Impacts of DNA methylation on H2A.Z deposition and nucleosome stability
- Laboratory of Chromosome and Cell Biology, The Rockefeller University, United States
- Basic Sciences Division, Fred Hutchinson Cancer Center, United States
Figures
Figure 1 with 2 supplements
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. 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) Root mean squared distance (RMSD) analysis comparing differences between unmethylated and methylated v1 Sat2R-P H2A.Z atomic models.
Figure 1—figure supplement 1
Workflow of Sat2R-P cryo-EM analysis.
(A) Native PAGE analysis of H2A.Z nucleosomes used for cryo-EM analysis. Bands visualized through SYBR Safe staining. (B) Native PAGE analysis of Sat2R-P DNA after digestion with BstBI to check for methylation status. (C) Diagram of structure analysis pipeline for either unmethylated (left) or methylated (right) H2A.Z SAT2R-P samples. Further details described in methods.
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Figure 1—figure supplement 1—source data 1
Labeled original gel images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Raw gel images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig1-figsupp1-data2-v1.zip
Figure 1—figure supplement 2
DNA atomic model generation for Sat2R-P H2A.Z nucleosome structures.
Overlays of electron density maps for the DNA with both generated atomic models for either the unmethylated (A) or methylated (C) Sat2R-P H2A.Z nucleosome structure. Zoom-in of dyad regions showing fitting of both DNA atomic models (v1 and v2) generated for either the unmethylated (B) or methylated (D) structure. Methylated cytosines are highlighted in green. Top panels are overlays of both models for the two structures. Positioning was determined by assessing densities around bases which retain purine/pyrimidine identity in both models vs those that switch. Bases that switched from purine to pyrimidine, and vice versa, showed more ambiguous densities than those that retained identity. Positioning was determined using the unmethylated structure and then used in modeling the methylated structure. (E) Comparison of predicted CpG positioning for the v1 (top) and v2 (bottom) atomic models generated for the methylated structure.
Figure 2 with 4 supplements
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 ~3 Å. 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.
Figure 2—figure supplement 1
Workflow of in silico mixing 3D classification analysis.
Particles from a merged batch of unmethylated and methylated Sat2R-P H2A.Z nucleosome micrographs were picked and fed through the standard analysis pipeline. Six 3D models were then generated representing various open, closed, or hexasome-containing states of the nucleosome. Merged particles were then sorted to each of the classes through CryoSPARC’s heterorefinement tool (n = 3 technical replicates).
Figure 2—figure supplement 2
Local resolution estimates for unmethylated and methylated Sat2R-P H2A.Z structures.
Left, local resolution estimates of DF1 for both unmethylated (top) and methylated (bottom) Sat2R-P density maps. Right, side view showing local resolution of the DNA backbone for both methylated and unmethylated density maps. Local resolution maps were generated in CryoSPARC using the local resolution estimation tool and overlaid on density maps filtered using CryoSPARC’s local filtering tool.
Figure 2—figure supplement 3
Cryo-EM structures of 601L H2A.Z nucleosomes show no DNA methylation-dependent differences.
(A) Diagram of the 205 bp palindromic 601L sequence. Triangle indicates sequence midpoint. CpGs are underlined. Final density maps of unmethylated (B) or methylated (C) H2A.Z nucleosomes. (D, E) Atomic models generated for (B, C). Methylated cytosines shown in green. (F) Root mean squared distance (RMSD) analysis comparing differences between methylated and unmethylated 601L H2A.Z atomic models.
Figure 2—figure supplement 4
Workflow of 601L H2A.Z nucleosome cryo-EM structures.
(A) Native PAGE analysis of 601L H2A.Z nucleosome samples used for cryo-EM. Bands visualized through SYBR Safe staining. (B) Native PAGE analysis of 601L DNA after digestion with HpaII to check for methylation status. (C) Diagram showing analysis pipeline for solving 601L nucleosome structures. Further details described in methods.
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Figure 2—figure supplement 4—source data 1
Labeled original gel images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig2-figsupp4-data1-v1.zip
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Figure 2—figure supplement 4—source data 2
Raw gel images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig2-figsupp4-data2-v1.zip
Figure 3 with 2 supplements
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.
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Figure 3—source data 1
Labeled original gel images of Figure 3C.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig3-data1-v1.zip
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Figure 3—source data 2
Raw gel images of Figure 3C.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig3-data2-v1.zip
Figure 3—figure supplement 1
Native PAGE and mass photometry analysis of nucleosomes used in HinfI digest assays.
(A) Native PAGE analysis of nucleosome samples. Bands visualized through SYBR Safe staining. Mass photometry analysis of methylated or unmethylated H2A (B) or H2A.Z (C) nucleosomes. Peaks around 200–220 kDa represent the fully formed nucleosome population. (D) BstBI digestion of 1HinfI_Sat2R DNA to verify methylation status. (E) HinfI digest time course of bare unmethylated or methylated DNA.
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Figure 3—figure supplement 1—source data 1
Labeled original gel images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Raw gel images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig3-figsupp1-data2-v1.zip
Figure 3—figure supplement 2
Replicate gels of data from Figure 3.
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Figure 3—figure supplement 2—source data 1
Labeled original gel images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig3-figsupp2-data1-v1.zip
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Figure 3—figure supplement 2—source data 2
Raw gel images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig3-figsupp2-data2-v1.zip
Figure 4 with 4 supplements
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. 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 (19x601) or 16-mer arrays of HSat2 (16xHSat2) were used. A representative blot of technical triplicates for each condition is presented. (D, F) Quantification of (C, 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).
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Figure 4—source code 1
CnT-BS analysis code.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig4-code1-v1.zip
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Figure 4—source data 1
Labeled original western blot images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig4-data1-v1.zip
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Figure 4—source data 2
Raw western blot images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig4-data2-v1.zip
Figure 4—figure supplement 1
Reproducibility comparison of CnT-BS samples.
(A) Pearson correlation plot comparing fragment counts mapped to 1000 bp genomic bins across samples. Bins containing zero fragments were first filtered out. Fragment counts were then normalized using DESeq2 to account for sequencing depth differences and underwent a variance stabilizing transformation (VST) before the correlation matrix was generated. (B) Pearson correlation plot comparing CpG methylation across samples. Percent methylation at individual CpG sites was calculated for high coverage CpGs (≥10 reads) and normalized using methylKit. The correlation matrix was then generated using pairwise observations across all eight samples. Both correlation matrices were ordered, and rectangles drawn (n = 4, user-specified), via hierarchical clustering.
Figure 4—figure supplement 2
H2A.Z localizes to hypomethylated transcription start sites (TSSs).
(A) Genomic annotation plots for the top quantile of 1000 bp genomic bins containing the most mapped reads averaged between replicates for each specified condition. Heatmaps of either H3 CpG methylation calls averaged over 500 bp tiles (left-most panel) or H2A.Z reads (right panels) from two biological replicates across a representation of all annotated Xenopus laevis genes from either sperm (B) or XTC-2 (C) samples. Corresponding H3 and H2A.Z data are shown on the same sets of genes sorted in the same order. (D) IGV snapshot of a region on chromosome 1L showcasing preferential H2A.Z deposition at hypomethylated TSSs. Average H3 methylation tracks were generated across 500 bp tiles for visualization purposes on a scale of 0–100% CpG methylation. Gray indicates regions with no methylation data availability. Two biological replicates of H2A.Z and H3 tracks are shown.
Figure 4—figure supplement 3
DNA methylation check and replicates of data from Figure 4.
(A) BstUI digestion of 19x601 DNA substrates to verify methylation status. Both 19x601 and 16xHSat2 DNA were methylated under identical conditions; however, the HSat2 sequence lacks appropriate cut sites for methylation-sensitive restriction enzymes. The 601 substrates were used to determine complete methylation status for each batch. (B) Replicate blots for data shown in Figure 4.
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Figure 4—figure supplement 3—source data 1
Labeled original gel and western blot images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig4-figsupp3-data1-v1.zip
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Figure 4—figure supplement 3—source data 2
Raw gel and western blot images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig4-figsupp3-data2-v1.zip
Figure 4—figure supplement 4
Both H2A.Z variants preferentially associate with unmethylated DNA substrates.
(A) Expression check of exogenously added mRNA expression for ALFA-tagged versions of both H2A.Z.1 and H2A.Z.2. Blots of DNA beads incubated with interphase extract expressing either ALFA-tagged H2A.Z.1 (B) and H2A.Z.2 (C). Presence of the specified H2A.Z variant on DNA beads was determined by blotting against the ALFA-tag with H4 shown as a loading control. (D) Quantification of (B, C). ALFA signals were normalized to H4 signals. Unique shapes denote results from three independent experiments (n = 3 biological replicates). Error bars represent SEM. (E) Additional replicate blots of data from (D).
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Figure 4—figure supplement 4—source data 1
Labeled original western blot images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig4-figsupp4-data1-v1.zip
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Figure 4—figure supplement 4—source data 2
Raw western blot images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig4-figsupp4-data2-v1.zip
Figure 5 with 4 supplements
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 (∆IgG) or SRCAP-depleted extract (∆SRCAP). 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 (19x601) 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.
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Figure 5—source data 1
Labeled original western blot images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig5-data1-v1.zip
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Figure 5—source data 2
Raw western blot images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig5-data2-v1.zip
Figure 5—figure supplement 1
SRCAP depletion efficiency and replicates of data from Figure 5.
(A) Quantification of SRCAP WB signal from egg extract after depletion with anti-IgG or anti-SRCAP antibody beads. Replicates from three independent experiments shown. Error bars represent SEM. (B) Replicate blots of data plotted in (A). (C) Replicate blots of Figure 5B–D. (D) Replicate blots of Figure 5E, F with Ku70 as a loading control.
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Figure 5—figure supplement 1—source data 1
Labeled original western blot images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Raw western blot images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig5-figsupp1-data2-v1.zip
Figure 5—figure supplement 2
Chromatinization assay with SRCAP depletion using a commercial antibody.
Western blots probing for H2A.Z (A) or H2A.X-F (C) signal along with respective loading controls bound to DNA beads incubated in interphase egg extract after SRCAP depletion using a commercial antibody raised against human SRCAP (Kerafast, Cat#: ESL103). (B, D) Quantification of (A, C). Results from three independent experiments plotted. Error bars represent SEM. (E) Replicate western blots.
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Figure 5—figure supplement 2—source data 1
Labeled original western blot images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig5-figsupp2-data1-v1.zip
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Figure 5—figure supplement 2—source data 2
Raw western blot images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig5-figsupp2-data2-v1.zip
Figure 5—figure supplement 3
TIP60-C does not display DNA methylation sensitivity.
Western blots of DNA bead pulldowns from interphase egg extract probing for endogenous p400 (ATPase for Tip60-C) along with respective loading controls.
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Figure 5—figure supplement 3—source data 1
Labeled original western blot image.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig5-figsupp3-data1-v1.zip
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Figure 5—figure supplement 3—source data 2
Raw western blot image.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig5-figsupp3-data2-v1.zip
Figure 5—figure supplement 4
SRCAP-C maintains unmethylated DNA binding bias in the absence of nucleosome loading.
(A) Western blot showing UBN2 depletion efficiency. (B) Western blot of SRCAP on DNA beads incubated in UBN2-depleted extract. Two replicates shown and total protein via ponceau staining used to assess loading. (C) Western blot of DNA beads incubated in IgG control- and UBN2-depleted extract. ZNHIT1, subunit of SRCAP-C, displays preference for unmethylated DNA across conditions. DNA binding proteins Dppa2 and xKID did not show any differences in chromatin accessibility between DNA methylation status. H3 shown to verify decreased nucleosome loading upon UBN2 depletion. Total protein via ponceau staining used to assess loading. One replicate of ∆IgG and two replicates of ∆UBN2 conditions shown.
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Figure 5—figure supplement 4—source data 1
Labeled original western blot images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig5-figsupp4-data1-v1.zip
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Figure 5—figure supplement 4—source data 2
Raw western blot images.
- https://cdn.elifesciences.org/articles/109762/elife-109762-fig5-figsupp4-data2-v1.zip
Figure 6
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.
Author response image 1
Verification of SRCAP depletion using DNA beads.
DNA beads were incubated in interphase-cycled Xenopus egg extract that had been depleted with either our custom SRCAP antibody or an IgG negative control. SRCAP and ZNHIT1 association was then assessed via Western Blot.
Author response image 2
Verification of 16xHSat2 methylation status via ZHX2/3 protein binding.
16xHSat2 DNA beads were incubated in Xenopus egg extract and endogenous ZHX2/3 protein binding assessed via Western Blot with a custom generated antibody that recognizes both ZHX2 and ZHX3.
Tables
Table 1
DNA sequences/oligos used in the study.
| Sequence name | Sequence | Vector | Plasmid name | Insert location | Source | Assay(s) used in | Additional notes |
|---|---|---|---|---|---|---|---|
| Sat2R-P | ATCATCAGATTCCATTCGAATCCATTCGAAAA TGATTACATTCGAATCCATTCGAAGATTCCAT TTGAGCCTGCTCGAGCAGGCTCAAATGGAA TCTTCGAATGGATTCGAATGTAATCATTTTCG AATGGATTCGAATGGAATCTGATGAT | pUC18 | RMSp81 | Between XbaI and BamHI sites | Generated and purchased from Integrated DNA Technologies (IDT) | Cryo-EM | Modified from the Sat2R sequence in Osakabe et al., 2015. |
| 601L | ATCACAATCCCGGTGCCGAGGCCGCTCAAT TGGTCGTAGACAGCTCTAGCACCGCTTAAA CGCACGTACGGAATCCGTACGTGCGTTTAA GCGGTGCTAGAGCTGTCTACGACCAATTGA GCGGCCTCGGCACCGGGATTGTGAT | pUC57 | 601L-145-8X | EcoRV | (601L-145-8X) was a gift from Curt Davey (Addgene plasmid # 158572; http://n2t.net/addgene:158572; RRID:Addgene_158572) | Cryo-EM | |
| 1HinfI_Sat2R | ATCATCGTTCCATTCGTGACTCCATTCGAAA ATGATTACATTCGTATCCATTCGAAGTTTCCA TTTGAGCCTGTTCGAAAATTCCATTTGTGTCC AACCAATGTTTCCATTCATTTCCATTCAATGTT TCCATTCGTATCCATTTGGATGAT | pUC18 | RMSp161 | Between XbaI and BamHI sites | Generated and purchased from Integrated DNA Technologies (IDT) | Restriction Digest Assay | DNA generated via PCR amplification using the following primers: /5Cy5/ATCATCGTTCCATTCGTGAC; ATCATCCAAATGGATACGAATGG |
| HSat2 arrays (1 repeat shown) | CGTTTGATTCCATTTGATGTTGATTCCATTCGA GTCCATTCGATGATAATTCCATTCGATTCTTT GCGATGATTCCATTCCTTTCCATTTGAGATGA TTCCATTCGAGACCATTCGATGATTGCATTCA ATTCATTCGATGACGATTATTCAATTCCGTTC AATGATTCCATTCGATTCCAATTGATGATGATT CCATTCGATTCCATTTGATGATGATTCCATGC GATTCCATTCGATGATGACTCCGATATCGGA TCTGATATC | pUC18 | RMSp64 | Between XbaI and BamHI sites | Generated and purchased from Integrated DNA Technologies (IDT) | Chromatinization Assays | GenBank: X06199.1 |
| 601 arrays (1 repeat shown) | CTGGAGAATCCCGGTGCCGAGGCCGCTCA ATTGGTCGTAGCAAGCTCTAGCACCGCTTAA ACGCACGTACGCGCTGTCCCCCGCGTTTTA ACCGCCAAGGGGATTACTCCCTAGTCTCCA GGCACGTGTCAGATATATACATCCTGTGCAT GTATTGAACAGCGACTCGGGTTATGTGATGG ACCCTATACGCGGCCGCC | pUC18 | pAS696 | EcoRI/XbaI | Gift from Aaron Straight | Chromatinization Assays | |
| Oligos for sequencing library preparation | |||||||
| Sequence name | Sequence | Source | Assay(s) used in | Additional notes | |||
| Tn5mC-Apt1 | T/iMe-dC/GT/iMe-dC/GG/iMe-dC/AG/iMe-dC/GT/iMe-dC/AGATGTGTATAAGAGA/iMe-dC/AG | Li et al., 2021; generated and purchased from Integrated DNA Technologies (IDT) | CnT-BS adapter oligo | Annealed with Tn5mC1.1-A1block to form adapter oligo | |||
| Tn5mC1.1-A1block | /5Phos/CTGTCTCTTATACA/3ddC/ | Li et al., 2021; generated and purchased from Integrated DNA Technologies (IDT) | CnT-BS adapter oligo | Annealed with Tn5mC-Apt1 to form adapter oligo | |||
| Tn5mC-ReplO1 | /5Phos//iMe-dC/TGT/iMe-dC/T/iMe-dC/TTATA/iMe-dC/A/iMe-dC/AT/iMe-dC/T/iMe-dC//iMe-dC/GAG/iMe-dC//iMe-dC//iMe-dC/A/iMe-dC/GAGA/iMe-dC//3InvdT/ | Li et al., 2021; generated and purchased from Integrated DNA Technologies (IDT) | CnT-BS | Used during oligonucleotide replacement step | |||
Table 2
Antibodies used in this study.
| Antigen | Host | Company | Catalog No. | Peptide used for generation | Dilution | Assay(s) used in |
|---|---|---|---|---|---|---|
| Xl H2A.Z | Rabbit | Custom | CIHKSLIGKKGQQKTV | 2 µg/ml | Western blots | |
| H2A.Z | Rabbit | Active Motif | Cat# 39013 | 1:10 | CnT-BS | |
| H3 | Mouse | Cosmo Bio USA | MABI0001-100-EX | 1:100 | CnT-BS | |
| H4 | Mouse | Active Motif | Cat# 61521 | 1:1000 | Western blots | |
| Xl H2A.X-F | Rabbit | Custom, gift from David Shechter | Shechter et al., 2009 | 1:1000 (of serum) | Western blots | |
| Xl Ku70 | Rabbit | Custom | Postow et al., 2008 | 2 µg/ml | Western blots | |
| Xl SRCAP | Rabbit | Custom | CPNSPSSHRVRKAKT | 0.8 µg per 1 µl of beads (depletions); 12 µg/ml (WB) | Depletions; western blots | |
| hsSRCAP | Rabbit | Kerafast | ESL103 | 0.8 µg per 1 µl of beads (depletions); 1:500 (WB) | Depletions; western blots | |
| ZNHIT1 | Rabbit | Proteintech | 16595-1-AP | 1:1000 | Western blots | |
| P400 | Rabbit | Bethyl | A300-541A | 1:1000 | Western blots | |
| ALFA tag | Alpaca (single domain antibody purified from E. coli) | NanoTag Biotechnologies | N1502-Li800-L | 1:1000 | Western blots | |
| Xl UBN2 | Rabbit | Custom | CGGQNKGDAKLPRKNR | 1.5 µg per 1 µl of beads (depletions); 2 µg/ml (WB) | Depletions; western blots | |
| xKID | Rabbit | Custom | Funabiki and Murray, 2000 | 1 µg/ml | Western blots | |
| Xl Dppa2 | Rabbit | Custom | Xue et al., 2013 | 5 µg/ml | Western blots | |
| Secondaries | ||||||
| IRDye 800CW anti-Rabbit IgG | Goat | LICORbio | 926-32211 | 1:10,000 | Western blots | |
| IRDye 680RD anti-Mouse IgG | Goat | LICORbio | 926-68070 | 1:10,000 | Western blots | |
| anti-Rabbit IgG | Guinea Pig | https://www.antibodies-online.com/ | ABIN101961 | 1:100 | CnT-BS | |
| anti-Mouse IgG | Rabbit | abcam | ab46540 | 1:100 | CnT-BS | |
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Transfected construct (X. laevis) | H2A.Z.1.l | GenBank | BC044011.1 | |
| Transfected construct (X. laevis) | H2A.Z.2.s | NCBI | NM_001092643.1 | |
| Strain, strain background (X. laevis, male) | XLa.J-StrainNXR | National Xenopus Resource | RRID:NXR_0024 | |
| Strain, strain background (X. laevis, male and female) | Xenopus laevis | Xenopus 1 | ||
| Cell line (X. laevis) | XTC-2 (Xenopus laevis fibroblast line) | Gift from David Shechter | RRID:CVCL_5610 | |
| Antibody | Anti-Xenopus laevis H2A.Z (Rabbit polyclonal) | This paper | Used at 2 µg/ml. Generated using the following peptide sequence: CIHKSLIGKKGQQKTV | |
| Antibody | Anti-H2A.Z (Rabbit polyclonal) | Active Motif | Cat# 39013; RRID:AB_2615081 | 1:10 |
| Antibody | Anti-H3 (Mouse monoclonal) | Cosmo Bio USA | MABI0001-100-EX; RRID:AB_10709243 | 1:100 |
| Antibody | Anti-H4 (Mouse monoclonal) | Active Motif | Cat# 61521; RRID:AB_2793667 | 1:1000 |
| Antibody | Anti-Xenopus laevis H2A.X-F (Rabbit polyclonal) | Gift from David Shechter (Shechter et al., 2009) | 1:1000 (of serum) | |
| Antibody | Anti-Xenopus laevis Ku70 (Rabbit polyclonal) | Postow et al., 2008 | 2 µg/ml | |
| Antibody | Anti-Xenopus laevis SRCAP (Rabbit polyclonal) | This paper | 0.8 µg per 1 µl of beads (depletions); 12 µg/ml (WB) | |
| Antibody | Anti-human SRCAP (Rabbit polyclonal) | Kerafast | ESL103; RRID:AB_3086743 | 0.8 µg per 1 µl of beads (depletions); 1:500 (WB) |
| Antibody | Anti-ZNHIT1 (Rabbit polyclonal) | Proteintech | 16595-1-AP; RRID:AB_2220382 | 1:1000 |
| Antibody | Anti-p400 (Rabbit polyclonal) | Bethyl | A300-541A; RRID:AB_2098208 | 1:1000 |
| Antibody | Anti-ALFA tag (Alpaca single domain antibody purified from E. coli) | NanoTag Biotechnologies | N1502-Li800-L; RRID:AB_3075985 | 1:1000 |
| Antibody | Anti-xlUBN2 (Rabbit polyclonal) | This paper | 1.5 µg per 1 µl of beads (depletions); 2 µg/ml (WB). Generated using the following peptide sequence: CGGQNKGDAKLPRKNR | |
| Antibody | Anti-xKID (Rabbit polyclonal) | Funabiki and Murray, 2000 | 1 µg/ml | |
| Antibody | Anti-xIDppa2 (Rabbit polyclonal) | Xue et al., 2013 | 5 µg/ml | |
| Antibody | IRDye 800CW anti-Rabbit IgG (Goat polyclonal) | LICORbio | 926-32211; RRID:AB_621843 | 1:10,000 |
| Antibody | IRDye 680RD anti-Mouse IgG (Goat polyclonal) | LICORbio | 926-68070; RRID:AB_10956588 | 1:10,000 |
| Antibody | anti-Rabbit IgG (Guinea Pig polyclonal) | https://www.antibodies-online.com/ | ABIN101961; RRID:AB_10775589 | 1:100 |
| Antibody | anti-Mouse IgG (Rabbit polyclonal) | abcam | ab46540; RRID:AB_2614825 | 1:100 |
| Recombinant DNA reagent | HSat2 array | This paper. Base sequence taken from GenBank: X06199.1 | RMSp64 | |
| Recombinant DNA reagent | 601 array | A gift from Aaron Straight (Guse et al., 2011) | pAS696 | |
| Sequence-based reagent | Sat2R-P | This paper. Modified from the Sat2R sequence in Osakabe et al., 2015 | RMSp81 | ATCATCAGAT TCCATTCGA ATCCATTCGA AAATGATTAC ATTCGAATCC ATTCGAAGATT CCATTTGAGCC TGCTCGAGCA GGCTCAAATGG AATCTTCGAAT GGATTCGAATG TAATCATTTT CGAATGGATTCG AATGGAATCTGA TGAT |
| Sequence-based reagent | 601L | A gift from Curt Davey (Addgene) | RRID:Addgene_158572 | |
| Sequence-based reagent | 1HinfI_sat2R | This paper | RMSp161 | ATCATCGTTCCAT TCGTGACTCCAT TCGAAAATGATTA CATTCGTATCCAT TCGAAGTTTCCAT TTGAGCCTGTTCG AAAATTCCATTTGT GTCCAACCAATGT TTCCATTCATTTCC ATTCAATGTTTCCA TTCGTATCCATTTG GATGAT |
| Sequence-based reagent | Tn5mC-Apt1 | Li et al., 2021 | CnT-BS adapter oligo | T/iMe-dC/GT/iMe-dC/GG/iMe-dC/AG/iMe-dC/GT/iMe-dC/AGATGTGTATAAGAGA/iMe-dC/AG |
| Sequence-based reagent | Tn5mC1.1-A1block | Li et al., 2021 | CnT-BS adapter oligo | /5Phos/CTGTCTCTTATACA/3ddC/ |
| Sequence-based reagent | Li et al., 2021 | CnT-BS replacement oligo | /5Phos//iMe-dC/TGT/iMe-dC/T/iMe-dC/TTATA/iMe-dC/A/iMe-dC/AT/iMe-dC/T/iMe-dC//iMe-dC/GAG/iMe-dC//iMe-dC//iMe-dC/A/iMe-dC/GAGA/iMe-dC//3InvdT/ | |
| Peptide, recombinant protein | pG-Tn5 | Soroczynski et al., 2024 | RRID:Addgene_198468 | |
| Peptide, recombinant protein | M.SssI CpG methyltransferase | New England Biolabs | Cat# M0226 | |
| Peptide, recombinant protein | HinfI | New England Biolabs | Cat# R0155 | |
| Peptide, recombinant protein | Ampligase | Lucigen (Biosearch Technologies) | A0102K | |
| Peptide, recombinant protein | T4 DNA polymerase | Thermo Scientific | EP0061 | |
| Peptide, recombinant protein | NEBNext Q5U Master Mix | New England Biolabs | Cat# M0597 | |
| Peptide, recombinant protein | Klenow Fragment (3′ → 5′ exo-) | New England Biolabs | Cat# M0212 | |
| Peptide, recombinant protein | Q5 DNA polymerase | New England Biolabs | Cat# M0491 | |
| Peptide, recombinant protein | hsH2A | This paper. Plasmids gifts from Shixin Liu | GenBank: AY131971.1 | |
| Peptide, recombinant protein | hsH2A.Z | This paper. Plasmids gifts from Shixin Liu | GenBank: M37583.1 | |
| Peptide, recombinant protein | hsH2B | This paper. Plasmids gifts from Shixin Liu | GenBank: AF531285.1 | |
| Peptide, recombinant protein | hsH3.2 | This paper. Plasmids gifts from Shixin Liu | GenBank: AF531307.1 | |
| Peptide, recombinant protein | hsH4 | This paper. Plasmids gifts from Shixin Liu | GenBank: M60749.1 | |
| Commercial assay or kit | Nuclei EZ Prep kit | Millipore | Cat# NUC101-1KT | |
| Commercial assay or kit | EZ DNA Methylation-Lightning kit | Zymo Research | Cat# D5030 | |
| Commercial assay or kit | MEGAscript SP6 transcription kit | Invitrogen | Cat# AM1330 | |
| Commercial assay or kit | M-280 Streptavidin Dynabeads | Invitrogen | Cat# 11206D | |
| Commercial assay or kit | Protein A Dynabeads | Invitrogen | Cat# 10008D | |
| Commercial assay or kit | CleanCap Reagent AG | TriLink | Cat# N-7113 | |
| Commercial assay or kit | MinElute PCR Purification kit | QIAGEN | Cat# 28004 | |
| Chemical compound, drug | Biotin-14-dATP | Jena Bioscience | Cat# NU-835-BIO14-S | |
| Software, algorithm | Relion v4 | Scheres, 2012 | RRID:SCR_01627 | |
| Software, algorithm | CryoSPARC v4.6 | Structura Biotechnology Inc | RRID:SCR_016501 | |
| Software, algorithm | Topaz v0.2 | Bepler et al., 2019 | ||
| Software, algorithm | Phenix v1.21 | Liebschner et al., 2019 | RRID:SCR_014224 | |
| Software, algorithm | Coot 0.9.8.7 | Emsley et al., 2010 | RRID:SCR_014222 | |
| Software, algorithm | PyMOL v2.5.7 | Schrödinger | RRID:SCR_000305 | |
| Software, algorithm | Fastp v0.24.0 | Chen, 2023 | RRID:SCR_016962 | |
| Software, algorithm | Bismark v0.24.2 | Krueger and Andrews, 2011 | RRID:SCR_005604 | |
| Software, algorithm | DESeq2 v1.42.1 | Love et al., 2014 | RRID:SCR_015687 | |
| Software, algorithm | SEACR v1.3 | Meers et al., 2019 | RRID:SCR_027011 | |
| Software, algorithm | methylKit v1.28.0 | Akalin et al., 2012 | RRID:SCR_005177 | |
| Software, algorithm | ChIPseeker v1.38.0 | Yu et al., 2015 | RRID:SCR_021322 | |
| Software, algorithm | deepTools v3.5.5 | Ramírez et al., 2016 | RRID:SCR_016366 |
Additional files
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Supplementary file 1
Collection and model statistics for the cryo-EM structures.
- https://cdn.elifesciences.org/articles/109762/elife-109762-supp1-v1.docx
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Supplementary file 2
Alignment statistics of CnT-BS sequencing libraries.
XTC reads were downsampled to 30 million reads to match sperm samples, then all raw reads were trimmed and deduplicated using fastp v0.24.0 and aligned to the Xenopus laevis genome (Xenla 10.1) or lambda genome (to assess for bisulfite conversion efficiency) using Bismark v0.24.2. Methylated CpG percentages for H2A.Z samples were determined for the top 2.5% of H2A.Z peaks by AUC (obtained using SEACR v1.3) while methylated percentages for H3 associated CpGs were conducted on regions outside of determined H2A.Z peaks. All CpGs met a cutoff of at least five reads to be counted. Methylated lambda CpG statistics were calculated from all mapped CpGs with no filtering.
- https://cdn.elifesciences.org/articles/109762/elife-109762-supp2-v1.docx
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Supplementary file 3
Fragment statistics of filtered genomic bins used for sequencing analysis.
A count matrix was generated from processed bam files over 1000 bp bins of the Xenopus laevis genome. Mitochondrial reads and bins containing fewer than 17 reads across less than 2samples were removed to filter out low signal areas. Replicates for each sample were averaged, and the resulting count matrix was used for genomic annotation analysis.
- https://cdn.elifesciences.org/articles/109762/elife-109762-supp3-v1.docx
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Supplementary file 4
Mass spectrometry validation data of generated custom anti-H2A.Z antibody.
- https://cdn.elifesciences.org/articles/109762/elife-109762-supp4-v1.xlsx
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Supplementary file 5
Mass spectrometry validation data of generated custom anti-SRCAP antibody.
- https://cdn.elifesciences.org/articles/109762/elife-109762-supp5-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/109762/elife-109762-mdarchecklist1-v1.pdf
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A two-part list of links to download the article, or parts of the article, in various formats.
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Impacts of DNA methylation on H2A.Z deposition and nucleosome stability
eLife 15:RP109762.
https://doi.org/10.7554/eLife.109762.3
