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. 2022 Oct 14;50(18):10680-10694.
doi: 10.1093/nar/gkac802.

Condensin I and condensin II proteins form a LINE-1 dependent super condensin complex and cooperate to repress LINE-1

Jacqueline R Ward  1 , Afshin Khan  1 , Sabrina Torres  1 , Bert Crawford  1 , Sarah Nock  2 , Trenton Frisbie  3 , John V Moran  3   4 , Michelle S Longworth  1   5
Affiliations

Condensin I and condensin II proteins form a LINE-1 dependent super condensin complex and cooperate to repress LINE-1

Jacqueline R Ward et al. Nucleic Acids Res. .

Abstract

Condensin I and condensin II are multi-subunit complexes that are known for their individual roles in genome organization and preventing genomic instability. However, interactions between condensin I and condensin II subunits and cooperative roles for condensin I and condensin II, outside of their genome organizing functions, have not been reported. We previously discovered that condensin II cooperates with Gamma Interferon Activated Inhibitor of Translation (GAIT) proteins to associate with Long INterspersed Element-1 (LINE-1 or L1) RNA and repress L1 protein expression and the retrotransposition of engineered L1 retrotransposition in cultured human cells. Here, we report that the L1 3'UTR is required for condensin II and GAIT association with L1 RNA, and deletion of the L1 RNA 3'UTR results in increased L1 protein expression and retrotransposition. Interestingly, like condensin II, we report that condensin I also binds GAIT proteins, associates with the L1 RNA 3'UTR, and represses L1 retrotransposition. We provide evidence that the condensin I protein, NCAPD2, is required for condensin II and GAIT protein association with L1 RNA. Furthermore, condensin I and condensin II subunits interact to form a L1-dependent super condensin complex (SCC) which is located primarily within the cytoplasm of both transformed and primary epithelial cells. These data suggest that increases in L1 expression in epithelial cells promote cytoplasmic condensin protein associations that facilitate a feedback loop in which condensins may cooperate to mediate L1 repression.

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Figures

Figure 1.
Figure 1.
Condensin II and GAIT proteins associate with the 3′UTR of L1 RNA. (A, B) HT-29 cells were transfected with either a full-length L1 (pJM101/L1.3) or a L1 harboring a deletion within its 3′UTR (pJM101/L1.3 3′UTRΔ) and RIP/qRT-PCR assays were conducted using either a (A) NCAPD3 antibody, or (B) EPRS antibody to immunoprecipitate L1 RNA. Reactions lacking an antibody (No Antibody IP) and reactions involving a non-specific, IgG antibody served as negative controls. Sequences within the neomycin resistance cassette present in pJM101/L1.3 and pJM101/L1.3 3′UTRΔ were used to design qRT-PCR primers to detect L1 RNA. The average levels of L1 RNA-associated protein were calculated as percentages of the input used for each immunoprecipitation; averages from three independent experiments are shown. (C) L1 transcripts in HT-29 cells transfected with either the pCEP4 empty vector, pJM101/L1.3 or pJM101/L1.3 3′UTRΔ were analyzed by qRT-PCR. Transcripts were normalized to actin transcript levels and the averages of four independent experiments are shown. (D) Representative immunoblot analysis of L1 ORF1p levels in HT-29 cells transfected with either the pCEP4 empty vector, pJM101/L1.3 or pJM101/L1.3 3′UTRΔ. (E) Band intensities from four biological replicates of the experiment shown in panel C were quantified from film and normalized to actin. (F) Retrotransposition assays performed using HT-29 cells transfected with either pJM101/L1.3 or pJM101/L1.3 3′UTRΔ. Quantification (right) of three independent experiments. P values were determined by performing unpaired t-tests. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Error bars indicate standard deviations from the mean.
Figure 2.
Figure 2.
Condensin I represses L1 retrotransposition and expression. (A) Schematic of the condensin I complex; the names of condensin I subunits are indicated by the different colored shapes. (B–D) HT-29 cells were induced to express either Non-target, control shRNA or NCAPD2 shRNA. (B) Cellular RNAs were analyzed by qRT-PCR to detect NCAPD2 transcript levels. Transcripts were normalized to actin transcript levels; the averages of three independent experiments are shown. Whole cell lysates were subjected to immunoblot analysis to detect (C) NCAPD2 protein or (D) endogenous ORF1p. Quantification of three independent experiments are shown to the right of each blot in panels C and D. Band intensities were quantified from film and normalized to actin. (E) HT-29 cells were induced to express either Non-Target shRNA or NCAPD2 shRNA and then were transfected with equal amounts of a pJM101/L1.3 expression vector to assess retrotransposition efficiency. Quantification of three independent experiments is shown. (F–H) HT-29 cells were induced to express either Non-target, control shRNA or NCAPG shRNA. (F) Cellular RNAs were analyzed by qRT-PCR to detect NCAPG transcript levels. Transcripts were normalized to actin transcript levels, and the averages of three independent experiments are shown. Whole cell lysates were subjected to immunoblot analysis to detect (G) NCAPG protein or (H) endogenous ORF1p. Quantification of three independent experiments is shown to the right of each blot in panels G and H. Band intensities were quantified from film and normalized to actin. (I) HT-29 cells were induced to express either Non-Target shRNA or NCAPG shRNA and then were transfected with equal amounts of pJM101/L1.3 to assess retrotransposition efficiency. Quantification of three independent experiments is shown. P values were determined by performing unpaired t-tests. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Error bars indicate standard deviations from the mean.
Figure 3.
Figure 3.
NCAPD2/ condensin I is required for EPRS and NCAPD3 association with L1 RNA. (A) HT-29 cells were transfected with either pJM101/L1.3 expression vector or a L1 construct harboring a deletion within its 3′UTR (pJM101/L1.3 3′UTRΔ) and RIP/qRT-PCR assays were conducted using a NCAPD2 antibody to immunoprecipitate L1 RNA. Reactions lacking an antibody (No Antibody IP) and reactions involving a non-specific, IgG antibody served as negative controls. Sequences within the neomycin resistance cassette present in pJM101/L1.3 and pJM101/L1.3 3′UTRΔ were used to design qRT-PCR primers to detect L1 RNA. The average levels of RNA-bound protein were calculated as percentages of the input used for each immunoprecipitation; averages from three independent experiments are shown. (B) EPRS co-IP/immunoblot experiments were conducted in HT-29 cells to detect association with NCAPD2. IPs using antibody only (ie. no lysate) and IPs using an IgG antibody served as negative controls. A representative blot of three independent experiments is shown. (C–E) HT-29 cells were induced to express either NT shRNA or NCAPD2 shRNA and then were transfected with pJM101/L1.3. RIP/qRT-PCR assays were conducted using either (C) NCAPD3 antibody, (D) EPRS antibody, or (E) eIF4G antibody to immunoprecipitate L1 RNA, as described for (A). P values were determined by performing unpaired t-tests. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Error bars indicate standard deviations from the mean.
Figure 4.
Figure 4.
NCAPD2 and NCAPD3 associate to form SCCs in a L1 RNA dependent manner. (A) Reciprocal NCAPD3 and NCAPD2 co-IP/immunoblots were conducted in HT-29 cells to detect an association between NCAPD3 and NCAPD2. Antibody only (i.e. no lysate) and IgG antibody IPs served as negative controls. Each experiment was performed three times and representative blots are shown. (B) NCAPG2 co-IP/immunoblot experiments were conducted in HT-29 cells to detect an association with NCAPD2 (middle panel) and with NCAPG (bottom panel). Antibody only (i.e. no lysate) and IgG antibody IPs served as negative controls. Each experiment was performed three times and representative blots are shown. (C) Proximity Ligation Assays (PLAs) were performed to detect an association between NCAPD2 and NCAPD3 in HT-29 cells transfected with control (siCTRL) or L1 siRNA (siL1). Single antibody controls were performed in parallel for each experiment. Images were taken using a confocal microscope with a 63x objective; maximum projections are shown. (D) Volocity imaging software was used to analyze confocal images and quantify the average number of nuclear and cytoplasmic PLA foci per cell. Each dot represents the average of 50–150 nuclei evaluated from a single image. Images were taken from three independent experiments. (E) PLAs were performed to detect associations between NCAPD3 and NCAPG2 in HT-29 cells transfected with siCTRL or siL1 and results were quantified as described in (D). P values were determined by performing unpaired t-tests. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Error bars indicate standard deviations from the mean.
Figure 5.
Figure 5.
NRTI Treatment reduces L1 transcripts and L1 retrotransposition events and decreases cytoplasmic SCC formation. (A) HT-29 cells were treated with either DMSO or DMSO containing Zidovudine and Didanosine at the indicated drug concentrations for 10 days then fixed and stained with crystal violet (CV) to measure cell toxicity. Shown are averages from three independent experiments. (B) Retrotransposition assays were performed using HT-29 cells that were transfected with pJM101/L1.3 and treated with either DMSO or a combination of the nucleoside reverse transcriptase inhibitors (NRTIs) Zidovudine and Didanosine; each drug was at a final concentration of 50 μM in tissue culture media. Quantification of retrotransposition assays from three independent experiments is shown. (C) qRT-PCR analysis of endogenous L1 RNA levels (using a 5′UTR primer pair; See Supplementary Figure S1 and Methods) in HT-29 cells treated with either DMSO or NRTIs. The average relative transcript levels for three independent experiments are shown. (D) PLA was performed to detect an association between NCAPD2 and NCAPD3 in HT-29 cells treated with DMSO or NRTIs at a final concentration of 50μM in tissue culture media. Single antibody controls were performed in parallel for each experiment. Images were taken using a confocal microscope with a 63x objective and maximum projections are shown. Volocity imaging software was used to analyze confocal images as noted in Figure 4 to quantify the average number of nuclear (left chart) and cytoplasmic (right chart) PLA foci per cell. P values were determined by performing unpaired t-tests. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ns = not significant. Error bars indicate standard deviations from the mean.
Figure 6.
Figure 6.
SCC formation occurs in primary cells expressing L1 transcripts. (A) qRT-PCR analysis of L1 transcripts in RPE-1 cells transfected with the pCEP4 empty vector or pJM101/L1.3 expression vector. Sequences within the neomycin resistance cassette present in pJM101/L1.3 were used to design qRT-PCR primers to detect L1 RNA. Transcript levels were normalized to actin and L1 transcript levels in pCEP4 transfected cells were set to 1. Shown is the average of three independent experiments. (B, C) IP/immunoblotting experiments for NCAPD2 and NCAPD3 were performed in RPE-1 cells transfected with (B) pCEP4 or (C) pJM101/L1.3. Antibody only (no lysate) and IgG antibody IPs served as controls. Asterisks indicate bands remaining after stripping the membrane that was immunoblotted for NCAPD3. Shown are representative blots of three independent experiments. (D) Average amounts of co-precipitated NCAPD2 protein, normalized to the amount of immunoprecipitated NCAPD3 protein, were quantified from the experiments in panels B and C. P values were determined by performing unpaired t-tests. *P ≤ 0.05, **P ≤ 0.01. Error bars indicate standard deviations from the mean.
Figure 7.
Figure 7.
The 3′UTR of L1 RNA antagonizes SCC formation. (A) HT-29 cells were transfected with the pCEP4 empty vector, pJM101/L1.3 expression vector, or L1 expression vector harboring a 3′UTR deletion (pJM101/L1.3 3′UTRΔ) and Proximity Ligation Assays were performed to detect association between NCAPD2 and NCAPD3. Single antibody controls were performed in parallel for each experiment. Images shown were taken on a confocal microscope with a 63x objective and maximum projections are shown. (B, C) Volocity imaging software was used to analyze confocal images as noted in Figure 4D to quantify the average number of nuclear (B) and cytoplasmic (C) PLA foci per cell. Images shown were taken from two independent experiments. P values were determined by performing unpaired t-tests. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ns = not significant. Error bars indicate standard deviations from the mean.
Figure 8.
Figure 8.
Working model for how condensin I and condensin II may affect L1 expression and retrotransposition. Condensin I and condensin II can primarily form cytoplasmic SCC complexes in a L1-dependent manner in both transformed and non-transformed human epithelial cells. We propose that condensins, either independently or following formation of the SCC, associate with GAIT complex proteins and the L1 RNA 3′ UTR to block translation of L1 mRNA (31) which subsequently leads to repression of L1 retrotransposition and prevents further increases in L1 RNA.
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References

    1. Hirano T. Condensins: organizing and segregating the genome. Curr. Biol. 2005; 15:R265–R275. - PubMed
    1. Ono T., Losada A., Hirano M., Myers M.P., Neuwald A.F., Hirano T.. Differential contributions of condensin i and condensin II to mitotic chromosome architecture in vertebrate cells. Cell. 2003; 115:109–121. - PubMed
    1. Ono T., Fang Y., Spector D.L., Hirano T.. Spatial and temporal regulation of condensins i and II in mitotic chromosome assembly in human cells. Mol. Biol. Cell. 2004; 15:3296–3308. - PMC - PubMed
    1. Hartl T.A., Sweeney S.J., Knepler P.J., Bosco G.. Condensin II resolves chromosomal associations to enable anaphase i segregation in drosophila male meiosis. PLoS Genet. 2008; 4:e1000228. - PMC - PubMed
    1. Longworth M.S., Herr A., Ji J.Y., Dyson N.J.. RBF1 promotes chromatin condensation through a conserved interaction with the condensin II protein dCAP-D3. Genes Dev. 2008; 22:1011–1024. - PMC - PubMed

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