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 immunoprecipitates (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 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 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 H3.1 and H3.3 K27 and K36 modifications in H3.1 and H3.3 immunoprecipitates (left panel). Middle and right panels represent H3.1 and H3.3 K27 and K36 modifications, respectively, as analyzed separately in H3.1 and H3.3 immunoprecipi-tates. (E) 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 immunoblotting.

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) (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. The X-axis from left to right (full dataset, no H2B variants, no H3 variants, no H2A variants, no histone modifications, and no histone variants (H2A/H3/H2B)).

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. 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 dotted 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. (G) Summary of the pull-down assay to identify binding regions in DDM1 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 supplements 3G and 3H.

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. (The groups are: no expression and expression divided into four quartiles where the 4th is the group with the highest expression.) Expressed TEs belong to the 3rd and 4th quartiles. n represents the number of TE genes in each group. (B-C) Stacked bar plots of the proportion of states in wild type (B) and in ddm1 (C) overlapping TE genes grouped by expression in ddm1. (D) Box plot showing the expression of TE genes overlapping the 16 concatenated model states in ddm1.