Transposable elements: an abundant and natural source of regulatory sequences for host genes.

The fact that transposable elements (TEs) can influence host gene expression was first recognized more than 50 years ago. However, since that time, TEs have been widely regarded as harmful genetic parasites-selfish elements that are rarely co-opted by the genome to serve a beneficial role. Here, we survey recent findings that relate to TE impact on host genes and remind the reader that TEs, in contrast to other noncoding parts of the genome, are uniquely suited to gene regulatory functions. We review recent studies that demonstrate the role of TEs in establishing and rewiring gene regulatory networks and discuss the overall ubiquity of exaptation. We suggest that although individuals within a population can be harmed by the deleterious effects of new TE insertions, the presence of TE sequences in a genome is of overall benefit to the population.

LIONS: analysis suite for detecting and quantifying transposable element initiated transcription from RNA-seq.

SUMMARY: Transposable elements (TEs) influence the evolution of novel transcriptional networks yet the specific and meaningful interpretation of how TE-derived transcriptional initiation contributes to the transcriptome has been marred by computational and methodological deficiencies. We developed LIONS for the analysis of RNA-seq data to specifically detect and quantify TE-initiated transcripts. AVAILABILITY AND IMPLEMENTATION: Source code, container, test data and instruction manual are freely available at www.github.com/ababaian/LIONS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Pluripotency and the endogenous retrovirus HERVH: Conflict or serendipity?

Remnants of ancient retroviral infections during evolution litter all mammalian genomes. In modern humans, such endogenous retroviral (ERV) sequences comprise at least 8% of the genome. While ERVs and other types of transposable elements undoubtedly contribute to the genomic "junk yard", functions for some ERV sequences have been demonstrated, with growing evidence that ERVs can be important players in gene regulatory processes. Here we focus on one particular large family of human ERVs, termed HERVH, which several recent studies suggest has a key regulatory role in human pluripotent stem cells. Remarkably, this is not the first instance of an ERV controlling pluripotency. We speculate as to why this convergent evolution might have come about, suggesting that it may reflect selection on the virus to extend the time available for transposition. Alternatively it may reflect serendipity alone.

Distinct isoform of FABP7 revealed by screening for retroelement-activated genes in diffuse large B-cell lymphoma.

Remnants of ancient transposable elements (TEs) are abundant in mammalian genomes. These sequences harbor multiple regulatory motifs and hence are capable of influencing expression of host genes. In response to environmental changes, TEs are known to be released from epigenetic repression and to become transcriptionally active. Such activation could also lead to lineage-inappropriate activation of oncogenes, as one study described in Hodgkin lymphoma. However, little further evidence for this mechanism in other cancers has been reported. Here, we reanalyzed whole transcriptome data from a large cohort of patients with diffuse large B-cell lymphoma (DLBCL) compared with normal B-cell centroblasts to detect genes ectopically expressed through activation of TE promoters. We have identified 98 such TE-gene chimeric transcripts that were exclusively expressed in primary DLBCL cases and confirmed several in DLBCL-derived cell lines. We further characterized a TE-gene chimeric transcript involving a fatty acid-binding protein gene (LTR2-FABP7), normally expressed in brain, that was ectopically expressed in a subset of DLBCL patients through the use of an endogenous retroviral LTR promoter of the LTR2 family. The LTR2-FABP7 chimeric transcript encodes a novel chimeric isoform of the protein with characteristics distinct from native FABP7. In vitro studies reveal a dependency for DLBCL cell line proliferation and growth on LTR2-FABP7 chimeric protein expression. Taken together, these data demonstrate the significance of TEs as regulators of aberrant gene expression in cancer and suggest that LTR2-FABP7 may contribute to the pathogenesis of DLBCL in a subgroup of patients.

Epigenetic mechanism causes Wnt9b deficiency and nonsyndromic cleft lip and palate in the A/WySn mouse strain.

BACKGROUND: The heritable multifactorial etiology of human nonsyndromic cleft lip with or without cleft palate (CL ± P) is not understood. CL ± P occurs in 15% of neonates in the homozygous A/WySn mouse strain, with a multifactorial genetic etiology, the clf1 and clf2 variant genes. Clf1 acts as a mutant allele of Wnt9b but its coding sequence is normal. An IAP (intracisternal A particle) retrotransposon inserted near the Wnt9b gene is associated with clf1. METHODS: Transcription of noncoding sequence between the IAP and the Wnt9b gene was examined in A/WySn embryos. The levels of Wnt9b transcript and of an "IAP antisense" transcript initiated in the IAP and extending into the noncoding interval were assayed in A/WySn and C57BL/6J whole embryos or heads across embryonic days 8 to 12. Methylation of the 5' LTR of the IAP was examined in E12 A/WySn embryo heads. RESULTS: Mean Wnt9b transcript levels were lower in A/WySn than in C57BL/6J at all ages examined and lower in CL ± P embryos than in their normal littermates. The "IAP antisense" transcript was found in all A/WySn embryos and was highest in CL ± P embryos. The IAP at Wnt9b was generally unmethylated in CL ± P embryos and approximately 50% methylated in normal littermates. CONCLUSION: The clf1 mutation in A/WySn is a "metastable epiallele", in which stochastic deficiency in some individuals of DNA methylation of a retrotransposon uniquely inserted near the Wnt9b gene allows transcriptional activity of the retrotransposon and interference with transcription from Wnt9b. Methylation of metastable epialleles should be investigated in human nonsyndromic CL ± P.

Lysine methyltransferase G9a is required for de novo DNA methylation and the establishment, but not the maintenance, of proviral silencing.

Methylation on lysine 9 of histone H3 (H3K9me) and DNA methylation play important roles in the transcriptional silencing of specific genes and repetitive elements. Both marks are detected on class I and II endogenous retroviruses (ERVs) in murine embryonic stem cells (mESCs). Recently, we reported that the H3K9-specific lysine methyltransferase (KMTase) Eset/Setdb1/KMT1E is required for H3K9me3 and the maintenance of silencing of ERVs in mESCs. In contrast, G9a/Ehmt2/KMT1C is dispensable, despite the fact that this KMTase is required for H3K9 dimethylation (H3K9me2) and efficient DNA methylation of these retroelements. Transcription of the exogenous retrovirus (XRV) Moloney murine leukemia virus is rapidly extinguished after integration in mESCs, concomitant with de novo DNA methylation. However, the role that H3K9 KMTases play in this process has not been addressed. Here, we demonstrate that G9a, but not Suv39h1 or Suv39h2, is required for silencing of newly integrated Moloney murine leukemia virus-based vectors in mESCs. The silencing defect in G9a(-/-) cells is accompanied by a reduction of H3K9me2 at the proviral LTR, indicating that XRVs are direct targets of G9a. Furthermore, de novo DNA methylation of newly integrated proviruses is impaired in the G9a(-/-) line, phenocopying proviral DNA methylation and silencing defects observed in Dnmt3a-deficient mESCs. Once established, however, maintenance of silencing of XRVs, like ERVs, is dependent exclusively on the KMTase Eset. Taken together, these observations reveal that in mESCs, the H3K9 KMTase G9a is required for the establishment, but not for the maintenance, of silencing of newly integrated proviruses.

Retrotransposon-induced heterochromatin spreading in the mouse revealed by insertional polymorphisms.

The "arms race" relationship between transposable elements (TEs) and their host has promoted a series of epigenetic silencing mechanisms directed against TEs. Retrotransposons, a class of TEs, are often located in repressed regions and are thought to induce heterochromatin formation and spreading. However, direct evidence for TE-induced local heterochromatin in mammals is surprisingly scarce. To examine this phenomenon, we chose two mouse embryonic stem (ES) cell lines that possess insertionally polymorphic retrotransposons (IAP, ETn/MusD, and LINE elements) at specific loci in one cell line but not the other. Employing ChIP-seq data for these cell lines, we show that IAP elements robustly induce H3K9me3 and H4K20me3 marks in flanking genomic DNA. In contrast, such heterochromatin is not induced by LINE copies and only by a minority of polymorphic ETn/MusD copies. DNA methylation is independent of the presence of IAP copies, since it is present in flanking regions of both full and empty sites. Finally, such spreading into genes appears to be rare, since the transcriptional start sites of very few genes are less than one Kb from an IAP. However, the B3galtl gene is subject to transcriptional silencing via IAP-induced heterochromatin. Hence, although rare, IAP-induced local heterochromatin spreading into nearby genes may influence expression and, in turn, host fitness.

Role of runt-related transcription factor 3 (RUNX3) in transcription regulation of natural cytotoxicity receptor 1 (NCR1/NKp46), an activating natural killer (NK) cell receptor.

Natural cytotoxicity receptor 1 (NCR1), also known as NKp46, is a natural killer (NK) lymphocyte-activating receptor. It is involved in major aspects of NK immune function and shows a high degree of lineage specificity in blood and bone marrow. The nature of its NK-restricted expression is not well understood. In this study, we confirm that human NCR1 NK-specific expression is achieved at the mRNA level. We found two key cis-regulatory elements in the immediate vicinity upstream of the gene. One element acts as an essential promoter, whereas the other acts as a tissue-dependent enhancer/repressor. This latter regulatory element contains a runt related-transcription factor (RUNX) recognition motif that preferentially binds RUNX3. Interfering with RUNX proteins using a dominant negative form results in decreased Ncr1 expression. RUNX3 overexpression had the opposite effect. These findings shed light on the role of RUNX3 in the control of an important NK-activating receptor.

Genistein versus ICI 182, 780: an ally or enemy in metastatic progression of prostate cancer.

BACKGROUND: Androgen signalling through the androgen receptor (AR) plays a critical role in prostate cancer (PCa) initiation and progression. Estrogen in synergy with androgen is essential for cell growth of the normal and malignant prostate. However, the exact role that estrogen and the estrogen receptor play in prostate carcinogenesis remains unclear. We have previously demonstrated the metastasis-promoting effect of an estrogen receptor beta (ERß) agonist (genistein) in a patient-derived PCa xenograft model mimicking localized and metastatic disease. METHODS: To test the hypothesis that the tumor-promoting activity of genistein was due to its estrogenic properties, we treated the xenograft-bearing mice with genistein and an anti-estrogen compound (ICI 182, 780) and compared the differential gene expression using microarrays. RESULTS: Using a second xenograft model which was derived from another patient, we showed that genistein promoted disease progression in vivo and ICI 182, 780 inhibited metastatic spread. The microarray analysis revealed that the metallothionein (MT) gene family was differentially expressed in tumors treated by these compounds. Using qRT-PCR, the differences in expression levels were validated in the metastatic and non-metastatic LTL313 PCa xenograft tumor lines, both of which were originally derived from the same PCa patient. CONCLUSIONS: Together our data provide evidence that genistein stimulates and ICI 182, 780 inhibits metastatic progression, suggesting that these effects may be mediated by ERß signalling.

Human Th1 and Th17 cells exhibit epigenetic stability at signature cytokine and transcription factor loci.

The linear model of Th cell lineage commitment is being revised due to reports that mature Th cells can trans-differentiate into alternate lineages. This ability of Th cells to reprogram is thought to be regulated by epigenetic mechanisms that control expression of transcription factors characteristic of opposing lineages. It is unclear, however, to what extent this new model of Th cell plasticity holds true in human Th cell subsets that develop under physiological conditions in vivo. We isolated in vivo-differentiated human Th1 and Th17 cells, as well as intermediate Th1/17 cells, and identified distinct epigenetic signatures at cytokine (IFNG and IL17A) and transcription factor (TBX21, RORC, and RORA) loci. We also examined the phenotypic and epigenetic stability of human Th17 cells exposed to Th1-polarizing conditions and found that although they could upregulate TBX21 and IFN-γ, this occurred without loss of IL-17 or RORC expression, and resulted in cells with a Th1/17 phenotype. Similarly, Th1 cells could upregulate IL-17 upon enforced expression of RORC2, but did not lose expression of IFN-γ or TBX21. Despite alterations in expression of these signature genes, epigenetic modifications were remarkably stable aside from the acquisition of active histone methylation marks at cytokine gene promoters. The limited capacity of human Th17 and Th1 cells to undergo complete lineage conversion suggests that the bipotent Th1/17 cells may arise from Th1 and/or Th17 cells. These data also question the broad applicability of the new model of Th cell lineage plasticity to in vivo-polarized human Th cell subsets.

Placenta-specific expression of the interleukin-2 (IL-2) receptor ß subunit from an endogenous retroviral promoter.

The long terminal repeat (LTR) sequences of endogenous retroviruses and retroelements contain promoter elements and are known to form chimeric transcripts with nearby cellular genes. Here we show that an LTR of the THE1D retroelement family has been domesticated as an alternative promoter of human IL2RB, the gene encoding the ß subunit of the IL-2 receptor. The LTR promoter confers expression specifically in the placental trophoblast as opposed to its native transcription in the hematopoietic system. Rather than sequence-specific determinants, DNA methylation was found to regulate transcription initiation and splicing efficiency in a tissue-specific manner. Furthermore, we detected the cytoplasmic signaling domain of the IL-2Rß protein in the placenta, suggesting that IL-2Rß undergoes preferential proteolytic cleavage in this tissue. These findings implicate novel functions for this cytokine receptor subunit in the villous trophoblast and reveal an intriguing example of ancient LTR exaptation to drive tissue-specific gene expression.

DNA methylation in ES cells requires the lysine methyltransferase G9a but not its catalytic activity.

Histone H3K9 methylation is required for DNA methylation and silencing of repetitive elements in plants and filamentous fungi. In mammalian cells however, deletion of the H3K9 histone methyltransferases (HMTases) Suv39h1 and Suv39h2 does not affect DNA methylation of the endogenous retrovirus murine leukaemia virus, indicating that H3K9 methylation is dispensable for DNA methylation of retrotransposons, or that a different HMTase is involved. We demonstrate that embryonic stem (ES) cells lacking the H3K9 HMTase G9a show a significant reduction in DNA methylation of retrotransposons, major satellite repeats and densely methylated CpG-rich promoters. Surprisingly, demethylated retrotransposons remain transcriptionally silent in G9a(-/-) cells, and show only a modest decrease in H3K9me2 and no decrease in H3K9me3 or HP1alpha binding, indicating that H3K9 methylation per se is not the relevant trigger for DNA methylation. Indeed, introduction of catalytically inactive G9a transgenes partially 'rescues' the DNA methylation defect observed in G9a(-/-) cells. Taken together, these observations reveal that H3K9me3 and HP1alpha recruitment to retrotransposons occurs independent of DNA methylation in ES cells and that G9a promotes DNA methylation independent of its HMTase activity.

Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency.

Induced pluripotent stem (iPS) cells may be of use in regenerative medicine. However, the low efficiency of reprogramming is a major impediment to the generation of patient-specific iPS cell lines. Here we report the first selection system for the isolation of human iPS cells. We developed the EOS (Early Transposon promoter and Oct-4 (Pou5f1) and Sox2 enhancers) lentiviral vector to specifically express in mouse and human embryonic stem cells but not in primary fibroblasts. The bicistronic EOS vector marked emerging mouse and human iPS cell colonies with EGFP, and we used puromycin selection to aid the isolation of iPS cell lines that expressed endogenous pluripotency markers. These lines differentiated into cell types from all three germ layers. Reporter expression was extinguished upon differentiation and therefore monitored the residual pluripotent cells that form teratomas. Finally, we used EOS selection to establish Rett syndrome-specific mouse and human iPS cell lines with known mutations in MECP2.

Distributions of transposable elements reveal hazardous zones in mammalian introns.

Comprising nearly half of the human and mouse genomes, transposable elements (TEs) are found within most genes. Although the vast majority of TEs in introns are fixed in the species and presumably exert no significant effects on the enclosing gene, some markedly perturb transcription and result in disease or a mutated phenotype. Factors determining the likelihood that an intronic TE will affect transcription are not clear. In this study, we examined intronic TE distributions in both human and mouse and found several factors that likely contribute to whether a particular TE can influence gene transcription. Specifically, we observed that TEs near exons are greatly underrepresented compared to random distributions, but the size of these "underrepresentation zones" differs between TE classes. Compared to elsewhere in introns, TEs within these zones are shorter on average and show stronger orientation biases. Moreover, TEs in extremely close proximity (<20 bp) to exons show a strong bias to be near splice-donor sites. Interestingly, disease-causing intronic TE insertions show the opposite distributional trends, and by examining expressed sequence tag (EST) databases, we found that the proportion of TEs contributing to chimeric TE-gene transcripts is significantly higher within their underrepresentation zones. In addition, an analysis of predicted splice sites within human long terminal repeat (LTR) elements showed a significantly lower total number and weaker strength for intronic LTRs near exons. Based on these factors, we selectively examined a list of polymorphic mouse LTR elements in introns and showed clear evidence of transcriptional disruption by LTR element insertions in the Trpc6 and Kcnh6 genes. Taken together, these studies lend insight into the potential selective forces that have shaped intronic TE distributions and enable identification of TEs most likely to exert transcriptional effects on genes.

The clf2 gene has an epigenetic role in the multifactorial etiology of cleft lip and palate in the A/WySn mouse strain.

BACKGROUND: The A/WySn mouse strain with 15 to 20% penetrance of cleft lip and palate (CLP) is an animal model for human multifactorial CLP. The CLP is due to two unlinked genes that interact epistatically, Wnt9b(clf1) and clf2, plus a maternal effect. The Wnt9b(clf1) mutation is an IAP transposon insertion. The clf2 gene, with unknown function, was located in a 13.6 Mb region of chromosome 13 containing 145 genes. METHODS: To reduce the clf2 candidate region, 1146 mice segregating for A/WySn and C57BL/6J alleles at clf2 were screened for recombinants by simple sequence-length polymorphism haplotypes; recombinants' testcross progeny were typed for CLP and simple-sequence length polymorphisms. To identify the function of clf2, the effect of clf2 genotype on risk of CLP was tested in Wnt9b(null/null) knockouts and in compound mutants (Wnt9b(clf1/null) ), and the methylation of the IAP at Wnt9b was assayed in the Wnt9b(clf1/null) mutants by combined bisulfite restriction analysis. RESULTS: The location of clf2 was redefined to 3.0 Mb between Cntnap3 and AK029746 containing 48 genes, of which 30 are Zfp genes. The clf2 genotype had no detectable effect on Wnt9b(null/null) embryos, but strongly affected risk of CLP and methylation of the IAP in Wnt9b(clf1/null) embryos. CLP was associated with low levels of methylation of the IAP. CONCLUSIONS: The clf2 gene is the first identified polymorphism that affects the epigenetic methylation and silencing of IAP retrotransposons. This CLP model raises the question of whether parallel epigenetic factors are involved in risk and environmental sensitivity of human CLP.

Creation of the two isoforms of rodent NKG2D was driven by a B1 retrotransposon insertion.

The mouse gene for the natural killer (NK) cell-activating receptor Nkg2d produces two protein isoforms, NKG2D-S and NKG2D-L, which differ by 13 amino acids at the N-terminus and have different signalling capabilities. These two isoforms are produced through differential splicing, but their regulation has not been investigated. In this study, we show that rat Nkg2d has the same splicing pattern as that of the mouse, and we mapped transcriptional start sites in both species. We found that the splice forms arise from alternative promoters and that the NKG2D-L promoter is derived from a rodent B1 retrotransposon that inserted before mouse-rat divergence. This B1 insertion is associated with loss of a nearby splice acceptor site that subsequently allowed creation of the short NKG2D isoform found in mouse but not human. Transient reporter assays indicate that the B1 element is a strong promoter with no inherent lymphoid tissue-specificity. We have also identified different binding sites for the ETS family member GABP within both the mouse and rat B1 elements that are necessary for high-promoter activity and for full Nkg2d-L expression. These findings demonstrate that a retroelement insertion has led to gene-regulatory change and functional diversification of rodent NKG2D.

Evidence for high bi-allelic expression of activating Ly49 receptors.

Stochastic expression is a hallmark of the Ly49 family that encode the main MHC class-I-recognizing receptors of mouse natural killer (NK) cells. This highly polygenic and polymorphic family includes both activating and inhibitory receptor genes and is one of genome's fastest evolving loci. The inhibitory Ly49 genes are expressed in a stochastic mono-allelic manner, possibly under the control of an upstream bi-directional early promoter and show mono-allelic DNA methylation patterns. To date, no studies have directly addressed the transcriptional regulation of the activating Ly49 receptors. Our study shows differences in DNA methylation pattern between activating and inhibitory genes in C57BL/6 and F1 hybrid mouse strains. We also show a bias towards bi-allelic expression of the activating receptors based on allele-specific single-cell RT-PCR in F1 hybrid NK cells for Ly49d and Ly49H expression in Ly49h(+/-) mice. Furthermore, we have identified a region of high sequence identity with possible transcriptional regulatory capacity for the activating Ly49 genes. Our results also point to a likely difference between NK and T-cells in their ability to transcribe the activating Ly49 genes. These studies highlight the complex regulation of this rapidly evolving gene family of central importance in mouse NK cell function.

Genome-wide assessments reveal extremely high levels of polymorphism of two active families of mouse endogenous retroviral elements.

Endogenous retroviral elements (ERVs) in mice are significant genomic mutagens, causing approximately 10% of all reported spontaneous germ line mutations in laboratory strains. The majority of these mutations are due to insertions of two high copy ERV families, the IAP and ETn/MusD elements. This significant level of ongoing retrotranspositional activity suggests that inbred mice are highly variable in content of these two ERV groups. However, no comprehensive genome-wide studies have been performed to assess their level of polymorphism. Here we compared three test strains, for which sufficient genomic sequence is available, to each other and to the reference C57BL/6J genome and detected very high levels of insertional polymorphism for both ERV families, with an estimated false discovery rate of only 0.4%. Specifically, we found that at least 60% of IAP and 25% of ETn/MusD elements detected in any strain are absent in one or more of the other three strains. The polymorphic nature of a set of 40 ETn/MusD elements found within gene introns was confirmed using genomic PCR on DNA from a panel of mouse strains. For some cases, we detected gene-splicing abnormalities involving the ERV and obtained additional evidence for decreased gene expression in strains carrying the insertion. In total, we identified nearly 700 polymorphic IAP or ETn/MusD ERVs or solitary LTRs that reside in gene introns, providing potential candidates that may contribute to gene expression differences among strains. These extreme levels of polymorphism suggest that ERV insertions play a significant role in genetic drift of mouse lines.

Expression of the leukemic prognostic marker CD7 is linked to epigenetic modifications in chronic myeloid leukemia.

BACKGROUND: Expression levels of the cell surface glycoprotein, CD7, and the serine protease, elastase 2 (ELA2), in the leukemic cells of patients with chronic myeloid leukemia (CML) have been associated with clinical outcome. However, little is known about the mechanisms that underlie the variable expression of these genes in the leukemic cells. RESULTS: To address this question, we compared the level of their expression with the DNA methylation and histone acetylation status of 5' sequences of both genes in leukemic cell lines and primitive (lin-CD34+) leukemic cells from chronic phase CML patients. DNA methylation of the ELA2 gene promoter did not correlate with its expression pattern in lin-CD34+ cells from chronic phase CML patient samples even though there was clear differential DNA methylation of this locus in ELA2-expressing and non-expressing cell lines. In contrast, we found a strong relation between CD7 expression and transcription-permissive chromatin modifications, both at the level of DNA methylation and histone acetylation with evidence of hypomethylation of the CD7 promoter region in the lin-CD34+ cells from CML patients with high CD7 expression. CONCLUSION: These findings indicate a link between epigenetic modifications and CD7 expression in primitive CML cells.