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. 2015 Dec 1;29(23):2449-62.
doi: 10.1101/gad.271353.115. Epub 2015 Nov 19.

Dynamic changes in histone modifications precede de novo DNA methylation in oocytes

Kathleen R Stewart  1 , Lenka Veselovska  1 , Jeesun Kim  2 , Jiahao Huang  1 , Heba Saadeh  3 , Shin-ichi Tomizawa  4 , Sébastien A Smallwood  1 , Taiping Chen  2 , Gavin Kelsey  5
Affiliations

Dynamic changes in histone modifications precede de novo DNA methylation in oocytes

Kathleen R Stewart et al. Genes Dev. .

Abstract

Erasure and subsequent reinstatement of DNA methylation in the germline, especially at imprinted CpG islands (CGIs), is crucial to embryogenesis in mammals. The mechanisms underlying DNA methylation establishment remain poorly understood, but a number of post-translational modifications of histones are implicated in antagonizing or recruiting the de novo DNA methylation complex. In mouse oogenesis, DNA methylation establishment occurs on a largely unmethylated genome and in nondividing cells, making it a highly informative model for examining how histone modifications can shape the DNA methylome. Using a chromatin immunoprecipitation (ChIP) and genome-wide sequencing (ChIP-seq) protocol optimized for low cell numbers and novel techniques for isolating primary and growing oocytes, profiles were generated for histone modifications implicated in promoting or inhibiting DNA methylation. CGIs destined for DNA methylation show reduced protective H3K4 dimethylation (H3K4me2) and trimethylation (H3K4me3) in both primary and growing oocytes, while permissive H3K36me3 increases specifically at these CGIs in growing oocytes. Methylome profiling of oocytes deficient in H3K4 demethylase KDM1A or KDM1B indicated that removal of H3K4 methylation is necessary for proper methylation establishment at CGIs. This work represents the first systematic study performing ChIP-seq in oocytes and shows that histone remodeling in the mammalian oocyte helps direct de novo DNA methylation events.

Keywords: ChIP-seq; DNA methylation; genomic imprinting; histone modifications; oocytes.

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Figures

Figure 1.
Figure 1.
Development of oocyte FACS suitable for ChIP-seq. (A) FACS strategy for E18.5 primary oocyte isolation using SYCP3. (B) FACS strategy for P10 growing oocyte nucleus isolation using DNA content and NOBOX. (C) Genome screenshots showing K4me3 signal (as corrected read count) in E18.5 ovarian somatic cells, starting from 25,000, 10,000, or 5000 cells. The 5000-cell track represents grouped biological replicates. Shown is a 1-kb window with a 1-kb step. (D) Peaks called in K4me3 ChIP-seqs starting from 25,000, 10,000, or 5000 cells show high agreement. The 5000-cell peaks were called from grouped biological replicates. (E) Replicate correlation over expected enrichment sites (log-transformed corrected read count) in the E18.5 and P10 oocyte data sets: 4 kb centered on promoters for K4me2 and K4me3 and gene bodies for K36me3. CGI promoters are indicated by blue dots, and non-CGI promoters are indicated by red dots. (F) K4me2 and K4me3 signal (corrected read count) at the non-CGI Oct4 and CGI Zp3 promoters reflects the silenced state of these genes at E18.5 (top tracks) and active state at P10 (bottom tracks). Shown is a 100-base-pair (bp) window with a 100-bp step. (G) Trend plots showing K4me2 and K4me3 signal (log-transformed corrected read count) over silent and active promoters (left panels; X-axis is in base pairs) and K36me3 signal across their associated gene bodies (right panels) as determined by RNA sequencing (RNA-seq). (TSS) Transcription start site; (TTS) transcription termination site.
Figure 2.
Figure 2.
H3K4 methylation dynamics at CGIs prior to de novo DNA methylation. (A) Density plot of CpG densities of GVmeth (blue) and GVunmeth (red) CGIs. (B) E18.5 and P10 K4me2 and K4me3 signal (log-transformed corrected read count) at 4 kb centered on CGIs, separated into GVmeth and GVunmeth CGIs and by CpG density. (Black line) Median; (dashed lines) 1.5× interquartile range. (C) Significance of K4me2 or K4me3 enrichment difference between GVmeth and matched GVunmeth CGIs within each CpG density subset at E18.5 and P10; unpaired one-sided Mann-Whitney test. (D) Scatter plots showing E18.5 versus P10 enrichment (log-transformed corrected read count) for K4me2 (top panels) and K4me3 (bottom panels) in GVmeth and GVunmeth CGIs. (Black dots) Imprinted gDMRs. (E) Histograms showing K4me2 and K4me3 enrichment (log-transformed corrected read count) at GVmeth CGIs that are intragenic at P10, separated by annotation at E18.5. (Magenta) Intergenic; (light blue) intragenic; (dark blue) TSS. Dashed lines indicate the mean. (F) Genome screenshots showing ChIP-seq signal (corrected read count) at E18.5 and P10 over the Peg13 and Impact imprinted gDMRs. Shown is a 100-bp window with a 100-bp step.
Figure 3.
Figure 3.
K36me3 is generally enriched, and K4me2/3 is depleted at certain GVmeth CGIs at P10. (A) Scatter plots showing E18.5 versus P10 enrichment (log-transformed corrected read count) for K36me3 in GVmeth and GVunmeth CGIs. (Black dots) Imprinted gDMRs. (B) Trend plots showing K36me3 signal (log-transformed corrected read count) at 4 kb centered on TSS, intragenic, and intergenic GVmeth and GVunmeth CGIs as determined by RNA-seq at E18.5 and P10. (C) Box plots showing K6me3 and H3 (log-transformed corrected read count) at P10 intragenic GVmeth and GVunmeth CGIs, parsed by expression level. (Black line) Median; (dashed lines) 1.5× interquartile range. (*) P < 1 × 10−10; (**) P < 1 × 10−25, Mood's median test. (D) Emission probabilities from a three-state segmentation of K4me2, K4me3, and K36me3 E18.5 and P10 signal over CGIs. (E) Bar chart showing the proportion of protective, permissive, and unenriched CGIs in GVmeth and GVunmeth CGIs at E18.5 and P10. (F) K36me3, K4me2, and K4me3 enrichment (log-transformed corrected read count) at 4 kb centered on GVmeth CGIs that transition from the protective to the permissive state between E18.5 and P10. (Black line) Median; (dashed lines) 1.5× interquartile range. (*) P < 1 × 10−10, Mood's median test. (G) Genome screenshot showing ChIP-seq signal (corrected read count) at E18.5 and P10 over the Zfp777 imprinted gDMR. Shown is a 100-bp window with a 100-bp step.
Figure 4.
Figure 4.
KDM1B, not KDM1A, predominantly affects CGI methylation. (A) Scatter plots showing methylation at CGIs in KDM1A-deficient (top) and KDM1B-deficient (bottom) oocytes, compared with controls. (Blue dots) Significantly hypomethylated CGIs (hypoCGIs); (red dots) significantly hypermethylated CGIs (hyperCGIs); (black dots) imprinted gDMRs; (WT) wild type; (CTRL) Kdm1aflox/+, ZP3-Cre- control. (B) Bar plots showing methylation levels at the 23 maternally imprinted gDMRs in KDM1A-deficient (top) and KDM1B-deficient (bottom) oocytes, compared with controls. (C) Genome screenshots showing K4me2 ChIP-seq signal (as corrected read coverage) in E18.5 and P10 wild-type oocytes and DNA methylation over the Grb10, Gnas, and Cdh15 gDMRs in normal and KDM1A- or KDM1B-deficient MII oocytes. Shown is a 100-bp window with a 100-bp step. (D) Box plots showing E18.5 and P10 K4me2 (log-transformed corrected read count) at 4 kb centered on CGIs that lose methylation in the absence of KDM1A, KDM1B, or both, compared with unaffected CGIs that gain full methylation in the knockouts. (Black line) Median; (dashed lines) 1.5× interquartile range. (*) P < 1 × 10−10; (**) P < 1 × 10−25, Mood's median test. (E) Stacked bar plot depicting the proportion of CGIs transitioning from protective to permissive states between E18.5 and P10 that lose methylation in the absence of KDM1A, KDM1B, or both. (Other) Either gain methylation in the knockout or are intermediately/unmethylated in the control. Shown are CGIs with adequate coverage in both the PBAT and the ChIP-seq data.
Figure 5.
Figure 5.
Stepwise modulation of CGI chromatin facilitates DNA methylation acquisition. At E18.5, CGIs destined for DNA methylation are marked with protective K4me2/K4me3 and lack permissive K36me3. In parallel with postnatal activation of the oocyte transcriptome, K36me3 accumulates specifically on CGIs destined for DNA methylation, and, at P10, these CGIs are marked with both K4me2/me3 and K36me3. During de novo DNA methylation between P10 and the GV stage, H3K4 methylation from CGIs must be removed, as evidenced by the methylation defects seen in mature oocytes in the absence of KDM1A and especially KDM1B.
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