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Deciphering fact from artifact when using reporter assays to investigate the roles of host factors on L1 retrotransposition

View Article: PubMed Central - PubMed

ABSTRACT

Background: The Long INterspersed Element-1 (L1, LINE-1) is the only autonomous mobile DNA element in humans and has generated as much as half of the genome. Due to increasing clinical interest in the roles of L1 in cancer, embryogenesis and neuronal development, it has become a priority to understand L1-host interactions and identify host factors required for its activity. Apropos to this, we recently reported that L1 retrotransposition in HeLa cells requires phosphorylation of the L1 protein ORF1p at motifs targeted by host cell proline-directed protein kinases (PDPKs), which include the family of mitogen-activated protein kinases (MAPKs). Using two engineered L1 reporter assays, we continued our investigation into the roles of MAPKs in L1 activity.

Results: We found that the MAPK p38δ phosphorylated ORF1p on three of its four PDPK motifs required for L1 activity. In addition, we found that a constitutively active p38δ mutant appeared to promote L1 retrotransposition in HeLa cells. However, despite the consistency of these findings with our earlier work, we identified some technical concerns regarding the experimental methodology. Specifically, we found that exogenous expression of p38δ appeared to affect at least one heterologous promoter in an engineered L1 reporter, as well as generate opposing effects on two different reporters. We also show that two commercially available non-targeting control (NTC) siRNAs elicit drastically different effects on the apparent retrotransposition reported by both L1 assays, which raises concerns about the use of NTCs as normalizing controls.

Conclusions: Engineered L1 reporter assays have been invaluable for determining the functions and critical residues of L1 open reading frames, as well as elucidating many aspects of L1 replication. However, our results suggest that caution is required when interpreting data obtained from L1 reporters used in conjunction with exogenous gene expression or siRNA.

No MeSH data available.


p38δ increases Fluc independent of a heterologous promoter. a Duplicate wells containing G418-resistant colonies resulting from transfection of HeLa cells with the L1 reporter JM101 in the presence of pcDNA mammalian expression vectors for: empty vector (EV), p38δ-WT (WT) or p38δ-F3324S (FS). b Mean Fluc (left) and Rluc (right) luminescence values obtained from lysates of HeLa cells transfected with the L1 reporter plasmid pYX014 in the presence of indicated pcDNA mammalian expression vectors. Averages were derived from raw data shown in (c) by first averaging technical replicates for each biological sample (n = 3), and averaging biological replicates; error bars represent SEM of biological samples, n = 2. c Individual luminescence values are shown for Fluc (blue) and Rluc (red) used to calculate averages in (b); technical replicates are side-by-side; biological replicates are indicated with subscripts
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Fig4: p38δ increases Fluc independent of a heterologous promoter. a Duplicate wells containing G418-resistant colonies resulting from transfection of HeLa cells with the L1 reporter JM101 in the presence of pcDNA mammalian expression vectors for: empty vector (EV), p38δ-WT (WT) or p38δ-F3324S (FS). b Mean Fluc (left) and Rluc (right) luminescence values obtained from lysates of HeLa cells transfected with the L1 reporter plasmid pYX014 in the presence of indicated pcDNA mammalian expression vectors. Averages were derived from raw data shown in (c) by first averaging technical replicates for each biological sample (n = 3), and averaging biological replicates; error bars represent SEM of biological samples, n = 2. c Individual luminescence values are shown for Fluc (blue) and Rluc (red) used to calculate averages in (b); technical replicates are side-by-side; biological replicates are indicated with subscripts

Mentions: The first and most obvious difference between the two reporters is that L1 is driven by a CMV promoter in JM101 but a CAG promoter in pYX017, though the CAG promoter contains a CMV enhancer element (Fig. 2). CMV promoters can be affected by some p38 isoforms [49–53], but we did not observe a significant effect of p38δ-WT or p38δ-F324S on EGFP, which is also driven by a CMV promoter. To address whether the increase in Fluc luminescence stemmed from effects of p38δ on the CAG promoter, we used the pYX014 construct, which is identical to pYX017 except that it relies on the native L1 promoter in the 5′ UTR for L1 expression instead of CAG (Fig. 2). Using JM101 in parallel with pYX014, we again found that p38δ-WT inhibited formation of G418-resistant colonies (Fig. 4a), while both p38δ-WT and p38δ-F324S increased Fluc luminescence from pYX014 by 1.5 and 2.2 fold, respectively (Figs. 4b left and c), compared to 1.3 and 1.5 fold from pYX017 (Fig. 3d left). Since p38δ-WT increased Fluc in both pYX014 and pYX017, the effect of p38δ-WT appears to be independent of the CAG promoter in pYX017. We eliminated p38δ-D176A from this and further experiments given its effect on cell growth (Fig. 3) as well as the report that, despite its inactivity in vitro, it can be activated in HEK293 cells [37], making its effects on L1 uninterpretable, particularly given the inhibitory effect of p38δ-WT on G418-resistant colony formation.Fig. 4


Deciphering fact from artifact when using reporter assays to investigate the roles of host factors on L1 retrotransposition
p38δ increases Fluc independent of a heterologous promoter. a Duplicate wells containing G418-resistant colonies resulting from transfection of HeLa cells with the L1 reporter JM101 in the presence of pcDNA mammalian expression vectors for: empty vector (EV), p38δ-WT (WT) or p38δ-F3324S (FS). b Mean Fluc (left) and Rluc (right) luminescence values obtained from lysates of HeLa cells transfected with the L1 reporter plasmid pYX014 in the presence of indicated pcDNA mammalian expression vectors. Averages were derived from raw data shown in (c) by first averaging technical replicates for each biological sample (n = 3), and averaging biological replicates; error bars represent SEM of biological samples, n = 2. c Individual luminescence values are shown for Fluc (blue) and Rluc (red) used to calculate averages in (b); technical replicates are side-by-side; biological replicates are indicated with subscripts
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Related In: Results  -  Collection

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Fig4: p38δ increases Fluc independent of a heterologous promoter. a Duplicate wells containing G418-resistant colonies resulting from transfection of HeLa cells with the L1 reporter JM101 in the presence of pcDNA mammalian expression vectors for: empty vector (EV), p38δ-WT (WT) or p38δ-F3324S (FS). b Mean Fluc (left) and Rluc (right) luminescence values obtained from lysates of HeLa cells transfected with the L1 reporter plasmid pYX014 in the presence of indicated pcDNA mammalian expression vectors. Averages were derived from raw data shown in (c) by first averaging technical replicates for each biological sample (n = 3), and averaging biological replicates; error bars represent SEM of biological samples, n = 2. c Individual luminescence values are shown for Fluc (blue) and Rluc (red) used to calculate averages in (b); technical replicates are side-by-side; biological replicates are indicated with subscripts
Mentions: The first and most obvious difference between the two reporters is that L1 is driven by a CMV promoter in JM101 but a CAG promoter in pYX017, though the CAG promoter contains a CMV enhancer element (Fig. 2). CMV promoters can be affected by some p38 isoforms [49–53], but we did not observe a significant effect of p38δ-WT or p38δ-F324S on EGFP, which is also driven by a CMV promoter. To address whether the increase in Fluc luminescence stemmed from effects of p38δ on the CAG promoter, we used the pYX014 construct, which is identical to pYX017 except that it relies on the native L1 promoter in the 5′ UTR for L1 expression instead of CAG (Fig. 2). Using JM101 in parallel with pYX014, we again found that p38δ-WT inhibited formation of G418-resistant colonies (Fig. 4a), while both p38δ-WT and p38δ-F324S increased Fluc luminescence from pYX014 by 1.5 and 2.2 fold, respectively (Figs. 4b left and c), compared to 1.3 and 1.5 fold from pYX017 (Fig. 3d left). Since p38δ-WT increased Fluc in both pYX014 and pYX017, the effect of p38δ-WT appears to be independent of the CAG promoter in pYX017. We eliminated p38δ-D176A from this and further experiments given its effect on cell growth (Fig. 3) as well as the report that, despite its inactivity in vitro, it can be activated in HEK293 cells [37], making its effects on L1 uninterpretable, particularly given the inhibitory effect of p38δ-WT on G418-resistant colony formation.Fig. 4

View Article: PubMed Central - PubMed

ABSTRACT

Background: The Long INterspersed Element-1 (L1, LINE-1) is the only autonomous mobile DNA element in humans and has generated as much as half of the genome. Due to increasing clinical interest in the roles of L1 in cancer, embryogenesis and neuronal development, it has become a priority to understand L1-host interactions and identify host factors required for its activity. Apropos to this, we recently reported that L1 retrotransposition in HeLa cells requires phosphorylation of the L1 protein ORF1p at motifs targeted by host cell proline-directed protein kinases (PDPKs), which include the family of mitogen-activated protein kinases (MAPKs). Using two engineered L1 reporter assays, we continued our investigation into the roles of MAPKs in L1 activity.

Results: We found that the MAPK p38δ phosphorylated ORF1p on three of its four PDPK motifs required for L1 activity. In addition, we found that a constitutively active p38δ mutant appeared to promote L1 retrotransposition in HeLa cells. However, despite the consistency of these findings with our earlier work, we identified some technical concerns regarding the experimental methodology. Specifically, we found that exogenous expression of p38δ appeared to affect at least one heterologous promoter in an engineered L1 reporter, as well as generate opposing effects on two different reporters. We also show that two commercially available non-targeting control (NTC) siRNAs elicit drastically different effects on the apparent retrotransposition reported by both L1 assays, which raises concerns about the use of NTCs as normalizing controls.

Conclusions: Engineered L1 reporter assays have been invaluable for determining the functions and critical residues of L1 open reading frames, as well as elucidating many aspects of L1 replication. However, our results suggest that caution is required when interpreting data obtained from L1 reporters used in conjunction with exogenous gene expression or siRNA.

No MeSH data available.