The temperature-dependent change in methylation of the Antirrhinum transposon Tam3 is controlled by the activity of its transposase

The Antirrhinum majus transposon Tam3 undergoes low temperature-dependent transposition (LTDT). Growth at 15 degrees C permits transposition, whereas growth at 25 degrees C strongly suppresses it. The degree of Tam3 DNA methylation is altered somatically and positively correlated with growth temperature, an exceptional epigenetic system in plants. Using a Tam3-inactive line, we show that methylation change depends on Tam3 activity. Random binding site selection analysis and electrophoretic mobility shift assays revealed that the Tam3 transposase (TPase) binds to the major repeat in the subterminal regions of Tam3, the site showing the biggest temperature-dependent change in methylation state. Methylcytosines in the motif impair the binding ability of the TPase. Proteins in a nuclear extract from plants grown at 15 degrees C but not 25 degrees C bind to this motif in Tam3. The decrease in Tam3 DNA methylation at low temperature also requires cell division. Thus, TPase binding to Tam3 occurs only during growth at low temperature and immediately after DNA replication, resulting in a Tam3-specific decrease in methylation of transposon DNA. Consequently, the Tam3 methylation level in LTDT is regulated by Tam3 activity, which is dependent on the ability of its TPase to bind DNA and affected by growth temperature. Thus, the methylation/demethylation of Tam3 is the consequence, not the cause, of LTDT.     

One of three nuclear localization signals of maize Activator (Ac) transposase overlaps the DNA-binding domain

The nuclear localization sequences (NLSs) of the Ac transposase (TPase) protein have been characterized by indirect immunofluorescence detection of TPase deletion derivatives and TPase/β-glucuronidase (GUS) fusion proteins in transiently transfected Petunia cells. The TPase contains three NLSs near its amino-terminal end, NLS(44–62), NLS(159–178) and NLS(174–206), each of which is sufficient to redirect GUS to the nucleus. Deletion of the N-terminal 102 TPase residues including NLS(44–62) results in strongly reduced nuclear import of the truncated TPase. NLS(44–62) and NLS(159–178) are bipartite NLSs, whereas the structure of NLS(174–206) does not allow a classification into one of the three major NLS categories. NLS(174–206) overlaps with the basic DNA-binding domain of TPase. A substitution of two amino acids in this segment (HiS₁₉₁→Arg and Arg₁₉₃→His) results in a total loss of DNA-binding activity, but retains reduced NLS activity. Accordingly, the two functions can be separated. In addition, we show that a NLS-deficient 71 kDa TPase derivative is co-imported into the nucleus in the presence of wildtype TPase.     

Evidence for a common evolutionary origin of inverted repeat transposons in Drosophila and plants: hobo, Activator, and Tam3.

We have sequenced HFL1 from D. melanogaster, the only cloned hobo element shown to have transposase activity. The 2959 bp HFL1 sequence predicts a 2.0 kb open reading frame (ORF1) with substantial amino acid similarity to the transposases of Activator (Ac) from maize (Zea mays) and Tam3 from snapdragon (Antirrhinum majus). Mutational analysis of a C-terminal region of ORF1 conserved with Ac and Tam3 indicates that it is essential for hobo transposase activity. This is an example of extensive amino acid sequence identity between short inverted repeat elements in different kingdoms. We discuss the possibility that the conservation of hobo, Ac, and Tam3 transposases represents an example of horizontal transmission of genetic information between plants and animals.     

Low transposition frequency of the medaka fish Tol2 element may be due to extranuclear localization of its transposase

Transposase proteins of some highly active DNA-based transposable elements, such as the maize Activator element, are known to possess nuclear localization signals (NLSs). We examined if this is also the case for the transposase of the medaka fish Tol2 element, a member of the hAT (hobo/Activator/Tam3) transposable element family, using human and mouse culture cells. Unexpectedly, the transposase-lacZ fusion protein, in which the lacZ is a location marker, was found to be present in the cytoplasm rather than in the nucleus, suggesting that the Tol2 transposase contains a signal for extranuclear localization. The same staining pattern was also observed with a fusion protein containing a 33-amino-acid region at about the center of the primary structure of the transposase. The Tol2 element might have a mechanism to control its transposition frequency that includes extranuclear localization of its transposase.     

Trans-activation of an artificial dTam3 transposable element in transgenic tobacco plants

In Antirrhinum majus only autonomous Tam3 transposons have been characterized. We investigated whether an artificial dTam3 element, with a deletion in the presumptive transposase coding region, can be trans-activated in tobacco by an activator Tam3 element, which was immobilized by the deletion of one inverted repeat. A phenotypic assay based on restored hygromycin resistance demonstrates that a dTam3 element harbouring a bacterial plasmid can be trans-activated with a low frequency. Molecular analysis confirms that the dTam3 element has been excised from the HPTII marker gene. Reintegration of the dTam3 element into the tobacco genome is detected only in one out of six hygromycin-resistant plants analysed. PCR analysis of empty donor sites shows that excision of the dTam3 element in tobacco results in rearrangements (deletions and additions), that have been shown to be characteristic of Tam3 excision in the original host Antirrhinum majus. This trans-activation assay allowed us to establish that, in contrast to what has been detected in Antirrhinum majus, a periodical temperature shift down to 15°C does not enhance dTam3 transposition in regenerating tobacco calli.     

In vivo aggregation of maize Activator (Ac) transposase in nuclei of maize endosperm and Petunia protoplasts

The transposase (TPase) of the maize transposon Activator (Ac) accumulates in the nuclei of maize endosperm and transfected Petunia protoplasts, where it aggregates into rod-like structures about 2 μm in length. In petunia protoplasts the amount of TPase aggregates increases with the strength of the promoter fused to the Ac-coding region. The excision frequency of a Ds element, however, does not increase proportionally. The data suggest that the aggregated TPase is not responsible for the mobilization of the Ds element, but rather is a transpositionally inactive form of the protein. In contrast to the full-length TPase, a functional, N-terminally truncated TPase derivative is inefficiently transported into the nucleus at high expression levels and aggregates predominantly in the cytoplasm. Accordingly, the N-terminus of the TPase is involved in nuclear localization and/or aggregation.     

DNA methylation is not necessary for the inactivation of the Tam3 transposon at non-permissive temperature in Antirrhinum

It has been proposed that DNA methylation plays an important role in the inactivation of transposons. This view stems from a comparison of the degree of methylation of transposons in the active and inactive state. However, direct evidence for the degree of methylation required for the suppression of transposition has not been reported. Transposon Tam3 in Antirrhinum majus undergoes somatic reversal of its transposition activity, which is tightly controlled by temperature: low temperature around 15 degrees C permits transposition, high temperatures around 25 degrees C strongly inhibits it. Our previous study had shown that the methylation state of the Tam3 end regions is negatively correlated with the Tam3 transposition frequency. The results of the present study reveal that the inactive state of Tam3 copies at high temperature is unlikely to be directly coupled to the methylation state. Treatment with methylation inhibitors (5-azacytidine or 5-azacytidine+ethionine) does not affect Tam3 excision frequency in calli derived from Antirrhinum hypocotyls. The results suggest that methylation is not essential for the suppression of Tam3 transposition at high temperature, but rather that some other mechanism(s) involved in the control of Tam3 transposition may be obscured by methylation.     

Structural conservation of the transposon Tam3 family in Antirrhinum majus and estimation of the number of copies able to transpose

We have investigated the organization of the transposon Tam3 family in Antirrhinum majus. Genomic hybridization experiments and characterization of 40 independent Tam3 clones isolated from an A. majus plant revealed that the Tam3 family is quite conserved and the copy sizes are uniform. We did not find any copy with a deleted internal sequence, unlike what is usually observed in other transposons. This exceptionally conserved structure of the Tam3 family was confirmed by PCR and sequencing analyses. Sequencing analysis identified eight copies with sequences completely identical to that of the Tam3 transposase gene. These results suggested that a considerable number of autonomous Tam3 copies are present in the genome of A. majus. Among 24 copies which are surrounded by single copy regions of the genome, 14 copies are present as specific insertions in the line which we used, but absent in other lines. These copies are therefore predicted to be movable. If this ratio is the same for all Tam3 copies in a genome, then a maximum of 60% of the copies are estimated to be movable in the genome. The relatively high frequency of gene tagged by Tam3 might reflect the large number of movable copies in the genome.     

Tam3 produces a suppressible allele of the DAG locus of Antirrhinum majus similar to Mu-suppressible alleles of maize

Tam3 from Antirrhinum majus belongs to the Ac/Ds family of transposable elements. An allele of the DAG locus of Antirrhinum ( dag ::Tam3), which is required for chloroplast development and leaf palisade differentiation, has been generated by Tam3 insertion into the untranslated leader sequence of the gene. This allele gives rise to a cold-sensitive phenotype, where mutant tissue containing wild-type revertant somatic sectors is observed in the leaves of plants grown at 15°C, while leaves of plants grown at 25°C appear near wild-type. The temperature sensitivity of dag ::Tam3 results from expression of the DAG locus responding to the activity of the transposable element, the transposition of which is very sensitive to growing temperature. Genetic suppression of Tam3 transposition, using the STABILISER locus, also results in suppression of the dag mutant phenotype. dag ::Tam3 represents a Tam3-suppressible allele similar to those described for Mu transposons in maize. Suppression of the dag mutant phenotype in response to element inactivation appears to result from use of an alternative promoter at the 3' end of the Tam3 element. The production of suppressible alleles by an Ac-like element is discussed in relation to the mutagenic potential of plant transposons in producing complex genetic diversity.     

Position effect of the excision frequency of the Antirrhinum transposon Tam3: implications for the degree of position-dependent methylation in the ends of the element

We identified eight independent Tam3 copies residing in the same Antirrhinum majus genome. All the copies showed excision at 15 °C, but not at 25 °C. Under conditions promoting excision, each copy appeared to transpose in the leaves and flower lobes with a nearly constant frequency, whereas individual transposition abilities varied widely: the most active copy had an excision frequency more than 100-fold greater than that of the least active one. Despite the different transposition abilities, the structures of the eight Tam3 copies were almost identical. These results made it clear that the transpositional ability of Tam3 is regulated by chromosomal position, but they do not imply position-dependent transposase activity. The position effect of the Tam3 transposition was found to be correlated to the methylation state of the copy's end regions: DNA methylation in the Tam3 end regions tended to suppress the excision activity, and the degree of methylation was dependent on the chromosomal position. Our results also provide evidence of de novo methylation provoked by transposition of the endogenous element. We propose a mechanism of transpositional regulation of plant transposons that responds to the degree of methylation as determined by chromosomal position.     

Resistance to gap repair of the transposon Tam3 in Antirrhinum majus: a role of the end regions

The extremely homogeneous organization of the transposon family Tam3 in Antirrhinum majus is in sharp contrast to the heterogeneity of the copies constituting many other transposon families. To address the issue of the Tam3 structural uniformity, we examined two possibilities: (1) recent invasion of Tam3 and (2) failure of gap repair, which is involved in conversion from autonomous forms to defective forms. The phylogenetic analysis of 17 Tam3 copies suggested that the invasion of Tam3 into the Antirrhinum genome occurred at least 5 mya, which is sufficiently long ago to have produced many aberrant copies by gap repair. Thus, we investigated gap repair events at the nivea(recurrens:Tam3) (niv(rec)::Tam3) allele, where Tam3 is actively excised. We show here that the gap repair of de novo somatic Tam3 excision was arrested immediately after initiation of the process. All of the identified gap repair products were short stretches, no longer than 150 bp from the ends. The Tam3 ends have hairpin structures with low free energies. We observed that the gap repair halted within the hairpin structure regions. Such small gap repair products appear to be distributed in the Antirrhinum genome, but are unlikely to be active. Our data strongly suggest that the structural homogeneity of Tam3 was caused by immunity to gap repair at the hairpins in both of the end regions. The frequency of extensive gap repair of de novo excision products in eukaryotic transposons was found to be correlated with the free energies of the secondary structures in the end regions. This fact suggests that the fates of transposon families might depend on the structures of their ends.     

Activity of the transposon Tam3 in Antirrhinum and tobacco: possible role of DNA methylation.

The transposon Tam3 from Antirrhinum majus can transpose in a heterologous host (Nicotiana tabacum); thus the element is autonomous, probably encoding the specific information required for its own transposition. In transgenic tobacco Tam3 rapidly becomes methylated at its ends whilst adjacent flanking sequences remain hypomethylated. This methylation may account for our failure to detect Tam3 transposition in the progeny of transgenic tobacco. Treatment with the inhibitor of cytosine methylation, 5 aza-cytosine appeared to induce transposon related activity at a low level. In Antirrhinum methylation also appears to be associated with inactivation of Tam3 copies.     

Large-scale chromosomal restructuring is induced by the transposable element tam3 at the nivea locus of antirrhinum majus

The transposable element, Tam3, gives rise to large-scale (greater than 1 kb) chromosomal rearrangements at a low frequency, when it is inserted at the nivea locus of Antirrhinum majus. Although some deletions may result from imprecise excision of Tam3, rearrangements involving deletion, dispersion and inverted duplication of flanking sequences, where Tam3 remains in situ, have also been identified. These rearrangements have been mapped at the molecular level, and the behavior of Tam3 following rearrangement has been observed. It is clear that Tam3 has enormous potential to restructure chromosomes through successive rounds of large-scale rearrangements. The mechanisms by which such rearrangements might arise are discussed.     

Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants

Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associated proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.     

Cytoplasmic PELP1 and ERRgamma Protect Human Mammary Epithelial Cells from Tam-Induced Cell Death

Tamoxifen (Tam) is the only FDA-approved chemoprevention agent for pre-menopausal women at high risk for developing breast cancer. While Tam reduces a woman''s risk of developing estrogen receptor positive (ER+) breast cancer, the molecular mechanisms associated with risk reduction are poorly understood. Prior studies have shown that cytoplasmic proline, glutamic acid and leucine rich protein 1 (PELP1) promotes Tam resistance in breast cancer cell lines. Herein, we tested for PELP1 localization in breast epithelial cells from women at high risk for developing breast cancer and found that PELP1 was localized to the cytoplasm in 36% of samples. In vitro, immortalized HMECs expressing a nuclear localization signal (NLS) mutant of PELP1 (PELP1-cyto) were resistant to Tam-induced death. Furthermore, PELP1-cyto signaling through estrogen-related receptor gamma (ERRγ) promoted cell survival in the presence of Tam. Overexpression of ERRγ in immortalized HMECs protected cells from Tam-induced death, while knockdown of ERRγ sensitized PELP1-cyto expressing HMECs to Tam. Moreover, Tam-induced HMEC cell death was independent of apoptosis and involved accumulation of the autophagy marker LC3-II. Expression of PELP1-cyto and ERRγ reduced Tam-induced LC3-II accumulation, and knockdown of ERRγ increased LC3-II levels in response to Tam. Additionally, PELP1-cyto expression led to the upregulation of MMP-3 and MAOB, known PELP1 and ERRγ target genes, respectively. Our data indicate that cytoplasmic PELP1 induces signaling pathways that converge on ERRγ to promote cell survival in the presence of Tam. These data suggest that PELP1 localization and/or ERRγ activation could be developed as tissue biomarkers for Tam responsiveness.     

Dephosphorylation at a Conserved SP Motif Governs cAMP Sensitivity and Nuclear Localization of Class IIa Histone Deacetylases

Histone deacetylase 4 (HDAC4) and its paralogs, HDAC5, -7, and -9 (all members of class IIa), possess multiple phosphorylation sites crucial for 14-3-3 binding and subsequent nuclear export. cAMP signaling stimulates nuclear import of HDAC4 and HDAC5, but the underlying mechanisms remain to be elucidated. Here we show that cAMP potentiates nuclear localization of HDAC9. Mutation of an SP motif conserved in HDAC4, -5, and -9 prevents cAMP-stimulated nuclear localization. Unexpectedly, this treatment inhibits phosphorylation at the SP motif, indicating an inverse relationship between the phosphorylation event and nuclear import. Consistent with this, leptomycin B-induced nuclear import and adrenocorticotropic hormone (ACTH) treatment result in the dephosphorylation at the motif. Moreover, the modification synergizes with phosphorylation at a nearby site, and similar kinetics was observed for both phosphorylation events during myoblast and adipocyte differentiation. These results thus unravel a previously unrecognized mechanism whereby cAMP promotes dephosphorylation and differentially regulates multisite phosphorylation and the nuclear localization of class IIa HDACs.     

Comparison of genetic behaviour of the transposable element Tam3 at two unlinked pigment loci in Antirrhinum majus

Summary In Antirrhinum majus the transposable element Tam3 has been described at two unlinked loci pallida and nivea, both of which are required for the production of anthocyanin pigment in flowers. In each case the element is inserted in the promoter region and gives a variegated phenotype. We show that the rate of Tam3 excision at both loci is greatly affected by temperature, being approximately 1000-fold higher at 15°C compared with 25°C. Tam3 is also controlled by an unlinked gene Stabiliser, which considerably reduces excision rate. We show that the high degree of sensitivity to temperature and Stabiliser is an intrinsic property of Tam3 which is not shared by an unrelated element, Tam1. The Tam3 insertion at nivea gives rise to a series of alleles which confer reduced pigmentation, novel spatial patterns and changed instability. These are probably a result of imprecise excision and rearrangements of the Tam3 element.