Abstract
BackgroundMammalian development is associated with extensive changes in gene expression, chromatin accessibility, and nuclear structure. Here, we follow such changes associated with mouse embryonic stem cell differentiation and X inactivation by integrating, for the first time, allele-specific data from these three modalities obtained by high-throughput single-cell RNA-seq, ATAC-seq, and Hi-C.ResultsAllele-specific contact decay profiles obtained by single-cell Hi-C clearly show that the inactive X chromosome has a unique profile in differentiated cells that have undergone X inactivation. Loss of this inactive X-specific structure at mitosis is followed by its reappearance during the cell cycle, suggesting a “bookmark” mechanism. Differentiation of embryonic stem cells to follow the onset of X inactivation is associated with changes in contact decay profiles that occur in parallel on both the X chromosomes and autosomes. Single-cell RNA-seq and ATAC-seq show evidence of a delay in female versus male cells, due to the presence of two active X chromosomes at early stages of differentiation. The onset of the inactive X-specific structure in single cells occurs later than gene silencing, consistent with the idea that chromatin compaction is a late event of X inactivation. Single-cell Hi-C highlights evidence of discrete changes in nuclear structure characterized by the acquisition of very long-range contacts throughout the nucleus. Novel computational approaches allow for the effective alignment of single-cell gene expression, chromatin accessibility, and 3D chromosome structure.ConclusionsBased on trajectory analyses, three distinct nuclear structure states are detected reflecting discrete and profound simultaneous changes not only to the structure of the X chromosomes, but also to that of autosomes during differentiation. Our study reveals that long-range structural changes to chromosomes appear as discrete events, unlike progressive changes in gene expression and chromatin accessibility.
Highlights
Mammalian development is associated with extensive changes in gene expression, chromatin accessibility, and nuclear structure
X chromosome inactivation (XCI) is initiated by Xist, a long non-coding RNA that coats in cis the future inactive X chromosome (Xi) and recruits/removes specific proteins or protein modifications to silence X-linked genes and condense the Xi into a heterochromatic structure [7, 8]
Uniform Manifold Approximation and Projection (UMAP) projections of contact decay profile (CDP) based on sci-Hi-C data clearly show a separation between the Xi from the Xa, except in a few cells in which there is an apparent lack of the characteristic Xi-specific structure, whereas, in contrast, projections CDPs for chr1 homologs show no separation (Fig. 2D, H)
Summary
Mammalian development is associated with extensive changes in gene expression, chromatin accessibility, and nuclear structure. We follow such changes associated with mouse embryonic stem cell differentiation and X inactivation by integrating, for the first time, allele-specific data from these three modalities obtained by high-throughput single-cell RNA-seq, ATAC-seq, and Hi-C. Xist RNA interacts with RNA-binding proteins and master structural proteins, which help reshape one of the two X chromosomes into a unique condensed bipartite structure [7, 9,10,11] In both human and mouse, the two superdomains of long-range chromatin interactions are separated by a hinge region containing the conserved lncRNA locus DXZ4/Dxz that represents a structural platform for frequent long-range contacts with multiple X-linked loci [12,13,14,15,16,17,18]. Studies of Xi-specific epigenetic and structural features point to mechanistic differences between establishment and maintenance of XCI [14, 18,19,20,21,22,23,24]
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