Biochemical analysis of the association between histone variants and histone marks.

(A) Histone H3.1 and H3.3 form homotypic and heterotypic nucleosomes. Spectral counts of H3.1– and H3.3-specific peptides in the respective immunoprecipitations (T – transgenic, E – endogenous H3.1 and H3.3). (B) H2A variants do not preferentially associate with H3.1– or H3.3– containing nucleosomes. HA-tagged H3.1 and H3.3 mononucleosomes were immunoprecipitated with HA agarose and analyzed for the presence of H2A variants by immunoblotting. (C) Histone H3 marks are present on both H3.1 and H3.3. HA-tagged H3.1 and H3.3 mononucleosomes were immunoprecipitated with HA agarose and analyzed for the presence of H3 marks by immunoblotting. Arrows indicate transgenic (T) and endogenous (E) H3. (D) MS analysis of cumulative H3K27, H3K36, and H3K37 modifications on H3.1 and H3.3. All measured spectra corresponding to H3.1 and H3.3 peptides from both IPs were used for analysis. (E) Relative abundance of H3K27, H3K36, and H3K37 modifications on H3.1 variant analyzed separately from MS data of H3.1 and H3.3 purified nucleosomes (left panel). Relative abundance of H3K27, H3K36, and H3K37 modifications on H3.3 variant analyzed separately from MS data of H3.1 and H3.3 purified nucleosomes (right panel). (F) Co-occurrence of H2A variants and H3 marks. Mononucleosomes were immunoprecipitated with the indicated antibodies and analyzed for the presence of H2A variants and H3 marks by western blotting.

Histone variants define chromatin states in Arabidopsis thaliana. (A) Bubble plot showing the emission probabilities for histone modifications/variants across the 26 chromatin states. (The size of the bubble represents the emission probability ranging from 0 to 1). The colors are ascribed for each type of chromatin. (B) Stacked bar plot showing the overlap between annotated genomic features and chromatin states. (C) Box plot showing the expression of protein-coding genes overlapping with each chromatin state in Transcripts per Million (TPM). (D) Box plot showing levels of CG methylation across chromatin states. (E) Box plot comparing DNase I-seq read coverage across chromatin states representing chromatin accessibility. (F) Heatmap showing the Jaccard similarity index between the states generated using the whole model and states using a subset of marks, i.e. excluding a set of marks and variants as indicated on the X-axis. The comparison with 9-state model (Sequeira-Mendes et al., 2014) did not include CG content, DNA methylation, H4K5ac and H3K4me2 which were not used in the 26-state model.

DDM1 loss of function disrupts chromatin states in Arabidopsis thaliana. (A) Heatmap showing the emission probability for each mark/variant across the 16 chromatin states of the concatenated wild type and ddm1 mutant model. The bar plot on the left represents the proportion of the genome covered by each state in wild type (green) and in ddm1 (red). (B) Bar plot showing the Jaccard indices between the state assignments in wild type and ddm1 mutant. (C) Bar plot showing the state assignment overlap between the wild type and ddm1 for each chromatin state. The red vertical line represents the genome wide overlap (62.2%). (D) Bar plot showing the log2 fold changes of proportion of genome covered by each state across the ddm1 genome compared to wild type. (E) Stacked bar plot showing the overlap between annotated genomic features and chromatin states from the concatenated model in wild type. (F) Stacked bar plot showing the overlap between annotated genomic features and chromatin states from the concatenated model in ddm1 mutant. (G) DDM1 interaction with H2A.W and H2A.Z. Coomassie stained 15% SDS-PAGE gel showing input protein samples (top panel) used for in vitro pull-down (bottom panel) with His6-tagged DDM1 and histone dimers. The lane ΔCTH2A.W shows that the deletion of the C-terminal tail of H2A.W does not influence binding to DDM1. (H) Summary of the pull-down assays to identify regions in DDM1 binding to H2A.W and H2A.Z. Blue and purple boxes indicate the H2A.W binding regions in DDM1 identified by previous work (Osakabe et al., 2021). Original gel pictures are shown in Figure 3 figure supplement 2A-C.

Impact of expression on chromatin states over TE genes in ddm1. (A) Enrichment profiles of H2A.W.6 and H2A.Z.9 over TE genes in ddm1. TE genes were grouped by expression in ddm1 mutant. Out of the 3901 TE genes in the Arabidopsis genome annotation, 497 were excluded because they showed expression in wild type, 2116 TE genes showed no expression in ddm1 (non-expressed TE genes) while 1288 TE genes were expressed. Because many of these TE genes showed very low expression levels, we divided the expressed TEs into 4 quartiles (322 TE genes each) based on their expression values where the 1st quartile contains TE genes with lowest expression and the 4th quartile contains TE genes with highest expression. Given that the TE genes in 1st and 2nd quartile showed nominal expression values, we placed only TE genes in 3rd and 4th quartile (644 TE genes) in the category of expressed TEs. n represents the number of TE genes in each group. (B) Stacked bar plots of the proportion of states in wild type (top panel) and in ddm1 (bottom panel) overlapping TE genes grouped by expression in ddm1. (C) Box plot showing the expression of TE genes overlapping the 16 concatenated model states in ddm1.