Cryo-EM structures of methylated and unmethylated Sat2R-P human H2A.Z nucleosomes.

(A) Diagram of the palindromic 152 bp Sat2R-P sequence. CpGs are underlined. Triangle denotes midpoint. (B and C) Atomic models of unmethylated (B) and methylated (C) Sat2R-P H2A.Z structures (the v1 model for both). Two face views (DF1 and DF2) and a side view (middle) are shown. (D) Left, overlay of unmethylated (UM) and methylated (Me) Sat2R-P DNA EM densities. Middle and right, overlays of DNA densities on top of the corresponding v1 DNA atomic models for either the unmethylated (middle) or methylated (right) structures. Arrows point to the visualizable end of the DF2 linker DNA for each structure. (E) RMSD analysis comparing differences between unmethylated and methylated v1 Sat2R-P H2A.Z atomic models.

DNA methylation reduces H3-DNA contacts, alters H4 N-terminal tail orientations, and forms open nucleosome structures.

(A) Density maps of unmethylated (UM) and methylated (Me) Sat2R-P H2A.Z nucleosomes filtered and resampled to ∼3Å. The face shown, and all following zoom-ins, is of DF2. Maps are overlaid onto each other. Boxed regions highlight major areas of difference shown in B-D. (B) Zoom-in of the H3 N-terminus on DF2 of both structures. Density maps are shown as a transparent overlay on top of the corresponding v1 atomic models. (C) Zoom-in of the DF2 acidic patch on both structures. Area corresponding to the extra density found in the Me structure labeled in red. (D) Zoom-in of the H4 N-terminal tail. Dotted lines show each structure’s preferred orientation (E) 3D Models used in the in silico mixing 3D classification analysis. Classes are as follows: (1 and 2) closed nucleosome structures with varying linker lengths; (3 and 6) open nucleosomes with highly flexible linkers; (4) open nucleosomes that have shifted position from center; (5) hexasomes. Both distal faces (DF1 and DF2) are shown of each model. (F) Results of classification analysis. Y-axis is the proportion of particles sorted into each class as a percent of the total input particles from each sample (UM or Me). Error bars represent SEM, n = 3 technical replicates.

DNA methylation increases H2A.Z nucleosome accessibility.

(A) Diagram of the 152 bp 1HinfI_Sat2R DNA sequence used. CpG sites underlined and sequence midpoint indicated by the triangle. HinfI recognition site highlighted in magenta. A Cy5 fluorophore is attached to the 5’ end nearest the HinfI site. (B) Schematic of 1HinfI_Sat2R H2A.Z nucleosome with HinfI recognition site demarcated in magenta and H2A.Z in yellow. (C) Native gel showing HinfI digestion time course of UM and Me human H2A and H2A.Z nucleosomes. SYBR Safe was used to stain total DNA. Digestion efficiency was assessed via loss of Cy5 signal and a downward shift in the total DNA band. (D) Quantification of (C). Cy5 signals were normalized to total SYBR Safe signal for each sample. Error bars represent SEM. Each shape represents data from one independent experiment, n = 5 experiments. Statistics comparing unmethylated and methylated samples at indicated timepoints were completed with a two-tailed t-test assuming unequal variance with p-values listed above each comparison.

Presence or absence of DNA methylation influences H2A.Z deposition.

(A) Percentage of methylated CpG sites (≥ 5x coverage) associated with H2A.Z peaks or H3 reads in sperm pronuclei incubated in interphase egg extract or XTC-2 nuclei following CnT-BS library preparation. H3 data is taken from regions outside of H2A.Z enriched peaks. Data points represent biological replicates (n = 2). (B) Schematic of chromatinization assay. Magnetic beads coated with DNA of interest were incubated in Xenopus egg extract with the addition of CaCl2 to induce cycling into interphase. After 60 min, DNA beads were isolated to assess for histone composition. (C and E) Western blot results of chromatinization assay probing for either H2A.Z (C) or H2A.X-F (E). Ku70 and H4 signals are shown as loading controls. Beads coated with unmethylated (UM) or methylated (Me) 19-mer arrays of Widom 601 (19×601) or 16-mer arrays of HSat2 (16xHSat2) were used. A representative blot of technical triplicates for each condition is presented. (D and F) Quantification of (C and E). H2A.Z and H2A.X-F signals were normalized to H4 intensity. Error bars represent SEM. Data points represent technical triplicates across 3 biological replicates with each shape representing data from a single independent experiment (n = 9).

The SRCAP complex mediates H2A.Z’s preferential association with unmethylated DNA.

(A) Western blot to detect SRCAP in control IgG-depleted Xenopus egg extract (ΔIgG) or SRCAP-depleted extract (ΔSRCAP). Bottom panel shows ponceau staining as a loading control. (B) Western blots of chromatinization assay in IgG control vs SRCAP depleted egg extract. Magnetic beads coated with 19-mer arrays of Widom 601 (19×601) or 16-mer arrays of HSat2 (16xHSat2) were incubated with interphase egg extract for 60 min, before their isolation. (C) Quantification of H2A.Z signal normalized to H4 from (B). (D) Quantification of H2A.X-F signal normalized to H4 from (B). Error bars in (C) and (D) represent SEM from n = 3 biological replicates and each shape represents data from one independent experiment. (E) Western blot staining for SRCAP on 16xHSat2 DNA beads incubated in interphase egg extract. (F) Western blot staining for ZNHIT1 on specified DNA beads incubated in interphase egg extract. Representative image shown from two independent experiments.

Schematic models for how DNA methylation influences physical nucleosome structure and SRCAP-mediated H2A.Z loading.

(A) DNA methylation has little effect on nucleosome openness/accessibility of H2A nucleosomes. H2A.Z nucleosomes are more open/accessible than H2A nucleosomes, and DNA methylation DNA slightly opens H2A.Z nucleosome further causing nucleosomal DNA ends to be more accessible. (B) SRCAP-C is capable of binding to unmethylated DNA and replacing H2A with H2A.Z on the nucleosome. SRCAP-C cannot bind to methylated DNA, and thus H2A.Z deposition is suppressed on methylated DNA. An SRCAP-independent mechanism (possibly via TIP60) deposits H2A.Z in a DNA methylation insensitive manner.

DNA sequences/oligos used in the study.

Antibodies used in this study.