Single cell multi-omics profiling reveals a hierarchical epigenetic landscape during mammalian germ layer specification

Abstract

Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan. Recent studies employing single cell RNAsequencing have identified major transcriptional changes associated with germ layer specification. Global epigenetic reprogramming accompanies these changes, but the role of the epigenome in regulating early cell fate choice remains unresolved, and the coordination between different epigenetic layers is unclear. Here we describe the first single cell tripleomics map of chromatin accessibility, DNA methylation and RNA expression during the exit from pluripotency and the onset of gastrulation in mouse embryos. We find dynamic dependencies between the different molecular layers, with evidence for distinct modes of epigenetic regulation. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of local lineagespecific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements, driven by loss of methylation in enhancer marks and a concomitant increase of chromatin accessibility. In striking contrast, the epigenetic landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or epigenetically remodelled prior to overt cell fate decisions during gastrulation, providing the molecular logic for a hierarchical emergence of the primary germ layers. Highlights• First map of mouse gastrulation using comprehensive single cell tripleomic analysis.• Exit from pluripotency is associated with a global repressive epigenetic landscape, driven by a sharp gain of DNA methylation and a gradual decrease of chromatin accessibility. • DNA methylation and chromatin accessibility changes in enhancers, but not in promoters, are associated with germ layer formation. • Mesoderm and endoderm enhancers become open and demethylated upon lineage commitment. • Ectoderm enhancers are primed in the early epiblast and protected from the global repressive dynamics, supporting a default model of ectoderm commitment in vivo . 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 82 84 86In mammals, specification of the basic body plan occurs during gastrulation, when a singlelayered blastula is reorganised to give rise to the three primary germ layers: the ectoderm, mesoderm and endoderm 1,2 . Recent advances in singlecell RNA sequencing have facilitated unbiased and comprehensive characterisation of such developmental trajectories in a variety of different systems 3-5 . However, while gene expression dynamics of mouse development have been characterized in detail 6-12 , the role of the epigenome in cell fate decisions in early development remains poorly understood 13 .The period of mouse development prior to gastrulation is characterised by extensive remodelling of the epigenome [14][15][16][17][18][19][20][21][22][23...

Xmas ESC: A new female embryonic stem cell system that reveals the BAF complex as a key regulator of the establishment of X chromosome inactivation

“…We have discovered that Smarcc1 is required to deplete promotors of nucleosomes on the future Xi at the very early stages of the establishment of XCI. A similar effect is observed on autosomes and can also be found in published NOMe-seq datasets from the equivalent stages of postimplantation embryos (Argelaguet et al, 2019), suggesting that nucleosome depletion at promoters may be a common occurrence during differentiation. Importantly, however, we have functionally linked this opening to gene silencing; cells with depleted Smarcc1 fail to open promotors and fail to establish XCI, with the resulting Xi following a similar trajectory to that of the Xa, both in terms of nucleosome positioning and gene silencing.…”

MOFA+: a probabilistic framework for comprehensive integration of structured single-cell data

“…As a final application, we considered a complex dataset with multiple sample groups and views. The dataset consists of a multi-omic atlas of mouse gastrulation where scNMT-seq was used to simultaneously profile RNA expression, DNA methylation and chromatin accessibility in 1,828 cells at multiple stages of development 41 . In this dataset MOFA+ provides a method for delineating coordinated variation between the transcriptome and the epigenome and for detecting at which stage(s) of development it occurs.…”

“…As input to the model we quantified DNA methylation and chromatin accessibility values over two sets of regulatory elements: gene promoters and enhancer elements (distal H3K27ac sites [41][42][43] ). RNA expression was quantified over protein-coding genes.…”

“…The gastrulation scNMT-seq multi-omics data was obtained from 41 and is available in the Gene Expression Omnibus under accession GSE121708 Gene expression counts were quantified over protein-coding genes using featureCounts 63 with the Ensembl gene annotation 87 64 . The read counts were log-transformed and size-factor adjusted, and modelled with a Gaussian likelihood.…”

“…The rates were subsequently transformed to M-values 62 and modelled with a Gaussian likelihood. As input to MOFA+ we filtered genomic features with low coverage (at least 3 CpG and 5 GpC measurements) and we selected the top 2500 most variable sites per combination of genomic context and data modality (see Figure S14 ) Details on the quality control and data preprocessing can be found in 41 .…”