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. 2020 May 28;16(5):e1008681.
doi: 10.1371/journal.pgen.1008681. eCollection 2020 May.

The domesticated transposase ALP2 mediates formation of a novel Polycomb protein complex by direct interaction with MSI1, a core subunit of Polycomb Repressive Complex 2 (PRC2)

Christos N Velanis  1 , Pumi Perera  1 , Bennett Thomson  2 , Erica de Leau  1 , Shih Chieh Liang  1 , Ben Hartwig  3 , Alexander Förderer  3 , Harry Thornton  1 , Pedro Arede  1 , Jiawen Chen  1 , Kimberly M Webb  4 , Serin Gümüs  5 , Geert De Jaeger  6   7 , Clinton A Page  8 , C Nathan Hancock  8 , Christos Spanos  4 , Juri Rappsilber  4   9 , Philipp Voigt  4 , Franziska Turck  3 , Frank Wellmer  2 , Justin Goodrich  1
Affiliations

The domesticated transposase ALP2 mediates formation of a novel Polycomb protein complex by direct interaction with MSI1, a core subunit of Polycomb Repressive Complex 2 (PRC2)

Christos N Velanis et al. PLoS Genet. .

Abstract

A large fraction of plant genomes is composed of transposable elements (TE), which provide a potential source of novel genes through "domestication"-the process whereby the proteins encoded by TE diverge in sequence, lose their ability to catalyse transposition and instead acquire novel functions for their hosts. In Arabidopsis, ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) arose by domestication of the nuclease component of Harbinger class TE and acquired a new function as a component of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a histone H3K27me3 methyltransferase involved in regulation of host genes and in some cases TE. It was not clear how ALP1 associated with PRC2, nor what the functional consequence was. Here, we identify ALP2 genetically as a suppressor of Polycomb-group (PcG) mutant phenotypes and show that it arose from the second, DNA binding component of Harbinger transposases. Molecular analysis of PcG compromised backgrounds reveals that ALP genes oppose silencing and H3K27me3 deposition at key PcG target genes. Proteomic analysis reveals that ALP1 and ALP2 are components of a variant PRC2 complex that contains the four core components but lacks plant-specific accessory components such as the H3K27me3 reader LIKE HETEROCHROMATION PROTEIN 1 (LHP1). We show that the N-terminus of ALP2 interacts directly with ALP1, whereas the C-terminus of ALP2 interacts with MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a core component of PRC2. Proteomic analysis reveals that in alp2 mutant backgrounds ALP1 protein no longer associates with PRC2, consistent with a role for ALP2 in recruitment of ALP1. We suggest that the propensity of Harbinger TE to insert in gene-rich regions of the genome, together with the modular two component nature of their transposases, has predisposed them for domestication and incorporation into chromatin modifying complexes.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. ALP2 acts in the same pathway as ALP1.
A. Rosette phenotypes of 9 week old plants grown in short days. B. Box plots showing median and inner quartiles of flowering time data (number of rosette leaves) of plants grown in long days. Whiskers delimit largest value within 1.5 times of inner quartiles. Statistical analysis was performed with analysis of variance (ANOVA) and Holm-Sidak multiple-testing correction. Different letters indicate distinct groups (two-tailed P values <0.05) C. Suppression of clf phenotype by alp2-1. D. alp1-1 and alp2-1 single mutants flower later than wild-type (Col-0) in long days but do not show gross phenotypic abnormalities. White arrow indicates inflorescence in Col-0, plants are four weeks old. E. alp2-1 and alp1-1 plant grown in short days show altered rosette morphology. F. Complementation of the alp2 mutant phenotype by an At5g24500 transgene. G. Complementation of the alp2 mutant phenotype by a 35S::GFP-ALP2 transgene.
Fig 2
Fig 2. Expression of PcG targets and ALP genes.
A. Real time RT PCR analysis of AG, SEP3, and FT expression in 12 day old seedlings grown in short days. Expression for each gene was normalised to the reference gene EiF4A1. Values are shown relative to the level in wild type (Col-0) seedlings (set to 1) and mean and standard deviation of three biological replicates. Asterisks indicate lhp alp sample means significantly different from lhp1-1 (** p < .01, * p < .05, Tukey’s pairwise comparisons). B. Expression of ALP1, ALP2 and CLF in different plant organs normalised to EiF4A1. Expression is shown relative to levels in roots, set to 1, and shows mean and standard deviation of two biological replicates.
Fig 3
Fig 3. ALP2 has similarity with Harbinger transposase DNA binding proteins.
Alignment using MAFTT of protein sequences of selected land plant ALP2 proteins (upper 8 sequences), Harbinger transposase DNA binding proteins and the domesticated transposase HDP2 (lower 9 sequences). The ALP2 sequences include a gymnosperm (Metasequioa glyptostroboides) and a leptosporangiate fern (Pilularia globifera). Full sequences and accession numbers are provided in S1 Text. Alignment of ALP2 proteins alone identified three regions of sequence conservation (I–III) indicated by the red lines above the alignments, these overlapped with three regions (A, B and C) of conservation between plant Harbinger DNA binding proteins (PONG class) previously identified [13] and indicated by green lines below the sequences. The three tryptophan residues that are highly conserved amongst Harbinger DNA binding proteins and HDP2 (but not in ALP2 proteins) are outlined with red boxes.
Fig 4
Fig 4. Volcano plot analysis of IP-MS data.
The relative abundance of proteins compared between two samples (log2 of fold difference, x axis) is plotted against statistical significance (-log10 of P-value, y axis) with each point representing a protein identified by 2 or more unique peptides. The curved lines on right hand side of plots delimit outliers with high relative abundance and significance in bait sample. PRC2 components are highlighted in blue, ALPs in red, for the full list of the most highly enriched proteins see S3 Dataset. A. Comparison of 35S::GFP-ALP2 and 35S:GFP IPs shows that ALP1 and PRC2 core components are highly enriched. B. Comparison of pALP1::ALP1-GFP with 35S::GFP IPs shows that ALP2 and PRC2 core components are highly enriched. C. Comparison of pALP1::ALP1-GFP IPs in ALP2+ and alp2-1 backgrounds shows that PRC2 core components are highly enriched in ALP2+ samples and depleted in alp2-1.
Fig 5
Fig 5. Interaction of ALP proteins in yeast two hybrid assays.
A. ALP1 and ALP2 protein interaction is abolished by the alp1-1 (G273E) missense mutation. B. A C-terminal region of ALP2 containing a conserved region is sufficient for the interaction with ALP1. C. The N-terminus of ALP2 interacts with MSI1. Although the N-ALP2 bait alone shows weak autoactivation ability (growth on -LWH), in the presence of MSI prey the more stringent ADE reporter is activated, allowing growth on -LWHA medium. In panel A, three independent transformants are shown, in B and C serial ten-fold dilutions of five pooled transformants.
Fig 6
Fig 6. Interactions of ALP proteins in BiFC assay.
A. ALP2 interacts with ALP1 but not with ALP1-1. B. ALP2 interacts with MSI1 but not with LHP1. C. ALP1 does not interact with MSI1 unless ALP2 is co-expressed. N. benthamiana leaves were transformed by infiltration with Agrobacterium and the epidermis viewed using confocal microscope. Images on the left show the YFP channel, and on the right a merged light field (showing cell outlines) and fluorescence channels. Chloroplasts present in stomata or underlying cell layers are autofluorescent and appear red. Low magnification images, showing larger numbers of cells, and further controls are shown in S5 Fig.
Fig 7
Fig 7. Pull down analysis of ALP-PRC2 protein interactions.
The ALP1 or ALP2 proteins were co-expressed in insect cell as fusions with streptavidin binding peptides (ALP1-S, S-ALP2) together with MSI1 tagged with 6 X His (H-MSI1). The Strep-tagged ALP1 or ALP2 proteins were pulled down from whole cell extracts using Strepavidin beads and the eluted proteins analysed in immunoblots using antisera directed against the Strep and 6 X His tags, respectively. H-MSI1 was pulled down with ALP2 but not with ALP1.
Fig 8
Fig 8. ChIP analysis of H3K27me3 enrichment at loci known to be affected by PRC2-CLF activity.
A. Schematics of genomic regions examined during ChIP experiments. Approximate regions amplified via real-time PCR are denoted by red bars. Blue bars: untranslated regions; black bars: exons; black lines: introns; arrow heads: direction of transcription. B. Immunoprecipitation of chromatin prepared from 10-day-old Col-0, alp1-1, alp2-1 and clf-28 seedlings. An H3K27me3 antibody was used to precipitate chromatin, and enrichments are displayed as percentage input. C. Immunoprecipitation of chromatin from clf-28, alp1-1 clf-28 and alp2-1 clf-28 10 day old seedlings. Three biological replicates were used for each genotype and error bars indicate the standard deviation from the mean. Primers that amplify regions in ACT7 (AT5G09810) and TUB2 (AT5G62690) were included as negative controls. In: intron; TSS: transcriptional start site.
Fig 9
Fig 9. Model for evolution of ALP-PRC2.
The ancestral transposases accumulated mutations that disabled their ability to catalyse transposition but maintained their interaction. The interaction of ALP2 with MSI1 displaced EMF1 and LHP1, which also interact with MSI1, leading to mutually exclusive PRC2 subcomplexes.
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