Argonautes team up to silence transposable elements in Arabidopsis

The de novo silencing of transposable elements in plants and animals is mediated in part by RNA‐directed chromatin modification. In flowering plants, AGO4 has been seen as the key argonaute protein in the RNA‐directed DNA methylation pathway that links the plant‐specific RNA polymerase V with the de novo DNA methyltransferase DRM2 (Zhong et al, 2014). Two recent papers in The EMBO Journal strongly implicate a role for the AGO6 protein in the process of de novo silencing.     

Arabidopsis Group IIId ERF proteins positively regulate primary cell wall-type CESA genes

Journal of Plant Research - The cell wall determines morphology and the environmental responses of plant cells. The primary cell wall (PCW) is produced during cell division and expansion,...     

Chromatin and siRNA pathways cooperate to maintain DNA methylation of small transposable elements in Arabidopsis

Background

DNA methylation occurs at preferred sites in eukaryotes. In Arabidopsis, DNA cytosine methylation is maintained by three subfamilies of methyltransferases with distinct substrate specificities and different modes of action. Targeting of cytosine methylation at selected loci has been found to sometimes involve histone H3 methylation and small interfering (si)RNAs. However, the relationship between different cytosine methylation pathways and their preferred targets is not known.

Results

We used a microarray-based profiling method to explore the involvement of Arabidopsis CMT3 and DRM DNA methyltransferases, a histone H3 lysine-9 methyltransferase (KYP) and an Argonaute-related siRNA silencing component (AGO4) in methylating target loci. We found that KYP targets are also CMT3 targets, suggesting that histone methylation maintains CNG methylation genome-wide. CMT3 and KYP targets show similar proximal distributions that correspond to the overall distribution of transposable elements of all types, whereas DRM targets are distributed more distally along the chromosome. We find an inverse relationship between element size and loss of methylation in ago4 and drm mutants.

Conclusion

We conclude that the targets of both DNA methylation and histone H3K9 methylation pathways are transposable elements genome-wide, irrespective of element type and position. Our findings also suggest that RNA-directed DNA methylation is required to silence isolated elements that may be too small to be maintained in a silent state by a chromatin-based mechanism alone. Thus, parallel pathways would be needed to maintain silencing of transposable elements.     

Strigolactones positively regulate chilling tolerance in pea and in Arabidopsis

Strigolactones (SL) fulfil important roles in plant development and stress tolerance. Here, we characterized the role of SL in the dark chilling tolerance of pea and Arabidopsis by analysis of mutants that are defective in either SL synthesis or signalling. Pea mutants (rms3, rms4, and rms5) had significantly greater shoot branching with higher leaf chlorophyll a/b ratios and carotenoid contents than the wild type. Exposure to dark chilling significantly decreased shoot fresh weights but increased leaf numbers in all lines. Moreover, dark chilling treatments decreased biomass (dry weight) accumulation only in rms3 and rms5 shoots. Unlike the wild type plants, chilling‐induced inhibition of photosynthetic carbon assimilation was observed in the rms lines and also in the Arabidopsis max3‐9, max4‐1, and max2‐1 mutants that are defective in SL synthesis or signalling. When grown on agar plates, the max mutant rosettes accumulated less biomass than the wild type. The synthetic SL, GR24, decreased leaf area in the wild type, max3‐9, and max4‐1 mutants but not in max2‐1 in the absence of stress. In addition, a chilling‐induced decrease in leaf area was observed in all the lines in the presence of GR24. We conclude that SL plays an important role in the control of dark chilling tolerance.     

Analysis of 3D genomic interactions identifies candidate host genes that transposable elements potentially regulate

Background

The organization of chromatin in the nucleus plays an essential role in gene regulation. About half of the mammalian genome comprises transposable elements. Given their repetitive nature, reads associated with these elements are generally discarded or randomly distributed among elements of the same type in genome-wide analyses. Thus, it is challenging to identify the activities and properties of individual transposons. As a result, we only have a partial understanding of how transposons contribute to chromatin folding and how they impact gene regulation.

Results

Using PCR and Capture-based chromosome conformation capture (3C) approaches, collectively called 4Tran, we take advantage of the repetitive nature of transposons to capture interactions from multiple copies of endogenous retrovirus (ERVs) in the human and mouse genomes. With 4Tran-PCR, reads are selectively mapped to unique regions in the genome. This enables the identification of transposable element interaction profiles for individual ERV families and integration events specific to particular genomes. With this approach, we demonstrate that transposons engage in long-range intra-chromosomal interactions guided by the separation of chromosomes into A and B compartments as well as topologically associated domains (TADs). In contrast to 4Tran-PCR, Capture-4Tran can uniquely identify both ends of an interaction that involve retroviral repeat sequences, providing a powerful tool for uncovering the individual transposable element insertions that interact with and potentially regulate target genes.

Conclusions

4Tran provides new insight into the manner in which transposons contribute to chromosome architecture and identifies target genes that transposable elements can potentially control.

     

MicroRNAs transcriptionally regulate promoter activity in Arabidopsis thaliana

Micro RNAs(mi RNAs) are vital regulators that repress gene expression in the cytoplasm in two main ways: m RNA degradation and translational inhibition. Several animal studies have shown that mi RNAs also target promoters, thereby activating expression.Whether this mi RNA action also occurs in plants is unknown. In this study, we demonstrated that several mi RNAs regulate target promoters in Arabidopsis thaliana. For example, mi R5658 was predominantly present in the nucleus and activated the expression of AT3 G25290 directly by binding to its promoter. Our observations suggest that this mode of action may be a general feature of plant mi RNAs, and thus provide insight into the vital roles of plant mi RNAs in the nucleus.     

Colonization of heterochromatic genes by transposable elements in Drosophila

As a further step toward understanding transposable element-host genome interactions, we investigated the molecular anatomy of introns from five heterochromatic and 22 euchromatic protein-coding genes of Drosophila melanogaster. A total of 79 kb of intronic sequences from heterochromatic genes and 355 kb of intronic sequences from euchromatic genes have been used in Blast searches against Drosophila transposable elements (TEs). The results show that TE-homologous sequences belonging to 19 different families represent about 50% of intronic DNA from heterochromatic genes. In contrast, only 0.1% of the euchromatic intron DNA exhibits homology to known TEs. Intraspecific and interspecific size polymorphisms of introns were found, which are likely to be associated with changes in TE-related sequences. Together, the enrichment in TEs and the apparent dynamic state of heterochromatic introns suggest that TEs contribute significantly to the evolution of genes located in heterochromatin.     

Population and evolutionary dynamics of Helitron transposable elements in Arabidopsis thaliana

Helitrons, a recently discovered superfamily of DNA transposons that capture host gene fragments, constitute up to 2% of the Arabidopsis thaliana genome. In this study, we identified 565 insertions of a family of nonautonomous Helitrons, known as Basho elements. We aligned subsets of these elements, estimated their phylogenetic relationships, and used branch lengths to yield insight into the age of each Basho insertion. The age distribution suggests that 87% of Bashos inserted within 5 Myr, subsequent to the divergence between A. thaliana and its sister species Arabidopsis lyrata. We screened 278 of these insertions for their presence or absence in a sample of 47 A. thaliana accessions. With both phylogenetic and population frequency data, we investigated the effects of gene density, recombination rate, and element length on Basho persistence. Our analyses suggested that longer Basho copies are less likely to persist in the genome, consistent with selection against the deleterious effects of ectopic recombination between Basho elements. Furthermore, we determined that 39% of Basho elements contain fragments of expressed protein-coding genes, but all of these fragments were explained by only 5 gene-capture events. Overall, the picture of A. thaliana Helitron evolution is one of rapid expansion, relatively few gene-capture events, and weak selection correlated with element length.     

Differential interactions of promoter elements in stress responses of the Arabidopsis Adh gene.

The Adh (alcohol dehydrogenase, EC 1.1.1.1.) gene from Arabidopsis thaliana (L.) Heynh. can be induced by dehydration and cold, as well as by hypoxia. A 1-kb promoter fragment (CADH: -964 to +53) is sufficient to confer the stress induction and tissue-specific developmental expression characteristics of the Adh gene to a beta-glucuronidase reporter gene. Deletion mapping of the 5' end and site-specific mutagenesis identified four regions of the promoter essential for expression under the three stress conditions. Some sequence elements are important for response to all three stress treatments, whereas others are stress specific. The most critical region essential for expression of the Arabidopsis Adh promoter under all three environmental stresses (region IV: -172 to -141) contains sequences homologous to the GT motif (-160 to -152) and the GC motif (-147 to -144) of the maize Adh1 anaerobic responsive element. Region III (-235 to -172) contains two regions shown by R.J. Ferl and B.H. Laughner ([1989] Plant Mol Biol 12: 357-366) to bind regulatory proteins; mutation of the G-box-1 region (5'-CCACGTGG-3', -216 to -209) does not affect expression under uninduced or hypoxic conditions, but significantly reduces induction by cold stress and, to a lesser extent, by dehydration stress. Mutation of the other G-box-like sequence (G-box-2: 5'-CCAAGTGG-3', -193 to -182) does not change hypoxic response and affects cold and dehydration stress only slightly. G-box-2 mutations also promote high levels of expression under uninduced conditions. Deletion of region I (-964 to -510) results in increased expression under uninduced and all stress conditions, suggesting that this region contains a repressor binding site. Region II (-510 to -384) contains a positive regulatory element and is necessary for high expression levels under all treatments.     

Identification of pregnane-X receptor target genes and coactivator and corepressor binding to promoter elements in human hepatocytes

Chromatin immunoprecipitation (ChIP) studies were conducted in human hepatocytes treated with rifampicin in order to identify new pregnane-X receptor (PXR) target genes. Genes, both previously known to be involved and not known to be involved in drug disposition, with PXR response elements (PXREs) located upstream, within or downstream from their potentially associated genes, were identified. Validation experiments identified several new drug disposition genes with PXR binding sites. Of these, only CYP4F12 demonstrated increased binding in the presence of rifampicin. The role of PXR in the basal and inductive response of CYP4F12 was confirmed in hepatocytes in which PXR was silenced. We also assessed the association of PXR-coactivators and -corepressors with known and newly identified PXREs. Both PXR and the steroid receptor coactivator (SRC-1) were found to bind to PXREs in the absence of rifampicin, although binding was stronger after rifampicin treatment. We observed promoter-dependent patterns with respect to the binding of various coactivators and corepressors involved in the regulation of CYP4F12, CYP3A4, CYP2B6, UGT1A1 and P-glycoprotein. In conclusion, our findings indicate that PXR is involved in the regulation of CYP4F12 and that PXR along with SRC1 binds to a broad range of promoters but that many of these are not inducible by rifampicin.     

Late-flowering genes interact with early-flowering genes to regulate flowering time in Arabidopsis thaliana.

To investigate the genetic mechanisms regulating the transition from the vegetative to reproductive growth in Arabidopsis, double mutants between three different early-flowering mutants, early flowering 1-1, 2-1, 3-1, (elf 1-1, 2-1, 3-1) and five different late-flowering mutants, gi-1, ft-1, fwa-1, ld-1, and fca-9, were constructed and phenotypes analyzed. Double mutants in all combinations displayed the late-flowering phenotypes which resembled their respective late-flowering parents in both flowering time and the number of vegetative leaves produced. The results indicate that five late-flowering mutants are epistatic to all three early-flowering mutants tested here. This epistatic relationship suggests that ELF1, ELF2, and ELF3 genes function upstream of these five late-flowering genes no matter if they are functioning in autonomous or photoperiod pathways. These three early-flowering genes may negatively modify the activity of most late-flowering genes to influence the time of the vegetative-to-reproductive transition in Arabidopsis.