Hontelez S et al. (2015), Embryonic transcription is controlled by matern...

XB-ART-51677

Nat Commun 2015 Dec 18;6:10148. doi: 10.1038/ncomms10148.

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Embryonic transcription is controlled by maternally defined chromatin state.

Hontelez S , van Kruijsbergen I , Georgiou G , van Heeringen SJ , Bogdanovic O , Lister R , Veenstra GJC .

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Histone-modifying enzymes are required for cell identity and lineage commitment, however little is known about the regulatory origins of the epigenome during embryonic development. Here we generate a comprehensive set of epigenome reference maps, which we use to determine the extent to which maternal factors shape chromatin state in Xenopus embryos. Using α-amanitin to inhibit zygotic transcription, we find that the majority of H3K4me3- and H3K27me3-enriched regions form a maternally defined epigenetic regulatory space with an underlying logic of hypomethylated islands. This maternal regulatory space extends to a substantial proportion of neurula stage-activated promoters. In contrast, p300 recruitment to distal regulatory regions requires embryonic transcription at most loci. The results show that H3K4me3 and H3K27me3 are part of a regulatory space that exerts an extended maternal control well into post-gastrulation development, and highlight the combinatorial action of maternal and zygotic factors through proximal and distal regulatory sequences.

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Species referenced: Xenopus laevis
Genes referenced: crebbp gas1 gata2 gbx2 gbx2.2 nodal1 nog sia1 sia2

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	Figure 1. Reference epigenome maps of Xenopus tropicalis development.(a) Genome-wide profiles were generated for stages 8 and 9 (blastula, before and after MBT), 10.5 and 12.5 (gastrula), 16 (neurula) and 30 (tailbud). Adapted from Tan, M.H. et al. Genome Res. 23, 201–216 (2013), under a Creative Commons License (Attribution-NonCommercial 3.0 Unported License), as described at http://creativecommons.org/licenses/by/3.0/. (b) Gata2 locus with late gastrula (stage 10.5) methylC-seq, ChIP-seq enrichment of histone modifications, RNAPII and p300 (cf. Supplementary Figs 1 and 2).
	Figure 2. Chromatin state dynamics.(a) Emission states (same for all developmental stages) of the hidden Markov model, identifying the 19 most prevalent combinations of histone modifications and bound proteins. From top to bottom: Polycomb (red), Poised enhancers and promoters (blue), Active Enhancers (gold), Transcribed (dark magenta), Promoter (green), Heterochromatin (purple) and unmodified (grey). (b) Alluvial plots of chromatin state coverage during development. Each plot shows the transitions (to and from the highlighted group of chromatin states) across developmental stages (stages 9–30). The height represents the base pair coverage of the chromatin state relative to the modified genome. The ‘modified genome' has a chromatin state other than unmodified in any of the stages 9–30. From top to bottom left: promoters (green), poised (blue), p300-bound enhancers (gold). From top to bottom right: transcribed (dark magenta), Polycomb (red) and heterochromatin (purple). Line plots: Chromatin state coverage per stage as a percentage of the modified genome.
	Figure 3. Developmental acquisition of chromatin states.(a) Inhibition of embryonic transcription with α-amanitin, adapted from Tan, M.H. et al. Genome Res. 23, 201–216 (2013), under a Creative Commons License (Attribution-NonCommercial 3.0 Unported License), as described at http://creativecommons.org/licenses/by/3.0/. (b) RNAPII on the TSS of genes in control and α-amanitin-injected embryos (stage 11). (c) Box plots showing RNA expression levels (RPKM) of maternal and embryonic transcribed genes in control and α-amanitin-injected embryos (stage 11). Box: 25th (bottom), 50th (internal band), 75th (top) percentiles. Whiskers: 1.5 × interquartile range of the lower and upper quartiles, respectively. (d) ChIP-sequencing on chromatin of α-amanitin-injected and control embryos reveals maternal and zygotic origins of H3K4me3, H3K27me3 or p300 binding. Data from two biological replicates, see Supplementary fig. 4.
	Figure 4. DNA methylation logic of maternally versus zygotically defined H3K4me3 and H3K27me3.(a) CpG density and methylation at stage 9 of promoters (H3K4me3: ±100 bp from TSS; H3K27me3: ±2.5 kb from TSS) that contain a zygotic defined (ZyD, lost in α-amanitin treated embryos, red) or maternal defined (MaD, maintained in α-amanitin treated embryos, grey) peak for H3K4me3 (left) or H3K27me3 (right) after inhibition of embryonic transcription. The size of the dot indicates the relative RPKM of the histone modification (background corrected). (b) Hoxd (MaD) and nodal1, -2 (ZyD) loci with stage 9 methylC-seq, H3K4me3 and H3K27me3 in control and α-amanitin-injected embryos. (c) Developmental profiles of H3K4me3 and H3K27me3 (median background corrected RPKM) at genes without detectable maternal mRNA do correlate with activation for methylated promoters (lower panels) but not for hypomethylated CpG island promoters (upper panels).
	Figure 5. Zygotically controlled p300 recruitment shapes enhancer clusters (EC) domains.(a) Modelled transcription factor motif activity to p300 enrichment (see Methods). Activity reflects modelled contributions in p300 peak RPKM. (b) Heatmaps of MaD (upper panel) and ZyD (lower panel) p300 binding sites in α-amanitin treated and control embryos. (c) Developmental increase in genomic coverage of the gas1 EC by acquisition of p300 binding at enhancers. (d) EC dynamics of p300 enrichment (left panel), percentage of total EC region identified in each stage based on stage-dependent p300 binding (middle panel) and number of p300 peaks (per 12.5 kb) in EC. (e) Percentage of zygotic defined (ZyD, lost in α-amanitin treated embryos) and maternal defined (MaD, maintained in α-amanitin treated embryos) p300 peaks that map to ECs. Asterisks indicate significance as more or less p300 peaks than expected by chance calculated using cumulative hypergeometric test: P=6E−14; *P=5E−29 (f) Percentage of ECs that have a MaD or ZyD seeding peak at stage 9. (g) Box plot showing the percentage of the EC region that is defined by MaD or ZyD p300 peaks. Box: 25th (bottom), 50th (internal band), 75th (top) percentiles. Whiskers: 1.5 × interquartile range of the lower and upper quartiles, respectively. Outliers are indicated with black dots.
	Figure 6. Maternal epigenetic control extends beyond gastrulation.Maternally defined (MaD) peaks emerge at or before stage 11 independent of embryonic transcription. Zygotically defined (ZyD) peaks appear before stage 11 and are lost in α-amanitin treated embryos, or emerge at or after stage 12. Not determined (ND) peaks are not consistently detected in replicates 1 and 2 and generally have low enrichment values. (a) Total number of genes with a MaD or ZyD peak in their promoter (H3K4me3 and H3K27me3), or total number of MaD and ZyD peaks per GREAT region (p300). ND peaks are not shown. (b) MaD and ZyD regulation of gastrula and neurula expressed genes. The pie charts show the number genes with a MaD or ZyD peak in their promoter (H3K4me3 and H3K27me3) or the number of MaD, ZyD and ND peaks per cis-regulatory region (p300). The H3K27me3 and p300 pie charts represent: Gastrula expressed genes with a MaD (far left) or ZyD (middle left) H3K4me3 peak; neurula expressed genes with a MaD (middle right) or ZyD (far right) H3K4me3 peak.
	Figure 7. Maternal and zygotic regulatory space separates early and late Wnt target genes.(a) The number of genes with MaD or ZyD H3K4me3 (pie charts) and relative RPKM (dot plots, horizontal line: median) of p300 in cis-regulatory regions of genes and H3K27me3 on promoters (±2.5 kb from TSS) at different developmental stages that have maternally or zygotically defined H3K4me3 at the promoter. Early targets sia1 and sia2 are not included, these genes lose H3K4me3 after stage 9 and cannot be assigned to MaD or ZyD space based on our stage 11 α-amanitin data. H3K4me3 on these genes is acquired at stage 8, before embryonic transcription. (b) Browser views of the early Wnt target nog (noggin) and the late Wnt targets gbx2.1 and gbx2.2 with ChIP-seq enrichment of H3K4me3, p300 and RNAPII on control and α-amanitin-injected embryos and RNAPII on stages 9 and 10.5.
	Figure 8. Model of maternal and zygotic regulatory space.This shows the segregation of maternal regulatory space, which contains hypomethylated promoters that are mainly controlled by maternal factors, and zygotic regulatory space, which includes methylated promoters and enhancers that are under zygotic control. Most p300-bound enhancers are in zygotic space, however, they can regulate promoters in both maternal and zygotic space, crossing the regulatory space border. This may contribute to varying degrees of permissiveness to transcriptional activation. Maternal regulatory space extends well into neurula and tailbud stages and includes many embryonic genes which are activated at specific stages of development. Zygotic regulatory space requires zygotic transcription, is established from the mid-blastula stage onwards but increases in relative contribution during development.

References [+] :

Akkers, A hierarchy of H3K4me3 and H3K27me3 acquisition in spatial gene regulation in Xenopus embryos. 2009, Pubmed, Xenbase