase insertion profile can be biased using a catalytically inactive Cas9 variant as a programmable DNA-binding module.
Abstract: SUMMARY Homology directed genome engineering is limited by transgene size. Although DNA transposons are more efficient with large transgenes, random integrations are potentially mutagenic. Catalytically inactive Cas9 is attractive candidate for targeting a transposase fusion-protein because of its high specificity and affinity for its binding site. Here we demonstrate efficient Cas9 targeting of a mariner transposon. Targeted integrations were tightly constrained at two adjacent TA dinucleotides about 20 bp to one side of the gRNA binding site. Biochemical analysis of the nucleoprotein complexes demonstrated that the transposase and Cas9 moieties of the fusion protein can bind their respective substrates independently. In the presence of the Cas9 target DNA, kinetic analysis revealed a delay between first and second strand cleavage at the transposon end. This step involves a significant conformational change that may be hindered by the properties of the interdomainal linker. Otherwise, the transposase behaved normally and was proficient for integration in vitro and in vivo.

1 citations


References
More filters

Journal ArticleDOI
Eric S. Lander1, Lauren Linton1, Bruce W. Birren1, Chad Nusbaum1  +245 moreInstitutions (29)
15 Feb 2001-Nature
TL;DR: The results of an international collaboration to produce and make freely available a draft sequence of the human genome are reported and an initial analysis is presented, describing some of the insights that can be gleaned from the sequence.
Abstract: The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

21,023 citations


"Development of a papillation assay ..." refers background in this paper

  • ...They are found in virtually all 78 organisms and are particularly present in eukaryotes where they can 79 represent a high percentage of the genome (1-3)....

    [...]


Journal ArticleDOI
Leslie E. Orgel1, Francis Crick1Institutions (1)
17 Apr 1980-Nature
TL;DR: The DNA of higher organisms usually falls into two classes, one specific and the other comparatively nonspecific, and it seems plausible that most of the latter originated by the spreading of sequences which had little or no effect on the phenotype.
Abstract: The DNA of higher organisms usually falls into two classes, one specific and the other comparatively nonspecific. It seems plausible that most of the latter originates by the spreading of sequences which had little or no effect on the phenotype. We examine this idea from the point of view of the natural selection of preferred replicators within the genome.

1,852 citations


"Development of a papillation assay ..." refers background in this paper

  • ...Originally described as 80 selfish elements due to their ability to replicate independently of their host, 81 TEs have now been shown to be important drivers of genome evolution (4, 5)....

    [...]


Journal ArticleDOI
TL;DR: This review focuses on DNA-mediated or class 2 transposons and emphasizes how this class of elements is distinguished from other types of mobile elements in terms of their structure, amplification dynamics, and genomic effect.
Abstract: Transposable elements are mobile genetic units that exhibit broad diversity in their structure and transposition mechanisms. Transposable elements occupy a large fraction of many eukaryotic genomes and their movement and accumulation represent a major force shaping the genes and genomes of almost all organisms. This review focuses on DNA-mediated or class 2 transposons and emphasizes how this class of elements is distinguished from other types of mobile elements in terms of their structure, amplification dynamics, and genomic effect. We provide an up-to-date outlook on the diversity and taxonomic distribution of all major types of DNA transposons in eukaryotes, including Helitrons and Mavericks. We discuss some of the evolutionary forces that influence their maintenance and diversification in various genomic environments. Finally, we highlight how the distinctive biological features of DNA transposons have contributed to shape genome architecture and led to the emergence of genetic innovations in different eukaryotic lineages.

928 citations


"Development of a papillation assay ..." refers background in this paper

  • ...They are found in virtually all 78 organisms and are particularly present in eukaryotes where they can 79 represent a high percentage of the genome (1-3)....

    [...]


Journal ArticleDOI
Hal S. Alper1, Curt R. Fischer1, Elke Nevoigt2, Elke Nevoigt1  +1 moreInstitutions (2)
TL;DR: The characterized library of promoters is used to assess the impact of phosphoenolpyruvate carboxylase levels on growth yield and deoxy-xylulose-P synthase Levels on lycopene production and is illustrated as being generalizable to eukaryotic organisms and thus constitutes an integral platform for functional genomics, synthetic biology, and metabolic engineering endeavors.
Abstract: Gene function is typically evaluated by sampling the continuum of gene expression at only a few discrete points corresponding to gene knockout or overexpression. We argue that this characterization is incomplete and present a library of engineered promoters of varying strengths obtained through mutagenesis of a constitutive promoter. A multifaceted characterization of the library, especially at the single-cell level to ensure homogeneity, permitted quantitative assessment correlating the effect of gene expression levels to improved growth and product formation phenotypes in Escherichia coli. Integration of these promoters into the chromosome can allow for a quantitative accurate assessment of genetic control. To this end, we used the characterized library of promoters to assess the impact of phosphoenolpyruvate carboxylase levels on growth yield and deoxy-xylulose-P synthase levels on lycopene production. The multifaceted characterization of promoter strength enabled identification of optimal expression levels for ppc and dxs, which maximized the desired phenotype. Additionally, in a strain preengineered to produce lycopene, the response to deoxy-xylulose-P synthase levels was linear at all levels tested, indicative of a rate-limiting step, unlike the parental strain, which exhibited an optimum expression level, illustrating that optimal gene expression levels are variable and dependent on the genetic background of the strain. This promoter library concept is illustrated as being generalizable to eukaryotic organisms (Saccharomyces cerevisiae) and thus constitutes an integral platform for functional genomics, synthetic biology, and metabolic engineering endeavors.

837 citations


"Development of a papillation assay ..." refers background or methods in this paper

  • ...To overcome these limitations, we synthesized a 144 set of five constitutive promoters (00, JJ, K, E, and W) based on (21)....

    [...]

  • ...456 Nomenclature (the letters indicated between brackets are from (21)), sequence, and 457 strength of the constitutive promoters used in this study....

    [...]

  • ...444 Constitutive promoters 445 Alper et al previously generated and characterized a set of constitutive 446 promoters based on pItetO ranging from strong down to very weak (21)....

    [...]


Journal ArticleDOI
TL;DR: Recent findings supporting the long-standing hypothesis that the waves of TE invasions endured by organisms for eons have catalysed the evolution of gene-regulatory networks are reviewed.
Abstract: Transposable elements (TEs) are widely known for their deleterious consequences of selfish propagation and mutagenesis. However, as described in this Review, TEs also provide hosts with rich, beneficial gene-regulatory machinery in the form of regulatory DNA elements and TE-derived gene products. The authors highlight the diverse regulatory contributions of TEs to organismal physiology and pathology, provide a framework for responsibly assigning functional roles to TEs and offer visions for the future.

714 citations


"Development of a papillation assay ..." refers background in this paper

  • ...Originally described as 80 selfish elements due to their ability to replicate independently of their host, 81 TEs have now been shown to be important drivers of genome evolution (4, 5)....

    [...]


Performance
Metrics
No. of citations received by the Paper in previous years
YearCitations
20194
20181
20031