The epigenetic regulator PLZF represses L1 retrotransposition in germ and progenitor cells.
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ABSTRACT:
SUBMITTER: Puszyk W
PROVIDER: S-EPMC3810588 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Long interspersed element 1 (LINE-1 or L1) retrotransposons have markedly affected the human genome. L1s must retrotranspose in the germ line or during early development to ensure their evolutionary success, yet the extent to which this process affects somatic cells is poorly understood. We previously demonstrated that engineered human L1s can retrotranspose in adult rat hippocampus progenitor cells in vitro and in the mouse brain in vivo. Here we demonstrate that neural progenitor cells isolated from human fetal brain and derived from human embryonic stem cells support the retrotransposition of engineered human L1s in vitro. Furthermore, we developed a quantitative multiplex polymerase chain reaction that detected an increase in the copy number of endogenous L1s in the hippocampus, and in several regions of adult human brains, when compared to the copy number of endogenous L1s in heart or liver genomic DNAs from the same donor. These data suggest that de novo L1 retrotransposition events may occur in the human brain and, in principle, have the potential to contribute to individual somatic mosaicism.
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Project description:Long interspersed element-1 (LINE-1 or L1) retrotransposition continues to affect human genome evolution. L1s can retrotranspose in the germline, during early development and in select somatic cells; however, the host response to L1 retrotransposition remains largely unexplored. Here we show that reporter genes introduced into the genome of various human embryonic carcinoma-derived cell lines (ECs) by L1 retrotransposition are rapidly and efficiently silenced either during or immediately after their integration. Treating ECs with histone deacetylase inhibitors rapidly reverses this silencing, and chromatin immunoprecipitation experiments revealed that reactivation of the reporter gene was correlated with changes in chromatin status at the L1 integration site. Under our assay conditions, rapid silencing was also observed when reporter genes were delivered into ECs by mouse L1s and a zebrafish LINE-2 element, but not when similar reporter genes were delivered into ECs by Moloney murine leukaemia virus or human immunodeficiency virus, suggesting that these integration events are silenced by distinct mechanisms. Finally, we demonstrate that subjecting ECs to culture conditions that promote differentiation attenuates the silencing of reporter genes delivered by L1 retrotransposition, but that differentiation, in itself, is not sufficient to reactivate previously silenced reporter genes. Thus, our data indicate that ECs differ from many differentiated cells in their ability to silence reporter genes delivered by L1 retrotransposition.
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Project description:Repetitive elements, including LINE-1 (L1), comprise approximately half of the human genome. These elements can potentially destabilize the genome by initiating their own replication and reintegration into new sites (retrotransposition). In somatic cells, transcription of L1 elements is repressed by distinct molecular mechanisms, including DNA methylation and histone modifications, to repress transcription. Under conditions of hypomethylation (e.g. in tumor cells), a window of opportunity for L1 derepression arises, and additional restriction mechanisms become crucial. We recently demonstrated that the microRNA miR-128 represses L1 activity by directly binding to L1 ORF2 RNA. In this study, we tested whether miR-128 can also control L1 activity by repressing cellular proteins important for L1 retrotransposition. We found that miR-128 targets the 3' UTR of nuclear import factor transportin 1 (TNPO1) mRNA. Manipulation of miR-128 and TNPO1 levels demonstrated that induction or depletion of TNPO1 affects L1 retrotransposition and nuclear import of an L1-ribonucleoprotein complex (using L1-encoded ORF1p as a proxy for L1-ribonucleoprotein complexes). Moreover, TNPO1 overexpression partially reversed the repressive effect of miR-128 on L1 retrotransposition. Our study represents the first description of a protein factor involved in nuclear import of the L1 element and demonstrates that miR-128 controls L1 activity in somatic cells through two independent mechanisms: direct binding to L1 RNA and regulation of a cellular factor necessary for L1 nuclear import and retrotransposition.
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Project description:Whether long interspersed element-1 (L1 or LINE-1) retrotransposition can occur in quiescent, nondividing, and/or terminally differentiated somatic cells has remained an unanswered fundamental question in human genetics. Here, we used a ubiquitously active phosphoglycerate kinase-1 promoter to drive the expression of a highly active human L1 element from an adenovirus-L1 hybrid vector. This vector system achieved retrotransposition in up to 91% of actively growing immortalized cells, and we demonstrated that L1 retrotransposition can be suppressed by the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine. This adenovirus vector enabled efficient delivery of the L1 element into differentiated primary human somatic cells and G1/S-arrested cells, resulting in retrotransposition in both cases; however, it was not detected in G0-arrested cells. Thus, these data indicate that L1 retrotransposition can occur in nondividing somatic cells.
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Project description:LINE-1 (L1) retrotransposons comprise approximately 17% of human DNA, yet little is known about L1 integration. Here, we characterized 100 retrotransposition events in HeLa cells and show that distinct DNA repair pathways can resolve L1 cDNA retrotransposition intermediates. L1 cDNA resolution can lead to various forms of genetic instability including the generation of chimeric L1s, intrachromosomal deletions, intrachromosomal duplications, and intra-L1 rearrangements as well as a possible interchromosomal translocation. The L1 retrotransposition machinery also can mobilize U6 snRNA to new genomic locations, increasing the repertoire of noncoding RNAs that are mobilized by L1s. Finally, we have determined that the L1 reverse transcriptase can faithfully replicate its own transcript and has a base misincorporation error rate of approximately 1/7,000 bases. These data indicate that L1 retrotransposition in transformed human cells can lead to a variety of genomic rearrangements and suggest that host processes act to restrict L1 integration in cultured human cells. Indeed, the initial steps in L1 retrotransposition may define a host/parasite battleground that serves to limit the number of active L1s in the genome.
| S-EPMC1190285 | biostudies-literature
Project description:Retrotransposons are currently active in the human and mouse genomes contributing to novel disease mutations and genomic variation via de novo insertions. However, little is known about the interactions of non-long terminal repeat (non-LTR) retrotransposons with the host DNA repair machinery. Based on the model of retrotransposition for the human and mouse LINE-1 element, one likely intermediate is an extension of cDNA that is heterologous to the genomic target, a flap intermediate. To determine whether a human flap endonuclease could recognize and process this potential intermediate, the genetic requirement for the ERCC1/XPF heterodimer during LINE-1 retrotransposition was characterized. Reduction of XPF in human cells increased retrotransposition whereas complementation of ERCC1-deficiency in hamster cells reduced retrotransposition. These results demonstrate for the first time that DNA repair enzymes act to limit non-LTR retrotransposition and may provide insight into the genetic instability phenotypes of ercc1 and xpf individuals.
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Project description:PLZF (promyelocytic leukemia zinc finger) is a transcription factor acting as a global regulator of hematopoietic commitment. PLZF displays an epigenetic specificity by recruiting chromatin-modifying factors but little is known about its role in remodeling chromatin of cells committed toward a given specific hematopoietic lineage. In murine myeloid progenitors, we decipher a new role for PLZF in restraining active genes and enhancers by targeting acetylated lysine 27 of Histone H3 (H3K27ac). Functional analyses reveal that active enhancers bound by PLZF are involved in biological processes related to metabolism and associated with hematopoietic aging. Comparing the epigenome of young and old myeloid progenitors, we reveal that H3K27ac variation at active enhancers is a hallmark of hematopoietic aging. Taken together, these data suggest that PLZF, associated with active enhancers, appears to restrain their activity as an epigenetic gatekeeper of hematopoietic aging.
| S-EPMC6511862 | biostudies-literature
Project description:Long Interspersed Element 1 (LINE-1 or L1) is capable of causing genomic instability through the activity of the L1 ORF2 protein (ORF2p). This protein contains endonuclease (EN) and reverse transcriptase (RT) domains that are necessary for the retrotransposition of L1 and the Short Interspersed Element (SINE) Alu. The functional importance of approximately 50% of the ORF2p molecule remains unknown, but some of these sequences could play a role in retrotransposition, or be necessary for the enzymatic activities of the EN and/or RT domains. Conventional approaches using the full-length, contiguous ORF2p make it difficult to study the involvement of these unannotated sequences in the function of L1 ORF2p. Our lab has developed a Bipartile Alu Retrotransposition (BAR) assay that relies on separate truncated ORF2p fragments: an EN-containing and an RT-containing fragment. We validated the utility of this method for studying the ORF2p function in retrotransposition by assessing the effect of expression levels and previously characterized mutations on BAR. Using BAR, we identified two pairs of amino acids important for retrotransposition, an FF and a WD. The WD appears to play a role in cDNA synthesis by the ORF2p molecule, despite being outside the canonical RT domain.
| S-EPMC4889948 | biostudies-literature
Project description:L1 retrotransposons comprise 17% of the human genome and are its only autonomous mobile elements. Although L1-induced insertional mutagenesis causes Mendelian disease, their mutagenic load in cancer has been elusive. Using L1-targeted resequencing of 16 colorectal tumor and matched normal DNAs, we found that certain cancers were excessively mutagenized by human-specific L1s, while no verifiable insertions were present in normal tissues. We confirmed de novo L1 insertions in malignancy by both validating and sequencing 69/107 tumor-specific insertions and retrieving both 5' and 3' junctions for 35. In contrast to germline polymorphic L1s, all insertions were severely 5' truncated. Validated insertion numbers varied from up to 17 in some tumors to none in three others, and correlated with the age of the patients. Numerous genes with a role in tumorigenesis were targeted, including ODZ3, ROBO2, PTPRM, PCM1, and CDH11. Thus, somatic retrotransposition may play an etiologic role in colorectal cancer.
| S-EPMC3514663 | biostudies-literature
Project description:Somatic LINE-1 (L1) retrotransposition has been detected in early embryos, adult brains, and the gastrointestinal (GI) tract, and many cancers, including epithelial GI tumors. We previously found numerous somatic L1 insertions in paired normal and GI cancerous tissues. Here, using a modified method of single-cell analysis for somatic L1 insertions, we studied adenocarcinomas of colon, pancreas, and stomach, and found a variable number of somatic L1 insertions in tumors of the same type from patient to patient. We detected no somatic L1 insertions in single cells of 5 of 10 tumors studied. In three tumors, aneuploid cells were detected by FACS. In one pancreatic tumor, there were many more L1 insertions in aneuploid than in euploid tumor cells. In one gastric cancer, both aneuploid and euploid cells contained large numbers of likely clonal insertions. However, in a second gastric cancer with aneuploid cells, no somatic L1 insertions were found. We suggest that when the cellular environment is favorable to retrotransposition, aneuploidy predisposes tumor cells to L1 insertions, and retrotransposition may occur at the transition from euploidy to aneuploidy. Seventeen percent of insertions were also present in normal cells, similar to findings in genomic DNA from normal tissues of GI tumor patients. We provide evidence that: 1) The number of L1 insertions in tumors of the same type is highly variable, 2) most somatic L1 insertions in GI cancer tissues are absent from normal tissues, and 3) under certain conditions, somatic L1 retrotransposition exhibits a propensity for occurring in aneuploid cells.
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