小鼠植入前胚胎的可接近染色质图谱

Result

1 小鼠植入前胚胎的可接近染色质图谱

Schematic of ATAC-seq and CARM for probing accessible chromatin in mouse preimplantation embryos.

The UCSC browser view shows enrichment of ATAC-seq in early embryos.

First view of chromatin landscape when life begins.

The overlap between ATAC-seq peaks and annotated promoters (TSS ± 0.5 kb) or distal DHSs (away from TSS ± 0.5 kb) in mESCs. A random set of peaks that match the lengths of individual ATAC-seq peaks on the same chromosomes were used as a control.

A large fraction of ATAC-seq peaks in early embryos overlapped with promoters and mESC distal DNase I hypersensitive sites (DHSs).

The UCSC browser views showing the ATAC-seq, RNA-seq and DNaseI-seq (mESC, ENCODE) enrichment near Pou5f1, Nanog, and Foxa1.

Heat maps show the expression (FPKM) of stage-specific genes (ZGA-only) (left) and the ATAC-seq enrichment (normalized reads per kilobase per million (RPKM) (middle) at their promoters (TSS ± 2.5 kb). Example genes are also listed (right).

The activation of genes at various stages was generally correlated with increased levels of promoter ATAC-seq signals.

2 开放染色质的allelic图谱

The UCSC browser view shows allelic ATAC-seq enrichment and DNA methylation. mCG/CG data from ref. 16.

To determine the global chromatin state on each parental genome, we measured allelic ATAC-seq enrichment using single-nucleotide polymorphisms (SNPs) present between the two parental strains.

Bar charts showing the percentages of ATAC-seq peaks that are biallelic, paternal- or maternal-specific. Only peaks covered by sufficient SNPs and allelic reads were considered (Methods).

Parental genomes showed comparable open chromatin as early as 2-cell stage.

Pie charts show the percentages of peaks (covered by SNPs) with biallelic or allele-specific accessible chromatin at the 4-cell stage.

The average ATAC-seq enrichment and DNA methylation levels on each allele around all promoter and distal ATAC-seq peaks (covered by SNPs).

Allele-specific expression of imprinted genes is controlled by differential DNA methylation. Therefore, we asked if allele-specific ATAC-seq peaks are associated with allelic DNA methylation. A global view showed that the comparable allelic landscape of accessible chromatin is in stark contrast to the distinct allelic DNA methylomes previously reported16 (Fig. 2a).
For both promoter and distal ATAC-seq peaks, DNA methylation shows preferential methylation on the maternal allele (Fig. 2d).
The differences in allelic DNA methylation at ATAC-seq peaks gradually decrease when development proceeds, as both alleles are progressively demethylated (Fig. 2d).

3 TESs和重复序列的可接近染色质

The average ATAC-seq enrichment (normalized) for the top 1,000 most active genes (ZGA-only genes) at each stage.

b, The UCSC browser view shows ATAC-seq enrichment near two active genes (Rps19 and Rps13) in embryos and somatic tissues (data from refs 9, 17). ATAC-seq peaks near TSSs and TESs are shaded.

Such enrichment decreases at late stages and is much weaker in somatic cells.

c, Enrichment of repeats in ATAC-seq promoter and distal peaks compared to that in random peaks for early embryos and somatic tissues. The enrichment was calculated as a log2 ratio for the numbers of observed peaks that overlap with repeats divided by the numbers for random peaks.

2-cell 期，ATAC-seq 更多地富集在了repeats；其中以 SINE（B1,B2,B4）和 ERVL 为主：

d, Heat maps show the enrichment (calculated similarly as in c) of repeat subfamily in ATAC-seq peaks in early embryos and mESCs.

说明，转座元件广泛塑造了早期胚胎中的开放染色质。

4 早期胚胎发育的调控网络

Identify key developmental regulators:
using motif analysis software HOMER we found the binding motifs for a set of transcription factors enriched in distal peaks in a highly stage-specific manner

a, TF motifs identified from distal ATAC-seq peaks.
每个 stage，各转录因子 motif 的enrichment。
Only TFs expressed at least at one stage (FPKM ≥ 5) and motif enrichment P value <1 × 10−10 at least at one stage were included.

b, Schematic of the Gata4 knockdown experiments. A Venn diagram shows the overlap between Gata4-knockdown downregulated genes and ICM-specific genes (compared to mESCs), with the P value (hypergeometric distribution) indicated.

c, Bar charts showing the relative gene expression with the GFP knockdown (KD) embryos normalized to 1. Error bars denote the standard errors of single-embryo RNA-seq FPKM values (n = 3 for GFP KD; n = 4 for Gata4 KD).

GATA4 KD后，PE的标记基因下调。
有趣的是，SOX2也下调。SOX2促进PE lineage，因此我们猜测：GATA4也调控SOX2的表达 。
结论：GATA4 is a regulator of ICM circuitry and its downregulation may have a role in resetting the transcription programs of ICMs to mESCs.

d, Key regulators for ICMs and TEs identified by MARINa. In each row, all genes were sorted (from left to right) by their differential expressions in ICM versus TE cells. Predicted positively and negatively regulated TF targets are marked as red or blue bars on the basis of their co-expression patterns with TFs across individual cells in blastocysts. The P values (FDR-corrected) represent the statistical significance of enrichment estimated by permutating the ICM and TE samples.

e, Heat maps show ICM to TE expression ratio (top) and Nr5a2 to GFP knockdown expression ratio in the 8-cell embryos (bottom).

We then knocked down Nr5a2 in zygotes and collected embryos at the 8-cell stage for RNA-seq analysis.
Indeed, ICM marker genes including Nanog, Pou5f, Tdgf1 and Tdh were downregulated.
By contrast, TE marker genes such as Id2, Gata3, Fabp3 and Krt8 were upregulated.
TFs that are expressed in both ICM and TE such as Gabpa, Ctcf and Sp1 were not affected.

以上这些数据表明，NR5A2 早在 8-cell 就开始调节向 ICM 的 transcription 了。

5 minor ZGA 过程中一种不同寻常的松散染色质状态

Previous studies suggested that enhancers may be dispensable for transcription of reporters in zygotes.

Instead, enhancers only become essential during the course of ZGA when chromatin is proposed to progressively adopt a repressive state.

To investigate the chromatin state before major ZGA in vivo, we examined early 2-cell embryos in which minor ZGA is most evident but preceding major ZGA.

Our RNA-seq analysis found only a few genes actively transcribed at this stage and a large fraction (48%) of them (such as Zscan4) reside in clusters in the genome (Extended Data Fig. 10a–c).

Consistent with limited gene activities in early 2-cell embryos, we obtained generally weak and noisy ATAC-seq enrichment, and the detected peaks are reduced for both number (Extended Data Fig. 10d) and genome coverage (Extended Data Fig. 10e) compared to those at the 2-cell stage.

These peaks are enriched for repetitive elements with classes similar to those found in 2-cell embryos (Extended Data Fig. 10f).

Interestingly, when searching for regions (100 kb bin) with strongest ATAC-seq enrichment in the genome, we discovered that a large fraction of them (52% of the top 100 regions) contain or are in the proximity (100 kb) of the full-length MERVLs.

a, The UCSC genome browser views show the promiscuous transcription41 and broad accessible chromatin domains downstream of MERVL or Zfp352. Zygote + DRB, zygote treated with transcription inhibitor DRB; APH, aphidicolin; alpha, alpha-amanitin.

Surprisingly, the open chromatin near MERVL is large in size (up to 117 kb; median length, 40 kb) and is specifically present at the 3′ downstream of active MERVLs

‘minor-ZGA-associated accessible chromatin domains’ (MAC domains).

Finally, we found that non-repeat early 2-cell genes are also preferentially associated with broad ATAC-seq domains, although at weaker levels than those near MERVLs (Extended Data Fig. 10i). These include Zfp352, which is the most highly expressed non-repeat gene in our data (Fig. 5a and Extended Data Fig. 10j).

Taken together, our study showed unique chromatin landscape in minor ZGA that features broad domains of open chromatin covering promiscuous transcription.

b, The average ATAC-seq enrichment around MERVLs. The MERVL gene itself is not shown owing to its highly repetitive nature and the resulting low mappability (the upstream and downstream regions of MERVL are mappable).

c, The average promiscuous transcription levels around MERVLs measured by total RNA-seq41.

d, A model shows the distinct transcription and chromatin states in pre-ZGA, minor ZGA and major ZGA.