Transposable elements are segments of DNA that can duplicate or move from one location in the genome to another. The initial understanding of transposons as junk DNA have now been overlooked and studies on transposable elements have highlighted their importance in the structure and evolution of the eukaryotic genome. Importantly, transposition should not be highly deleterious to the host. Do the transposable elements confer some selective advantage on the host or merely exist as selfish parasites, only to increase the number of copies of the element? This critical issue is a continuing controversy. When transposable elements are active, they cause DNA damage, causing new mutations by inserting into essential protein coding genes or by promoting rearrangements and genome instability. To suppress the inherent mutagenic potential of transposable elements, the host genome has evolved a number of defense mechanisms which are interdependent pathways such as noncoding small RNAs, DNA methylation and chromatin modifications to protect the harmful effects of TEs (Slotkin and Martienssen, 2007). The chemistry of DNA transposition reactions is relatively well characterized (Kennedy et al., 2000), including the regulatory mechanism that keeps a check on the frequency of transposition. There is a constant battle between the host and the transposons and more research should be carried out to identify and characterize the host factors that regulate the expression of DNA transposon and its mobilization in higher eukaryotes.
Transcriptional control of Sleeping Beauty (SB) transposase by host factors
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The main aim of this project was to study the host transposon interaction between Sleeping Beauty (SB) transposon and its host encoded factor, HMG2L1. The molecular reconstruction of multiple inactive Tc1/mariner elements in fish resulted in the resurrection of an active DNA transposon named Sleeping Beauty (SB) mobilized through the cut and paste mechanism of transposition (Ivics et al., 1997). SB is the most active and studied transposon in vertebrates and it gives us an opportunity to understand the host transposon regulation in a variety of vertebrate species (Ivics et al., 2004). SB transposase has a wide range of activity in vertebrates with different efficiency and among cells of different tissues of the same species (Ivics et al., 2004). Possible explanation for such difference in the efficiency can be attributed to the interaction of the transposition machinery with host factors. It has been shown that the host encoded HMG-box-containing cellular protein [high-mobility group 2-like 1 (HMG2L1)] functions as a positive transcriptional regulator of SB transposase (Walisko et al., 2008). The biological consequence of such host regulation can result in spatial and temporal targeting of transposase expression. Past works from this lab have investigated the interacting partners of HMG2L1 protein, as it is a poorly characterized protein, to understand its molecular function. This led to the identification of two specific protein interacting partners namely SUMO1 and PIAS1. But the main question was to understand the SUMO1 modification of HMG2L1 in the context of SB transposase regulation. Previous work has shown that HMG2L1 upregulates transcription from the 5' UTR of SB 10-15-fold in a reporter assay and the induction of transcription by HMG2L1 is specific to SB transposon (Walisko et al., 2008). SUMOylation is a post-translational modification involved in various cellular processes that are covalently attached to and detached from other proteins in cells to modify their function (Johnson, 2004). SUMOylation of transcription factors have been shown to have various effects on their activity, including modifying their transcription activity (Gill, 2004). In this study, HMG2L1 is shown to be modified in vivo by all members of SUMO family (Figure 4.2 A) and its specific interaction with PIAS1 protein (Figure 4.6 A). It is well proven that PIAS1 can function as an E3 ligase (Sharrocks, 2006) and through series of point mutations on E3 ligase motif which will make them mutant for their E3 ligase function. In this study, it is shown that HMG2L1 can be modified by SUMO1 independent of PIAS1 E3 ligase function (Figure 4.6 B). SUMOylation is a highly dynamic process controlled by a group of cysteine protease that specifically targets members of the small ubiquitin-like modifier (SUMO) protein family (Gill, 2004). This protease regulates SUMO pathways by deconjugating SUMOylated proteins (Shen et al., 2006). This study shows that both SENP 1 and 2 regulates the SUMOylation of HMG2L1, and SENP2 completely deconjugated the SUMO1 from HMG2L1 (Figure 4.5). Taken together, these results show that HMG2L1 is modified by SUMO1 and it is interesting to find out the effect on the expression of SB transposase.
Effect of SUMOylated HMG2L1 on intrinsic promoter activity of Sleeping Beauty left terminal inverted repeat
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Transposon mobilization is often controlled by the proteins encoded by host organisms. On the level of transcriptional regulation, multiple factors were found to influence transposon proteins expression. A large fraction of the human genome is covered by interspersed repeats among them the human L1 retroelement. The 5ï‚¢ UTR of the L1 element contains an internal promoter that contributes to transcriptional activation (Babushok and Kazazian, 2007). Several proteins were found to bind the promoter sequence in the 5ï‚¢ UTR of L1 and regulate its activity. Transcription factor Yin Yang-1 (YY1) binds to base pairs 13 and 21 (Becker et al., 1993) and increases promoter activity (Singer et al., 1993). Members of the SRY family of transcription factors, particularly the SOX factors can bind at two sites of the 5ï‚¢ UTR. Using luciferase assays Sox11 was shown to activate promoter activity of L1 up to 10-fold. HMG2L1, a host-cell protein enhances expression of SB transposase from an intrinsic promoter located in SB left IR with moderate activity (Walisko et al., 2008). To understand the effect of SUMO1 modification on HMG2L1, the transcriptional activity of HMG2L1 was checked in the presence and absence of SUMO1 by a transient luciferase assay in HeLa cells in which 5'UTR Transposon sequences including the left inverted repeat (LIR) was fused to a luciferase gene. As shown in the (Figure 4.9), the result clearly demonstrated that LIR exhibits moderate promoter activity on their own but gets upregulated substantially upon the expression of HMG2L1 but in the presence of SUMO1, the transcriptional activity of HMG2L1 on the LIR promoter was downregulated upon SUMO1 modification leading to transcriptional repression. Importantly, the SUMO1 mutant form of HMG2L1 did not show repression in the presence of SUMO1, clearly highlighting the fact that the transcriptional repression of HMG2L1 is mediated by SUMO1. It had been shown that IR's intrinsic promoter activity was sufficient to drive transposition of SB on their own (Walisko et al., 2008). To examine the effect of SUMO1 and PIAS1 activity on HMG2L1 induced transcription of SB promoter at the level of transposition (Figure 4.9), a transposition assay was carried out in HeLa cells, in which SB transposases driven by its own promoter and neomycin selection marker cloned between SB transposon along with PIAS1, SUMO1, wild-type and SUMO1 mutant form of HMG2L1, followed by G418 selection. Similar results were obtained to the one that were observed in the luciferase assay. Taken together, these two experiments shows that SUMO1 modification of HMG2L1 downregulated the effect on transcription activity of HMG2L1 on the 5'UTR of SB transposases. The molecular mechanism associated to SUMO1 on inhibition of transcription-enhancing activity is still unclear. It is shown in this study that this inhibition was s not related to changes in the stability or DNA-binding activity of HMG2L1. It might be that the repression could be linked to SUMO itself, because SUMO possesses an intrinsic repressive ability when fused to the DNA binding of Gal4 (Seeler and Dejean, 2003). Such a repressive mechanism could involve interactions between HMG2L1 and transcriptional co-repressors. It has been shown that SUMOylation alter the subcellular localization of many target proteins, such as RanGAP1 (Mahajan et al., 1997) and PML protein (Zhong et al., 2000). Another possibility is that SUMOylated forms of HMG2L1 become localized into an inactive subnuclear compartment. In conclusion, the data in this study support the notion that SUMO1 attachment to HMG2L1 provides a fine-tuning mechanism in regulating the expression of Sleeping Beauty transposase.
SUMO1 alters HMG2L1 subcellular localization
The spatial organization of vertebrate cells, the presence of physical boundaries and distinct cellular compartments facilitate a regulation of various biological processes including protein homeostasis and the control over the proteome quality. PML and NBs were proposed to fine-tune wide variety of processes, through facilitation of partner protein posttranslational modifications (notably SUMOylation itself) resulting in partner sequestration, activation, or degradation (Lallemand-Breitenbach and de Thé, 2010). PML NBs can act as depot for SUMO1 modified proteins and given that most of proteins modified by SUMO1 are localized in PML-containing NBs (Zhong et al., 2000). It was logical to study the localization of the Sleeping Beauty transposase in the presence of HMG2L1 with or without SUMO1. The results obtained from immunofluorescent microscopy experiments showed that wild-type HMG2L1 colocalized with SB exclusively in the nucleus, where it was concentrated in NBs containing PML. Similar pattern of colocalization was observed for wild type HMG2L1 with PML NBs in the presence of SUMO1 whereas SUMOylation deficient mutant HMG2L1 fail to colocalize both with SB and SUMO1 in PML bodies indicating that SUMO1 is prerequisite for the localization of HMG2L1 in PML NBs (figure 4.13). The localization of transcription factors within defined subnuclear structures has been of considerable interest, although its functional implications are unknown. Taken together, the results from the localization experiment of SB transposase with wild type and the SUMOylation deficient mutant HMG2L1 partially accounted for the observed difference in the transcriptional activity of HMG2L1 as PML-NBs are a hub for numerous proteins and are involved in a range of functions and a large amount of PML partners have been described in literature as SUMOylated (Van Damme et al., 2010). In addition, PML bodies may form scaffolds onto which co-regulator complexes, including both coactivators and repressors, are assembled to be utilized by transcription factors, and SUMO modifications can act as determinants in these assemblies (Lallemand-Breitenbach and de Thé, 2010). Our results suggest that SUMOylation status of HMG2L1 determines its subnuclear localization. Thus, movable HMG2L1 may influence transcription by modulating SUMO-directed trafficking of coactivator or corepressor proteins between nucleoplasm and PML NBs.
Protein Interactome network of HMG2L1 proteins
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Many cellular processes are regulated by the coordination of several post-translational modifications that allow a very fine modulation of substrates. This study investigated the protein interactome of both wild type and SUMOylated form of HMG2L1 in the presence and absence of SUMO1 and SB by stable isotope labeling. It is one of the powerful methods in quantitative proteomics and has also been used to identify post-translational modifications in proteins, identify components of DNA-protein and protein-protein complexes. A triple SILAC pullâ€down experiments was performed to investigate the interaction partners of wild type HMG2L1-HA tag and SUMOylation-deficient mutant HMG2L1-HA tag in the presence/absence of Sleeping Beauty. HMG2L1 was identified as a reader of H3K4me3 histone mark, which is found in actively transcribed promoters (Vermeulen et al., 2010). From the results, it was shown that both wild type HMG2L1-HA tag and SUMOylation-deficient mutant HMG2L1-HA tag had high enrichment of proteins involved in transcriptional and translational process (Data not shown). It was interesting to understand the protein interactome network of both wild type and SUMOylation-deficient mutant HMG2L1 protein in PML-NBs (Figure 4.15) and the results obtained showed that RPL11 is upregulated by SUMOylated HMG2L1. Previous studies have shown that RPL11 physically associates with PML protein and enhances p53 stability by sequestering ubiquitin-ligase Mdm2 (Bernardi et al., 2004). This may explain the fact PML-NB represents a site for SUMO1 modification of HMG2L1 protein and all these proteins participate in recruiting coregulator complexes accounting for the down regulation of SB transposase expression. In line with the results from this study, another interesting data came out of this experiment showing that the SUMOylation-deficient mutant HMG2L1 upregulated SENP3 compared to wild type HMG2L1. It was reported that SENP3 co-localize with PML bodies and reduced the number of PML bodies in SENP3-dependent manner (Han et al., 2010). The PML bodies are normally separated from the SUMO proteases, but they undergo an intensive de-SUMOylation upon stress (Han et al., 2010). SENP3-mediated de-SUMOylation of PML might contribute to the deSUMOylation of HMG2L1 protein in order to maintain fine balance between SUMOylated vs unSUMOylated HMG2L1 proteins for different cellular functions. Taken together the results gave an understanding of the molecular effects of SUMOylation on HMG2L1 protein and its relation with Sleeping Beautytransposase expression.
HMG2L1 is an evolutionarily conserved protein
Positive promoter regulation of SB left IR by human HMG2L1 was first shown by (Walisko et al., 2008). The experiments were performed in HeLa cells, thus promoter activation of SB was assayed in a human background. Since Sleeping Beautytransposase enzyme was resurrected originally from salmonid fish (Ivics et al., 1997) questions on significance of its induction are raised. An alignment of HMG2L1 amino acid sequences from different vertebrate species (e.g. Danio rerio) showed high conservations that share 49% protein identity to the hHMG2L1 sequence. A fish ortholog for hHMG2L1 was cloned for the present study, zHMG2L1. In order to show the function of HMG2L1 on SB left IR and interaction with SB transposase is conserved in diverse vertebrate species and to demonstrate the relevance and analogous inductive effect with a related protein of vertebrate origin, similar luciferase reporter assay and transposition assay were performed with zHMG2L1 and SB left IR. It was found that zHMG2L1 induces promoter activity of SB left IR up to 3-fold, compared to hHMG2L1 and the identical results were obtained for transposition assay. Asshown that hHMG2L1 is modified by SUMO1 protein which attenuates its activity as a transcription factor on the 5' UTR of SB, it was worth to check whether the sequence similarities between human and zebrafish HMG2L1 contributed to its functional conservation. This study showed that zHMG2L1 was modified by SUMO1 by in vivo SUMOylation assay and its shows similar transcriptional repression like hHMG2L1 by both luciferase and transposition assays. Based on the results, the function of HMG2L1 conserved across species was confirmed and the zHMG2L1 was found to have higher activity compared to hHMG2L1. The observed high activity of zHMG2L1 was mainly due to its close relationship with Sleeping Beauty transposase as SB is a fish protein and successful utilization of host factors contributed to the successful propagation of the transposase.
HMG2L1 is expressed in germ cells and throughout early embryogenesis
Conventional wisdom of considering transposons as ancient relics has been challenged with the startling evidence that certain class of TEs can hop around genome (Akagi et al., 2013). DNA transposons are not actively mobilized when compared to reterotransposons, but the recent activity of DNA transposon in Myotis lucifugus has highlighted the importance to study the impact of this class of elements on mammalian genome evolution (Ray et al., 2008). The separation of germ-line and somatic cells in metazoans facilitates the transposon to minimize the chance of a potentially harmful and evolutionary unproductive integration event in somatic cells. Keeping the views mentioned, it was interesting to study the germline versus somatic mobilization of Sleeping Beauty transposase by following its expression in a cell dependent manner. Given the interaction between 5'UTR of SB transposon and human HMG2L1 observed in mammalian cells, the activity of pLIR (5'UTR transposon sequences fused to a luciferase reporter gene) and Pâˆ†LIR (a 65-bp deleted region abolishing HMG2L1 binding) were checked by microinjecting into a single-cell fertilized zebra fish embryos to follow the regulation of tissue-specific expression of SB transposase in developing zebrafish embryos. A twofold higher luciferase activity of pLIR was observed when compared to Pâˆ†LIR in developing zebrafish embryos highlighting the presence of host encoded transcriptional factors that can control the expression of SB transposase in a tissue specific manner. This observation prompted to analyze the temporal expression of zHMG2L1 in developmental stages of zebrafish. This study shows that zHMG2L1 is maternally expressed and the expression level went down during the maternal to zygotic transition accounting for the reduced activity of Pâˆ†LIR in luciferase assay with zebrafish embryos. Evidences presented in this study indicate that the zHMG2L1 expression controls the transcription of SB transposase in a developmental manner accounting for new somatic insertions. In contrast with the germline TEs insertions, somatic insertions are not meiotically heritable from one generation to the next. But the promoters within the Sleeping Beauty transposable element can initiate variable transcription of adjacent genes resulting in novel transcripts contributing to genome plasticity at a tissue specific level. Significant germline transposition in mammals has contributed in the retention of exapted or exonized TEs that may increase divergence and help drive speciation (Akagi et al., 2013). A classical example is the syncytin gene, evolved from the envelope gene of the human endogenous defective retrovirus HERV-W shown to be playing an important role in human placental morphogenesis (Mi et al., 2000). The very ability of transposons to evolve rapidly and quickly mobilize have made the host genome to use small RNAs efficiently to control transposition activity of most transposons but such an immune system is not always applicable against a new transposon's invasions (Rozhkov et al., 2013). All these observations raised an important question, whether Sleeping Beauty transposase, a synthetic DNA transposon resurrected from fish can evade the host surveillance mechanism in germ cells. Toward this goal, the expression of HMG2L1 was investigated first, as it was shown to be positive regulator of SB transposase in rat spermatogonial stem cells (SSCs), a germ cell model and its differentiated form by an in vivo culture technique and showed that the expression of rat HMG2L1 was upregulated in SSCs, which might indicate that the SB transposase can undergo successful transposition in germ cells. It has been shown that human L1 RNA is transcribed in both male and female germ cells which can be carried over through fertilization and integrate during embryogenesis contributing to somatic mosaicism in humans (Kano et al., 2009)
To summarize the findings, it has been shown that HMG2L1, a host-cell protein enhances expression of SB transposase from an intrinsic promoter located in SB left IR with moderate activity (Walisko et al., 2008). Thus, the action of HMG2L1 could effect in a temporary increase in transposase production but transposon mobilization is often subjected to the control of proteins encoded by host organisms. Host factor HMG2L1 is a novel protein with uncharacterized function, which led to the investigation of the protein-protein interactions that can influence the molecular properties of HMG2L1 in distinct ways. This study shows that HMG2L1 undergoes a covalent modification by SUMO1, a reversible post-translational modification that has been shown to regulate the activity of many transcriptional factors. Upon SUMOylation, the SUMO1 modified forms of HMG2L1 are compartmentalized exclusively in the PML nuclear bodies (NBs) that attenuates their transcriptional activity and in turn downregulate the expression of SB transposase. Further, the results of this work shows that HMG2L1 function is highly conserved protein iterating the fact contributing to the successful colonization of SB transposase in various species. Life cycle of Tc1/mariner elements includes colonization of host germ line, proliferation by transposition and spread by sexual reproduction (Miskey et al., 2005). Higher expression of HMG2L1 in germ cells could initiate the expression of SB transposase resulting in new insertions in germ cells and are likely passed to the next generations, although, much is not known about the underlying mechanisms regulating transposons and transposition reactions. Results presented in this study can broaden the knowledge on the host factors regulating the SB transposition and thus can be adapted for beneficial application of the SB system in future.
Targeted Transgenic in Rat Model
The laboratory rat (Rattus norvegicus) is an important model for biomedical research. Hundreds of unique rat models have been developed to mimic pathological and physiological human clinical conditions, especially in the case of complex diseases. A standardized approach for routine and reproducible rat transgenesis will make rat models more accessible to the research community. Traditional ways to induce expression of foreign genes in vertebrates rely on microinjection of nucleic acids into oocytes or fertilized eggs. But main limitations of these methods are low rates of genomic integration and muticopy transgene concatemer resulting in transgene silencing (Mátés, 2011). The problem was circumvented by hyperactive Sleeping Beautytransposon system, SB100X which was shown to mediate 14-72 % transgenic founders in rat (Katter et al., 2012). The SB100X-mediated transgene integrations are less prone to genetic mosaicism and gene silencing, and can be used to generate founders with single-copy integrations (Mátés, 2011). In this study, Sleeping Beauty mediated single-copy transgene integration was combined with recombinase-mediated cassette exchange for targeted transgenesis. Firstly, efficacy of this study was analysed by establishing two stable cell lines (single copy and double copy transgene) mediated by Sleeping BeautyTransposon system expressing a Venus protein containing heterologous loxP sites. The efficiency of RMCE and site-specific exchange were checked by a transient transfection of a targeting vector (pT2/Neo RMCE) flanking an analogous loxp sites and plasmid expressing Cre protein in the stable cell lines and the loss of Venus expression was measured both by colony forming assay and FACS analysis. Outcome of the experiment clearly established the fact that the rate of exchange in RMCE is proportional to the copy number and the concentration of Cre protein is very critical for targeted exchange. Now, this part of the study would be extended to animal models. The laboratory rat was chosen for two main reasons, firstly, ratt is the most preferred animal model over mouse in many human diseases and secondly, the afe Harbor Locus is not well characterized. Moreover, transposon mediated transgenesis have provided the great advantage to scan the genome for Safe Harbor Loci in rat due to their inherent capacity of random integration and co-injection of engineered transposons with in vitro transcribed transposase messenger RNA (mRNA) helps to overcome the problem of genetic mosaicism because only the translation of synthesized transposase mRNA is sufficient for transposase protein to shift the window of transposon-mediated integration events to early stages in order to promote successful germ line transmission of the transgene to the next generation in spite of the actual transcriptional quiescent state of the zygote.
A model transgenic rat model with a single copy transgene expressing Venus protein flanked by heterospecific loxP sites using Sleeping Beauty transposon system was established. Germ line competency and genomic insertion site was characterized for the transgenic rat. In a proof of principle experiment, one cell embryos from the homozygous transgenic rat carrying single transposon integration were used for targeted transgenesis by RMCE, and animals were successfully reconstituted from the intact zygotes after pronuclear injection, demonstrating the feasibility of an experimental pipeline of targeted transgenesis into transposon-tagged genomic loci. An efficiency exchange of 3.5% was observed by microinjection, though the rate of exchange can be improved by carefully titrating the concentration of Cre protein and by assessing the epigenetic status of the genomic loci, where transgene is integrated. The accessibility of the region will be critical for efficient RMCE by pronuclear injection in zygotes. In summary, this study shows the use of SB transposon system for efficient rat transgenesis and the animals carrying single transposon integration were used for targeted transgenesis by RMCE. Analysis from this work demonstrates that that Cre-mediated cassette exchange is possible and can be highly efficient in rat fertilized oocytes using heterospecific lox sites. These results demonstrate the general applicability of the system and provide a basis for further investigations.