LINE-1 Based Insertional Mutagenesis Screens to Identify Genes Involved in Embryonic Stem Cell Differentiation

Background: Knowledge of the intrinsic properties of embryonic stem cells is essential before the possibility of using these cells for therapeutic purposes becomes a reality. Insertional mutagenesis screens are widely used to identify genes sufficient to confer a particular phenotype; however, applications of such approaches are limited to mouse models. Thus, there is a need to develop a new DNA transposon system that allows gene discovery approaches in stem cells. Methods: This study was performed to explore the possibility of using the long interspersed nuclear element 1 (LINE -1) retrotransposon as a gene trap vector to identify genes involved in the maintenance and differentiation of mouse embryonic stem cells. Results: We developed an episomal, nonviral LINE-1 retrotransposon system using the scaffold/matrix attachment regions in the backbone of our vector. This gene trap vector harbors a GFP marker whose expression occurs only after successful insertional mutagenesis. By utilizing this vector, coupled with GFP expression, we have successfully isolated four individual embryonic stem cell clones that display disrupted genes, including two known genes. We then confirmed the identity of these genes using an inverse PCR approach and verified their function in cell differentiation using shRNAs and undifferentiated markers of embryonic stem cells. Conclusions: The ease of using this insertional mutagen and the simplicity of identifying the cells with disrupted genes by GFP expression make this LINE-1 vector a promising tool for embryonic stem cell and cancer stem cell gene discovery

Silencing of LINE-1 retrotransposons contributes to variation in small noncoding RNA expression in human cancer cells

Noncoding RNAs are key players in the maintenance of genomic integrity, particularly in silencing the expression of repetitive elements, some of which are retrotransposable and capable of causing genomic instability. Recent computational studies suggest an association between L1 expression and the generation of small RNAs. However, whether L1 expression has a role in the activation of small RNA expression has yet to be determined experimentally. Here we report a global analysis of small RNAs in deep sequencing from L1-active and L1-silenced breast cancer cells. We found that cells in which L1 expression was silenced exhibited greatly increased expression of a number of miRNAs and in particular, members of the let-7 family. In addition, we found differential expression of a few piRNAs that might potentially regulate gene expression. We also report the identification of several repeat RNAs against LTRs, LINEs and SINE elements. Although most of the repeat RNAs mapped to L1 elements, in general we found no significant differences in the expression levels of repeat RNAs in the presence or absence of L1 expression except for a few RNAs targeting subclasses of L1 elements. These differentially expressed small RNAs may function in human genome defence responses

Passenger or Driver: Can Gene Expression Profiling Tell Us Anything about LINE-1 in Cancer?

LINE-1 retrotransposons are expressed in epithelial cancers but not normal adult tissues. Previously, we demonstrated repression of cell proliferation, migration, and invasion genes in L1-reverse transcriptase-inhibited T47D cells, while genes involved in cell projection, formation of vacuolar membranes, and intercellular junctions were upregulated. Extending this, we examined microarray data from L1-silenced and Efavirenz-treated T47D cells by Weighted Gene Correlation Network Analysis and literature mining. Hub genes in the most significant module comparing L1-silenced and untreated controls included HSP90AB2p, DDX39A, PANK2, MT1M, and LIMK2. HSP90AB2p is related to HSP90, a master regulator of cancer, cancer evolvability and chemo-resistance. DDX39A is a known cancer driver gene while PANK2 and MT1M affect multiple pathways. LIMK2 and SYBL1 impact actin cytoskeletal dynamics and the cofilin pathway, cancer cell motility, and the epithelial-to-mesenchymal transition. Also affected were signal transduction, HIF1 pathways, iron/redox metabolism, stress granules and cancer stem cell-related metabolic reprogramming and the eIF4F translation initiation complex. Hub genes in other modules, including BTRC, MDM2, and FBXW7, stabilize oncoproteins like MYC, p53, and NOTCH1 or reflect CXCL12–CXCR4 signalling. Our findings support mounting evidence that L1 activity is a cause, rather than a consequence of oncogenesis, with L1 affecting the formation of cancer stem cells

Control of chicken CR1 retrotransposons is independent of Dicer-mediated RNA interference pathway

BACKGROUND: Dicer is an RNase III-ribonuclease that initiates the formation of small interfering RNAs as a defence against genomic parasites such as retrotransposons. Despite intensive characterization in mammalian species, the biological functions of Dicer in controlling retrotransposable elements of the non-mammalian vertebrate are poorly understood. In this report, we examine the role of chicken Dicer in controlling the activity of chicken CR1 retrotransposable elements in a chicken-human hybrid DT40 cell line employing a conditional lossof- Dicer function. RESULTS: Retrotransposition is detrimental to host genome stability and thus eukaryotic cells have developed mechanisms to limit the expansion of retrotransposons by Dicer-mediated RNAi silencing pathways. However, the mechanisms that control the activity and copy numbers of transposable elements in chicken remain unclear. Here, we describe how the loss of Dicer in chicken cells does not reactivate endogenous chicken CR1 retrotransposons with impaired RNAi machinery, suggesting that the control of chicken CR1 is independent of Dicer-induced RNAi silencing. In contrast, upon introduction of a functionally active human L1 retrotransposable element that contains an active 5' UTR promoter, the Dicer-deficient chicken cells show a strong increase in the accumulation of human L1 transcripts and retrotransposition activity, highlighting a major difference between chicken CR1 and other mammalian L1 retrotransposons. CONCLUSION: Our data provide evidence that chicken CR1 retrotransposons, unlike their mammalian L1 counterparts, do not undergo retrotransposition because most CR1 retrotransposons are truncated or mutated at their 5'UTR promoters and thus are not subjected to Dicer-mediated RNAi-silencing control

Analysis of Epigenetic Factors in Mouse Embryonic Neural Stem Cells Exposed to Hyperglycemia

BACKGROUND Maternal diabetes alters gene expression leading to neural tube defects (NTDs) in the developing brain. The mechanistic pathways that deregulate the gene expression remain unknown. It is hypothesized that exposure of neural stem cells (NSCs) to high glucose/hyperglycemia results in activation of epigenetic mechanisms which alter gene expression and cell fate during brain development. METHODS AND FINDINGS NSCs were isolated from normal pregnancy and streptozotocin induced-diabetic pregnancy and cultured in physiological glucose. In order to examine hyperglycemia induced epigenetic changes in NSCs, chromatin reorganization, global histone status at lysine 9 residue of histone H3 (acetylation and trimethylation) and global DNA methylation were examined and found to be altered by hyperglycemia. In NSCs, hyperglycemia increased the expression of Dcx (Doublecortin) and Pafah1b1 (Platelet activating factor acetyl hydrolase, isoform 1b, subunit 1) proteins concomitant with decreased expression of four microRNAs (mmu-miR-200a, mmu-miR-200b, mmu-miR-466a-3p and mmu-miR-466 d-3p) predicted to target these genes. Knockdown of specific microRNAs in NSCs resulted in increased expression of Dcx and Pafah1b1 proteins confirming target prediction and altered NSC fate by increasing the expression of neuronal and glial lineage markers. CONCLUSION/INTERPRETATION This study revealed that hyperglycemia alters the epigenetic mechanisms in NSCs, resulting in altered expression of some development control genes which may form the basis for the NTDs. Since epigenetic changes are reversible, they may be valuable therapeutic targets in order to improve fetal outcomes in diabetic pregnancy.This study is supported by the NUS bridging fund R-181-000-130-720. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

The Impact of CpG Island on Defining Transcriptional Activation of the Mouse L1 Retrotransposable Elements

BACKGROUND: L1 retrotransposable elements are potent insertional mutagens responsible for the generation of genomic variation and diversification of mammalian genomes, but reliable estimates of the numbers of actively transposing L1 elements are mostly nonexistent. While the human and mouse genomes contain comparable numbers of L1 elements, several phylogenetic and L1Xplore analyses in the mouse genome suggest that 1,500-3,000 active L1 elements currently exist and that they are still expanding in the genome. Conversely, the human genome contains only 150 active L1 elements. In addition, there is a discrepancy among the nature and number of mouse L1 elements in L1Xplore and the mouse genome browser at the UCSC and in the literature. To date, the reason why a high copy number of active L1 elements exist in the mouse genome but not in the human genome is unknown, as are the potential mechanisms that are responsible for transcriptional activation of mouse L1 elements. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the promoter sequences of the 1,501 potentially active mouse L1 elements retrieved from the GenBank and L1Xplore databases and evaluated their transcription factors binding sites and CpG content. To this end, we found that a substantial number of mouse L1 elements contain altered transcription factor YY1 binding sites on their promoter sequences that are required for transcriptional initiation, suggesting that only a half of L1 elements are capable of being transcriptionally active. Furthermore, we present experimental evidence that previously unreported CpG islands exist in the promoters of the most active T(F) family of mouse L1 elements. The presence of sequence variations and polymorphisms in CpG islands of L1 promoters that arise from transition mutations indicates that CpG methylation could play a significant role in determining the activity of L1 elements in the mouse genome. CONCLUSIONS: A comprehensive analysis of mouse L1 promoters suggests that the number of transcriptionally active elements is significantly lower than the total number of full-length copies from the three active mouse L1 families. Like human L1 elements, the CpG islands and potentially the transcription factor YY1 binding sites are likely to be required for transcriptional initiation of mouse L1 elements

Histone Variant H2A.Z Can Serve as a New Target for Breast Cancer Therapy

Histone H2A variant, H2A.Z, plays an essential role in transcriptional activation of ERa-dependent genes, cell proliferation, development, and differentiation. High expression of H2A.Z is ubiquitously detected in the progression of breast cancer, and is significantly associated with lymph node metastasis and patient survival. This makes H2A.Z an excellent target for diagnostic and therapeutic interventions. A recent study provides a new insight into the role of H2A.Z within the context of cancer-related genes and further corroborates the emerging link between dysfunction of this histone variant and cancer. Interestingly, the depletion of H2A.Z also causes defective in the stability and integrity of the human genome. These abnormalities include defective chromosome segregation, activation of LINE-1 retrotransposable elements, and changes in cell cycle-related genes. This article also presents the molecular pathways linking H2A.Z to breast cancer and mechanisms have been proposed to explain how altered H2A.Z leads to tumorigenesis. Two strategies are proposed here for anti-tumor treatments of H2A.Z-defective breast cancer. One is to restore H2A.Z function by targeting c-Myc transcription factor and the other is to find potential drug treatment by blocking the activity of H2A.Z-remodelling complex, p400/Tip60

Distinctive patterns of epigenetic marks are associated with promoter regions of mouse LINE-1 and LTR retrotransposons

Background: The long terminal repeat (LTR) retrotransposons and the non-LTR retrotransposons (LINE-1 or L1) make up more than one-third of the mouse genome. Because of their abundance, the retrotransposons are the major players in genomic structure and function. While much attention has been focused on the biology of retrotransposons, little is known about the chromatin structure of these elements or the potential role of epigenetic marks on the regulation of retrotransposon expression. Findings. Using sequential chromatin immunoprecipitation analysis, we analyzed the cohabitation of several post-translational histone modifications in the promoter regions of mouse L1 and LTR retrotransposons. We show here that the variant histone H2A.Z selectively present in L1 promoters. Notably, H2A.Z and trimethylated histone H3 (H3K9me3) co-localize in the same genomic location of the L1 promoter along with heterochromatin-binding protein HP1. In contrast, MmERV and intracisternal A-particle (IAP) classes of LTR promoters are enriched with core histone H2A and heterochromatic trimethylated histone H4 (H4K20me3). These distinctive patterns of chromatin modifications are relatively consistent irrespective of cell type. Conclusions: Chromatin structure regulates the expression of retrotransposons. LINE-1 elements are associated with H2A.Z and HP1-containing constitutive heterochromatin, while the LTR elements are enriched with H2A and the H4K20me3-type of facultative heterochromatin. Our findings demonstrate that different epigenetic mechanisms operate in the mouse genome to silence different classes of retrotransposons