Author response: LINE-1 protein localization and functional dynamics during the cell cycle (2017)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract LINE-1/L1 retrotransposon sequences comprise 17% of the human genome. Among the many classes of mobile genetic elements, L1 is the only autonomous retrotransposon that still drives human genomic plasticity today. Through its co-evolution with the human genome, L1 has intertwined itself with host cell biology. However, a clear understanding of L1’s lifecycle and the processes involved in restricting its insertion and intragenomic spread remains elusive. Here we identify modes of L1 proteins’ entrance into the nucleus, a necessary step for L1 proliferation. Using functional, biochemical, and imaging approaches, we also show a clear cell cycle bias for L1 retrotransposition that peaks during the S phase. Our observations provide a basis for novel interpretations about the nature of nuclear and cytoplasmic L1 ribonucleoproteins (RNPs) and the potential role of DNA replication in L1 retrotransposition. https://doi.org/10.7554/eLife.30058.001 eLife digest Only two percent of our genetic material or genome are occupied by genes, while between 60-70 percent are made up of hundreds of thousands of copies of very similar DNA sequences. These repetitive sequences evolved from genetic elements called transposons. Transposons are often referred to as ‘jumping genes’, as they can randomly move within the genome and thereby create dangerous mutations that may lead to cancer or other genetic diseases. LINE-1 is the only remaining active transposon in humans, and it expands by copying and pasting itself to new locations via a process called 'retrotransposition'. To do so, it is first transcribed into RNA – the molecules that help to make proteins – and then converted back into identical DNA sequences. Previous research has shown that LINE-1 can form complexes with a series of proteins, including the two encoded by LINE-1 RNA itself: ORF1p and ORF2p. The LINE-1 complexes can enter the nucleus of the cell and insert a new copy of LINE-1 into the genome. However, until now it was not known how they do this. To investigate this further, Mita et al. used human cancer cells grown in the lab and tracked LINE-1 during the different stages of the cell cycle. The results showed that LINE-1 enters the nucleus as the cell starts to divide and the membrane of the nucleus breaks down. The LINE-1 complexes are then retained in the nucleus while the membrane of the nucleus reforms. Later, as the cell duplicates its genetic material, LINE-1 starts to copy and paste itself. Mita et al., together with another group of researchers, also found that during this process, only LINE-1 RNA and ORF2p were found in the nucleus. This shows that the cell cycle dictates both where the LINE-1 complexes gather and when LINE-1 is active. A next step will be to further investigate how the ‘copy and paste’ mechanisms of LINE-1 and the two LINE-1 proteins are regulated during the cell cycle. In future, this may help to identify LINE-1’s role in processes like aging or in diseases such as cancer. https://doi.org/10.7554/eLife.30058.002 Introduction Retrotransposons are genetic elements that move within the host genome through a ‘copy and paste’ process utilizing an RNA intermediate. 17% of the human genome is made up of copies of the Long Interspersed Nuclear Element-1 (LINE-1 or L1) retrotransposon (Lander et al., 2001). L1 is the only autonomous human retrotransposon able to ‘jump’ through a process called retrotransposition. The full length, active L1 retrotransposons consists of a 5’ untranslated region (UTR) containing a bidirectional promoter (Speek, 2001; Swergold, 1990), two open reading frames (ORFs), ORF1 and ORF2, separated by a short inter-ORF region and a 3’ UTR with a weak polyadenylation signal (Doucet et al., 2015; Dombroski et al., 1991; Burns and Boeke, 2012; Burns, 2017). A short, primate specific, third ORF of unknown function called ORF0 was also described in the 5’UTR of L1 with an antisense orientation compared to ORF1 and ORF2 (Denli et al., 2015). Upon transcription by RNA polymerase II, the 6 kb bicistronic L1 mRNA is poly-adenylated and exported into the cytoplasm. LINE-1 exhibits ‘cis-preference’ (Wei et al., 2001), the mechanism of which is unknown. A popular model posits that newly translated ORF1 and/or ORF2 proteins preferentially bind the mRNA molecule that encoded them (Boeke, 1997). Interestingly, translation of the second ORF, ORF2p, is mediated and regulated by an unconventional process that remains poorly understood (Alisch et al., 2006). ORF1p is an RNA binding protein with chaperone activity (Martin and Bushman, 2001; Khazina et al., 2011) while ORF2p contains domains with endonuclease (EN) and reverse transcriptase (RT) activity (Feng et al., 1996; Mathias et al., 1991; Weichenrieder et al., 2004). It is believed that ribonucleoprotein particles (RNPs), consisting of many ORF1p molecules, as few as one or two ORF2ps and one L1 mRNA (Khazina et al., 2011; Basame et al., 2006; Dai et al., 2014) form in the cytoplasm. The RNP is then imported into the nucleus through a still uncharacterized process. L1 RNPs accumulate in cytoplasmic stress granules (SGs) and processing bodies (PBs) (Goodier et al., 2007; Hu et al., 2015; Doucet et al., 2010), but the role of these cellular structures in the L1 lifecycle is still controversial. L1 RNPs accumulate mainly in the cytoplasm but nuclear ORF1p was also observed in a certain percentage of cells using cell lines and cancer specimens (Sokolowski et al., 2013; Sharma et al., 2016; Doucet et al., 2016; Goodier et al., 2007; Harris et al., 2010). While several specific antibodies against ORF1p have been raised (Taylor et al., 2013; Wylie et al., 2016; Doucet-O'Hare et al., 2015), a highly effective antibody against ORF2 protein that would allow definitive observation of protein localization is still lacking. Antibodies against LINE-1 ORF2p have been recently developed (De Luca et al., 2016; Sokolowski et al., 2014) but the much lower amount of expressed ORF2p compared to ORF1p makes the study of ORF2p expression and localization difficult. To overcome these difficulties, tagged ORF2ps have been employed (Taylor et al., 2013; Doucet et al., 2010). In the nucleus, L1 endonuclease nicks the DNA at A/T rich consensus target sites (5’-TTTT/AA-3’) (Feng et al., 1996) and through a process called TPRT (Target Primed Reverse Transcription)(Cost et al., 2002; Luan et al., 1993) inserts a DNA copy into the new genomic target locus. During TPRT, ORF2p EN domain nicks the DNA and the newly formed 3’OH end is then used by the RT domain of ORF2p to prime the synthesis of a complementary DNA using the L1 mRNA as template. A second strand of cDNA is then synthetized and joined to adjacent genomic DNA. L1 lifecycle is extensively entwined with host cellular processes. Several proteins and cellar pathways have been shown to restrict or support L1 retrotransposition and life cycle (Goodier, 2016). RNA metabolism (Belancio et al., 2008; Dai et al., 2012), DNA damage response (Servant et al., 2017) and autophagy (Guo et al., 2014) are a few cellular processes shown to affect LINE-1 retrotransposition. Progression through the cell cycle was shown by several groups to promote L1 retrotransposition (Shi et al., 2007; Xie et al., 2013) but a molecular understanding of this aspect has been elusive. Because of the importance of the cell cycle in efficient retrotransposition, it has been proposed that, as for some exogenous retroviruses (Goff, 2007; Suzuki and Craigie, 2007), nuclear breakdown during mitosis could represent an opportunity for entrance of L1 into the nucleus (Xie et al., 2013; Shi et al., 2007). This hypothesis, never directly tested previously, was challenged by studies demonstrating effective retrotransposition in non-dividing and terminally differentiated cells (Kubo et al., 2006; Macia et al., 2017). To shed light on the role of the cell cycle on different aspects of L1 life cycle we explored the nuclear localization of L1 proteins and the retrotransposition efficiency of L1 in different stages of the cell cycle in rapidly dividing cancer cells. Here, we use imaging, genetic and biochemical approaches to show that in these cells, the L1 lifecycle is intimately coordinated with the cell cycle. LINE-1 encoded proteins enter the nucleus during mitosis and retrotransposition appears to occur mainly during S phase. Results Analysis and quantification of ORF1p and ORF2p expression and cellular localization Previous works (Doucet et al., 2016; Luo et al., 2016; Sokolowski et al., 2013; Goodier et al., 2007; Goodier et al., 2004; Sharma et al., 2016; Rodić et al., 2014; Taylor et al., 2013; Branciforte and Martin, 1994) have shown varying localization of ORF1 protein in cells growing in culture or in mammalian specimens from various organs and tumors. In particular, the nuclear localization of LINE-1 encoded proteins has been sparsely studied and the mechanisms driving import of L1 retrotransposition intermediates into the nucleus are largely unknown. We therefore set out to characterize the cellular localization of L1 ORF1p and ORF2p in human cells overexpressing recoded (ORFeus) or non-recoded L1 (L1rp) with a 3xFlag tag on the ORF2p C-terminus (Taylor et al., 2013). Immuno-fluorescence staining of HeLa-M2 cells (Hampf and Gossen, 2007) overexpressing ORFeus, showed clear expression of ORF1p and ORF2p (Figure 1A and D and Figure 1—figure supplements 1,2, Videos 1 and 2). As previously observed (Taylor et al., 2013), ORFeus ORF1p was detected in virtually all the cells (≅ 97%) whereas ORF2p, encoded by the same bicistronic L1 mRNA also expressing ORF1p, was detected in just a subset of cells (≅10% using a rabbit anti-Flag antibody and ≅20% using a more sensitive mouse anti-Flag antibody) (Figure 1B, bar graph and inset). This pattern of expression is most likely due to an unknown mechanism controlling ORF2p translation (Alisch et al., 2006). Interestingly, when non-recoded L1 was over-expressed, only 44% of cells displayed ORF1p expression. Overexpression of ORFeus-Hs and L1rp with or without the L1 5’ untranslated region (5’ UTR) excluded the possibility that the reduced expression of L1rp compared to ORFeus was due to the presence of the 5’ UTR (Figure 1—figure supplement 3) (Chen et al., 2012). Due to the overall lower expression of L1rp, ORF2p was barely observable in cells expressing non-recoded L1 (Figure 1A and B). ORF1p was mainly present in the cytoplasm but some cells (≅26%) also showed clear nuclear staining (Figure 1A, Figure 1D, white arrowheads, Figure 1—figure supplements 1,2, Videos 1 and 2). Z-stack movies of cells expressing L1 and stained for recoded and non-recoded ORF1p and ORF2p are also presented (Videos 1 and 2). Confocal images recapitulated our observations and confirmed our conclusions (Figure 1—figure supplement 2). Interestingly, the cells with nuclear ORF1p fluorescence were usually observed as pairs of cells in close proximity. This observation suggested to us that cells with nuclear ORF1p may have undergone mitotic division immediately prior to fixation and observation. In HeLa cells, this observation was made more evident by the fact that daughter cells displaying nuclear ORF1p were sometimes connected by intercellular bridges often containing filaments of DNA and persisting from incomplete cytokinesis during the previous mitosis (Figure 1D, lower panels) (Steigemann et al., 2009; Carlton et al., 2012). To quantify our initial qualitative observation of closer cell proximity for cells displaying nuclear ORF1p, we employed automated image acquisition of HeLa cells expressing recoded L1 and stained for ORF1p (see ‘quantification and statistical analysis’ section). We compared the distance of pairs of cells expressing nuclear ORF1p versus the distance of the same number of cell pairs expressing ORF1p just in the cytoplasm. This ‘proximity analysis’, described in more details in the methods section, clearly shows that cells with nuclear ORF1p are statistically closer to each other than cells expressing ORF1p exclusively in the cytoplasm (Figure 1C). Figure 1 with 4 supplements see all Download asset Open asset L1 protein expression and localization (A) Representative pictures of immunostained HeLa M2 cells expressing a recoded L1 (ORFeus) or a non-recoded L1 (L1rp). Antibody target names are reported atop the corresponding pictures and colored according to the colors used in the merged pictures. Scale bar = 100 μm. (B) Quantification of ORF1p and ORF2p expression in the cytoplasm and nucleus of HeLa cells expressing the indicated L1 element (error = S.E.M.) of at least 10 20X fields with about 100 cells each. The inset shows quantification of ORF2p stained using an anti-FLAG mouse antibody (SIGMA-M2 F1804). (C) Proximity analysis of cells with (purple) or without (yellow) nuclear ORF1p is reported. HeLa cells expressing Flag tagged ORFeus for 24 hr, were stained with anti-ORF1p JH74 Ab and DAPI. Pictures were collected using an Arrayscan microscope and analysis was performed as described in the Materials and Methods. ***p<0.001 (p=4.351e−13) (D) Immunostaining of ORF1p and ORF2p using, respectively, rabbit monoclonal Ab JH74 and mouse monoclonal antibody M2 against FLAG. Scale bar = 20 μm. White arrowheads indicate cells with nuclear ORF1p. https://doi.org/10.7554/eLife.30058.003 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg ORFeus ORF1p/ORF2p Z stack. Z-stacks of recoded L1 (ORFeus) ORF1p (left) and ORF2p (right) expressed in HeLa-M2 treated for 24 hr with doxycycline 0.1 μg/ml. Red arrows points to cells expressing cytoplasmic and nuclear ORF2p and ORF1p; the green arrow points to a cell expressing cytoplasmic and nuclear ORF2p and just cytoplasmic ORF1p. https://doi.org/10.7554/eLife.30058.008 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg L1rp ORF1p/ORF2p Z stack. Z-stacks of L1rp ORF1p (left) and ORF2p (right) expressed in HeLa-M2 treated for 24 hr with doxycycline 0.1 μg/ml. https://doi.org/10.7554/eLife.30058.009 The cytoplasmic ORF1p localization pattern was often dominated by previously described cytoplasmic foci (Doucet et al., 2010; Goodier et al., 2007). The formation of these foci is particularly enhanced by L1 overexpression. The nature and role of these cytoplasmic structures is still debated (Goodier et al., 2007; Martin and Branciforte, 1993; Guo et al., 2014) and in our system they may be induced by non-physiological expression levels of ORF1p. We therefore performed the localization experiments reported here using Tet-inducible constructs, and we induced L1 expression at lower concentrations of doxycycline (0.1 μg/ml) compared to the concentrations typically used to induce full induction of retrotransposition or production of RNP intermediates for proteomic studies (1 μg/ml) (Taylor et al., 2013). We observed very heterogeneous localization of ORF1p and ORF2p in cells overexpressing L1 upon treatment with 0.1 μg/ml doxycycline for 24 hr. ORF1p is observed to be only cytoplasmic (Figure 1D top panel), or both cytoplasmic and nuclear (Figure 1D, top panel) but is never exclusively nuclear when immunostaining is performed using 4H1, 4632 and JH74 antibodies (Figure 1—figure supplements 1,2). ORF2p also had heterogeneous nuclear/cytoplasmic localization with cells displaying ORF2p only in the cytoplasm (Figure 1A and Figure 1D middle panel), only in the nucleus (Figure 1D, top panel) or in both the cytoplasm and the nucleus (Figure 1D, lower panel). Nuclear ORF2p had a punctate pattern (Figure 1D top panel) and nuclear ORF1p was usually excluded from nucleoli (Figure 1D, arrowheads). Also, a small population of cells (usually less than 0.1% of L1 expressing cells) showed a strong and clear nuclear signal of ORF2p in the absence of nuclear ORF1p (Figure 1D, top panel and Figure 1—figure supplement 2) consistent with a possible nuclear form of L1 RNPs that lacks ORF1p (see also accompanying paper by Taylor et al., 2018). On the contrary, in the cytoplasm, ORF2p always co-localized with ORF1p (Figure 1A and D). We compared several antibodies against ORF1p that confirmed a consistent pattern of nuclear/cytoplasmic localization of ORF1p (Figure 1—figure supplement 1). The corresponding secondary-only controls and IF staining of cells not expressing L1 are presented in Figure 1—figure supplement 4. The mouse 4H1 monoclonal antibody (mAb) recognizing the N-terminus of ORF1p and the rabbit JH74 antibody against the C-terminus of ORF1p have an identical pattern. The polyclonal 4632 antibody (pAb) displayed a high nuclear non-specific signal that renders this antibody less sensitive than 4H1 and JH74 (Figure 1—figure supplements 1,4). The JH73g rabbit Ab was distinct from the other antibodies and recognized mainly nuclear ORF1p (Figure 1—figure supplement 1A–B and Figure 4—figure supplement 1). This nuclear form of ORF1p is also recognized by 4H1 and JH74 antibodies but is much more easily observed using JH73g because of its higher affinity for nuclear ORF1p and much lower affinity for cytoplasmic ORF1p. Indeed, quantification of nuclear ORF1p upon staining with JH74 and JH73g revealed a comparable percentage of cells displaying nuclear ORF1p using the two antibodies (≅14–17%). The percentage of cells detected overall as expressing ORF1p using JH73g Ab is much lower than the percentage recognized as ORF1p expressing cells by JH74 Ab (37% versus 93% respectively) because JH73g is able to recognize mainly the nuclear form (Figure 1—figure supplement 1B). This unusual staining pattern suggests that the nuclear form of ORF1p may be highly enriched for a conformation specifically recognized by JH73g. Staining of L1rp expressing cells, on the other hand, reveals a lower threshold of sensitivity for the JH73g antibody that is unable to detect the lower amount of non-recoded ORF1p. Interestingly, ORF1p immunoprecipitated with JH73g Ab is impaired in binding to ORF2p protein, consistent with the possibility that most of the nuclear ORF1p species fail to bind ORF2p (Figure 1—figure supplement 1D). ORF1p enters the nucleus during mitosis Our immunofluorescence staining of ORF1p and ORF2p suggests that L1 RNPs enter the nucleus during mitosis, when the nuclear membrane breaks down. To better explore this hypothesis, previously put forward by other studies (Xie et al., 2013), we exploited well-characterized markers of the cell cycle: geminin, expressed only in S/G2/M phases, and Cdt1, which specifically marks the G1 phase (Arias and Walter, 2007). Co-staining of ORF1p with geminin and Cdt1 clearly showed that ORF1p is nuclear in cells expressing Cdt1 (G1 phase), and completely cytoplasmic in cells expressing geminin (Figure 2A and quantification in B). Confocal images of Cdt1, geminin and ORF1p staining confirmed our conclusions (Figure 2—figure supplement 1). We also verified these results using the FUCCI system (Fluorescent Ubiquitin Cell Cycle Indicator), that exploits Cdt1 and geminin fragments fused to mAG and mKO2 (Monomeric Azami-Green and 2 et al., of an in cells treated with the tag et al., shows that ORF1p is nuclear only in cells in G1 phase with from expression of Cdt1 (Figure In this of cells with nuclear and green nucleus in were observed number of cells = cells with nuclear ORF1p and green = cells with nuclear ORF1p and = was expressed in cells the cell cycle that displayed or green (Figure 2—figure supplement 2). This suggests that ORF2p expression is not cell cycle our that ORF1p, most likely together with ORF2p, enters into the nucleus during mitosis and is retained when the nuclear membrane cell Figure 2 with 2 supplements see all Download asset Open asset ORF1p the nucleus immediately (A) Representative pictures of of ORF1p with Cdt1 of G1 or geminin of S/G2/M DNA was stained with DAPI. Red arrowheads = cells with nuclear ORF1p, green arrowheads = cells without nuclear ORF1p. (B) Quantification of cells expressing nuclear ORF1p and Cdt1 top and or top or HeLa M2 cells expressing ORFeus or L1rp are shown in the two top or The antibodies used for staining and quantification are indicated at the of the (error = (C) Representative pictures of cell lines expressing and an recoded L1 with ORF1p tagged with an et al., et al., was used to The proteins are reported to of images in colors used in the merged panel). To into the nuclear/cytoplasmic of L1 we also performed by immunofluorescence staining of ORF1p and ORF2p in cells expressing recoded (ORFeus) or non-recoded L1 (L1rp) (Figure As the detected L1 mRNA co-localized with ORF1p staining in both ORFeus and L1rp (Figure and images in Figure supplement Interestingly, and in with our results on ORF1p localization (Figure and Figure nuclear signal of L1 mRNA was particularly in cells mitosis by cytoplasmic bridges still the two daughter cells (Figure supplement 1B, Figure supplement 2 and Videos and To that high nuclear L1 mRNA was detected in cells in G1 we performed of ORFeus by Cdt1 and (Figure and Figure supplement 2). As shown for ORF1p and in with our hypothesis, nuclear L1 mRNA co-localized with cells with and higher Cdt1 These results confirmed our ORF1p localization and that L1 RNPs formed by ORF1p, L1 mRNA most ORF2p enter the nucleus during mitosis and in the nucleus upon nuclear membrane in G1 phase. Figure with 2 supplements see all Download asset Open asset Analysis of nuclear/cytoplasmic (A) HeLa-M2 cells expressing ORFeus L1rp or not expressing L1 2 and were stained for using against ORFeus or IF of ORF1p using rabbit JH74 Ab and ORF2p using mouse Ab was performed Z-stack images were collected using a The images of each fluorescence are shown in together with the image in (B) L1 mRNA was stained using together with IF staining of Cdt1 and geminin, of G1 and S/G2/M HeLa-M2 cells expressing recoded L1 were A merged image of Cdt1 and geminin IF is shown in the top image while L1 is presented in in the Red arrowheads indicate cells that show strong nuclear L1 mRNA signal and are in G1 phase high arrowheads indicate cells that show only cytoplasmic L1 mRNA signal and that are in S/G2/M phase pictures of some cells by and green arrows are presented in Figure supplement (C) Quantification of the of nuclear L1 mRNA G1 or S/G2/M cells. (error bar = = is reported. Video Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg ORFeus mRNA Z stack. Z-stacks of ORF2p ORF1p and L1 ORFeus mRNA in HeLa-M2 cells expressing ORFeus and treated for 24 hr with doxycycline 0.1 μg/ml. The images of these cells is presented in Figure Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg L1rp Z stack. Z-stacks of ORF2p ORF1p and L1rp mRNA in HeLa-M2 cells expressing L1rp and treated for 24 hr with doxycycline 0.1 μg/ml. The image of these cells is presented in Figure 4. A of these conclusions would be that expression of L1 in dividing cells lead to a population with all cells displaying nuclear ORF1p because a of L1 induction will allow all the cells to mitosis while expressing ORF1p. To this hypothesis, we the percentage of cells displaying nuclear ORF1p 24 and hr of L1 that HeLa cell is about 24 hr. and nuclear/cytoplasmic analysis cell analysis was as for proximity We set very fluorescence in Figure from cells not treated with doxycycline and therefore not expressing as described in the methods These were necessary to of the strong ORF1p cytoplasmic signal with the of nuclear ORF1p The analysis of ORF1p nuclear and cytoplasmic showed that the percentage of cells with nuclear ORF1p does not but hr of L1 induction compared to the 24 hr (Figure The in nuclear ORF1p hr induction is due to the of a more cell The absence of an of cells with nuclear ORF1p with of L1 suggests that, the nucleus in nuclear ORF1p is and/or exported from the nucleus during or G1 phase. Figure 4 with 2 supplements see all Download asset Open asset ORF1p nuclear localization upon (A) HeLa M2 cells expressing a recoded L1 (ORFeus) with Flag tagged ORF2p were treated for 24 or hr with or without 0.1 μg/ml doxycycline on cells were in and stained with JH74 antibody and DAPI. were with Arrayscan and using The number of cells with cytoplasmic ORF1p as the number of ORF1p expressing cells) and nuclear ORF1p are reported as as amount of cells from quantification of cells expressing nuclear ORF1p in HeLa M2 cells treated with or without 10 or 20 for hr. treatment cells were and ORF1p stained using the indicated are reported for (error = S.E.M.) of ORF1p nuclear/cytoplasmic during the cell cycle. ORF1p nuclear localization is upon treatment To better explore potential of ORF1p and ORF2p we of a known of 1 We treated HeLa cells expressing LINE-1 with for hr. different concentrations of were used and several antibodies were to detect ORF1p in immunofluorescence (Figure both and using of the recognizing ORF1p we observed an number of cells with nuclear ORF1p that at least a subset of ORF1p is exported from the nucleus in a (Figure As a known regulated protein et al., 2014) tagged with was used to show nuclear upon treatment (Figure 4—figure supplement 2). LINE-1 retrotransposition