Abstract
Epigenetic gene silencing induced by expanded repeats can cause diverse phenotypes ranging from severe growth defects in plants to genetic diseases such as Friedreich’s ataxia in humans. The molecular mechanisms underlying repeat expansion-induced epigenetic silencing remain largely unknown. Using a plant model with a temperature-sensitive phenotype, we have previously shown that expanded repeats can induce small RNAs, which in turn can lead to epigenetic silencing through the RNA-dependent DNA methylation pathway. Here, using a genetic suppressor screen and yeast two-hybrid assays, we identified novel components required for epigenetic silencing caused by expanded repeats. We show that FOURTH ULP GENE CLASS 1 (FUG1)—an uncharacterized SUMO protease with no known role in gene silencing—is required for epigenetic silencing caused by expanded repeats. In addition, we demonstrate that FUG1 physically interacts with ALFIN-LIKE 3 (AL3)—a histone reader that is known to bind to active histone mark H3K4me2/3. Loss of function of AL3 abolishes epigenetic silencing caused by expanded repeats. AL3 physically interacts with the chromodomain protein LIKE HETEROCHROMATIN 1 (LHP1)—known to be associated with the spread of the repressive histone mark H3K27me3 to cause repeat expansion-induced epigenetic silencing. Loss of any of these components suppresses repeat expansion-associated phenotypes coupled with an increase in IIL1 expression with the reversal of gene silencing and associated change in epigenetic marks. Our findings suggest that the FUG1–AL3–LHP1 module is essential to confer repeat expansion-associated epigenetic silencing and highlight the importance of post-translational modifiers and histone readers in epigenetic silencing.
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Data availability
All sequencing data are available at the National Center for Biotechnology Information short reads archive. The ChIP–seq reads are available in Bioproject ID PRJNA1080228, the small RNA sequence from fug1 is in Bioproject ID PRJNA1080246 and the RNA-seq data are in Bioproject ID PRJNA1080085. Source data are provided with this paper.
Code availability
The python script that we used to extract allele frequencies is available upon request to the corresponding authors.
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Acknowledgements
We thank R. Clark (Utah, USA) for help with sequencing the fug1 mutant, A. Seleznev for help with bioinformatic analysis and H. Sako for initial characterization of suppressors. We thank J. Bowman, J. Coutts, Y. Guo, D. Smyth and L. Wong for discussions and comments on this manuscript. This work is supported by a Canadian Institute for Health Research grant PJT-178112 (E.R.), Department of Science and Technology – Science and Engineering Research Board Core grant (R.K.Y.), a National Health and Medical Research Council project grant APP1182090 (S.B. and S.S.), Australian Research Council Discovery Projects DP1095325 (S.B.) and DP190101818 (S.B.), and Australian Research Council Future Fellowships FT100100377 (S.B.), FT190100403 (S.S.), Australia–India Strategic Research Fund–Early and Mid-Career Fellowship (S.S.) and a Monash Larkins Fellowship (S.B.).
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Conceptualization: S.S. and S.B.; methodology: S.S., H.L., S.A., A.S., E.R. and S.B.; software: C.I.D., S.S. and S.B.; formal analysis: S.S., C.B., J.L., C.I.D., P.K.B., S.M., R.S., C.A., H.M.Y., N.S., G.F., P.T., A.S.Y., B.G.B., S.K., P.S., O.S.G. and S.B.; investigation: S.S., J.L., S.M., R.S., C.A., H.M.Y., N.S. and C.B.; writing—original draft: S.S.; writing—review and editing: S.S., A.S., S.A., H.L. and S.B.; visualization: S.S., S.A., H.M.Y. and S.B.; supervision: S.S., R.K.Y., S.A., A.S., H.L. and S.B.; project administration: S.S. and S.B.; funding acquisition: S.S. and S.B.
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Extended data
Extended Data Fig. 1 Genetic mapping of the suppressors of Bur-0 by SHOREMAP.
Allele frequency of EMS-type SNPs in the fraction of F2 pooled plants that display the suppression of the iil phenotype. a) 49-9 b) 61-7 and c) 57-3. High frequency alleles (>0.85) are coloured red and red crosses show the putative causal alleles.
Extended Data Fig. 2 fug1 mutant suppresses the temperature-dependent iil phenotype despite the presence of repeat expansion.
a) fug1 phenotype compared with Bur-0 at 23 °C and 27 °C. b) Suppression of the iil phenotype in fug1 is not due to the loss of a repeat expansion. Gels showing retention of the expanded repeat in fug1 plants compared with Bur-0 plants. The red boxes display the typical banding pattern of the repeat expansion. 1.5Kb band on the 1Kb plus (Invitrogen) is shown as the molecular marker. Each lane represents DNA analysed from individual plants with the Col-0 control in the first lane after the marker showing a non-expanded repeat. This is analysed multiple times with similar results.
Extended Data Fig. 3 FUG1 is localised to the nucleus.
Representative maximum intensity projections (MIPs) of protoplasts of transgenic Bur-0 leaves harbouring either GFP (left) or GFP-FUG1 (right). Autofluorescence (purple) was separated using fluorescence life time. Scale bar=10 µM. The experiment is replicated more than twice with similar results.
Extended Data Fig. 4 Analysis of AL3 expression in transgenic lines.
a) Knocking down AL3 leads to increase in IIL1 expression. Independent plants representing multiple T2 lines (shown in X-axis) were analysed for IIL1 expression. Data were analysed through ANOVA and the statistical significance was tested with Tukey’s posthoc test. Different labels indicate significant differences (p < 0.05) in gene expression n = 5 in Bur-0, n = 4 in T2 Line 28 and n = 3 in all other T2 lines. b) Comparison of the expression levels of various fusion constructs of GFP-AL3. Primers against the GFP region allowed the measurement of transgenic AL3 levels in different backgrounds. Anlysis by ANOVA revealed that there were no significant differences in gene expression n = 3 in all samples except 35 S::GFP-AL3, where n = 6. Error bars represent standard errors of mean.
Extended Data Fig. 5 Perturbing the SUMOylation site of AL3 affects its sub-cellular localisation.
a) A schematic of the Fluorescence Lifetime Imaging (FLIM) assay using transformed protoplasts. Scale bar = 5 μm b) Representative maximum intensity projections showing the localisation of GFP or GFP-AL3 or AL3 harbouring K178R mutation (GFP-AL3-K178R) in Bur-0 and fug1 mutant backgrounds 2.5 μm. c) Box and whisker plots of nuclear:cytoplasm ratio of GFP (blue, n = 6 in both Bur-0 and fug1) or GFP-AL3 (cyan, n = 16 in Bur-0 and n = 24 in fug1) or GFP-AL3-K178R (orange, n = 8 in Bur-0 and n = 14 in fug1) in Bur-0 and fug1 mutant backgrounds. Box plots show the 50th percentile and the whiskers show the max and minimum with the mean shown by a line in the graph. Each dot represents the quantification from an individual protoplast expressing the corresponding transgene. Statistical comparisons were done with a two sided Student’s t-test. P-values: **<0.01, ***<0.001, ****<0.0001. The first lane of panel b and the left half (Bur) of panel c are also used in the Main Fig. 3(h & i). The protoplast experiments were carried out at least twice with similar results.
Extended Data Fig. 6 XVE::amiR-LHP1 lines show an increase in IIL1 levels upon estradiol induction.
a) Relative IIL1 expression levels. Comparison is made between Bur-0, empty vector controls as well as mock treated plants with that of estradiol treated samples in one-way ANOVA with the samples being separated as controls vs test. b) Comparison of IIL1 expression in plants that show the suppression of the iil phenotype and those that do not analysed together independent of the specific transgenic lines in one-way ANOVA c) Comparison of IIL1 expression in plants that show the suppression of the iil phenotype and those that do not from independent T2 lines. Expression analysis is from 2–3 independent plants as shown by individual data points on each of the bar graphs. The percentage of plants that show the suppression and the number of plants analysed are shown above each of the transgenic lines. The comparison of gene expression in this dataset is by two-sided Student’s T-test. Error bars represent standard errors of mean. p-values: *<0.05, **<0.01, ***<0.001, ****<0.0001.
Extended Data Fig. 7 Abundance of siRNAs that map to IIL1 locus in fug1 mutant.
Small RNAs that are typically found in Bur-0 at 27 °C (Fig. 2 of Eimer et al, Cell, 20184 were not observed in the fug 1 mutant. Small RNA profiles are generated as previously described4 using Small Complementary RnA Mapper (SCRAM)37. The genic region is shown in the bottom with the black boxes and lines representing exons and introns, respectively. Normalised coverage along with standard error shown as shadows is shown for different types of small RNAs as indicated by the colour code. Only small RNAs that mapped to the non-triplet repeat sequences of IIL1 are shown. Quantification of the sense and antisense small RNAs are shown in the positive and negative dimensions, respectively, along the y-axis. The green arrow indicates the position of the GAA/TTC repeat in the intron 3 of IIL1.
Extended Data Fig. 8 Effects of fug1 on gene silencing cannot be explained by changes in the RNA expression levels of the RdDM components.
A volcano plot depicting the changes in the RNA expression levels of genes in the RdDM pathway. Genes that show significant differential expression are marked in red. Differential expression was assessed by DESeq2 and only samples with an adjusted p-value after multiple correction (p-adj) are shown. An example for upregulated (AGO4) and downregulated (AGO9) are highlighted in the figure.
Supplementary information
Supplementary Information
Supplementary Tables 1–4.
Source data
Source Data Fig. 2
Unprocessed actin blot of the lower panel of Fig. 2c.
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Sureshkumar, S., Bandaranayake, C., Lv, J. et al. SUMO protease FUG1, histone reader AL3 and chromodomain protein LHP1 are integral to repeat expansion-induced gene silencing in Arabidopsis thaliana. Nat. Plants (2024). https://doi.org/10.1038/s41477-024-01672-5
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DOI: https://doi.org/10.1038/s41477-024-01672-5
