Biology subjects

Disteche, C. M.

Publications and source records attributed to Disteche, C. M..

18 recordsLinked to original sources

bioRxiv2026

Reference-guided pseudotime inference across species and biological contexts

Cells collected at the same chronological age can vary substantially in biological age due to the heterogeneity in the timing of differentiation, speed of maturation, and degeneration. However, existing pseudotime inference methods either disregard chronological time information, or rely on accurate time-series labels within similar species or biological conditions of interest. As a result, both types of strategies often fail to faithfully order cells from biological contexts without reliable time labels, along the desired axis of interest such as human embryonic development or disease progression. Here, we propose Cavebear, a machine learning framework that enables pseudotime inference in a query species or condition guided by scRNA-seq time-series profiles from a reference species or condition. Cavebear achieves more accurate developmental pseudotime inference than existing methods and provides in vivo temporal mapping for in vitro experiments. Furthermore, we illustrate the potential of Cavebear to study cellular-level disease progression in human patients using mouse cancer development models as references. By transferring temporal information across species and conditions, Cavebear enables systematic investigation of biological variation in contexts where such annotations were previously unattainable.

bioinformatics↗

bioRxiv2026

Embryo-scale Visual Cell Sorting reveals a conserved transcriptomic signature of nucleolar size linked to proteostasis

Nucleoli, nuclear speckles and other compartments regulate transcription, RNA processing, and chromatin organization within the nucleus, yet the relationship of their morphology to developmental gene expression programs in vivo is poorly understood. Here, we develop a high-throughput Visual Cell Sorting (VCS) workflow for fixed cells and nuclei that combines antibody-based photoconversion; GPU-accelerated, real-time image analysis; and three-level single-cell combinatorial indexing RNA-seq (sci-RNA-seq3) to link nuclear compartment morphology to single-nucleus transcriptomes at embryo scale. We use VCS to analyze and sort over 1 million mouse embryo-derived nuclei by nucleolar, nuclear speckle, or nuclear size and construct a transcriptional atlas annotated with nuclear compartment phenotypes. Nuclear compartment size varies both between and within lineages and is shaped by proliferation and differentiation. In extracellular matrix protein-producing cell types such as fibroblasts, chondrocytes, and osteoblasts, nucleolar enlargement is uncoupled from cell cycle, and in erythroid cells exhibit a sharp nucleolar contraction preceding cell-cycle exit. We identify a 41-gene transcriptional signature whose expression tracks nucleolar size, enriched for ribosome biogenesis, mitochondrial metabolism, unfolded protein response, stress granule, and ubiquitin-proteasome pathway components. We used this nucleolar transcriptional signature to annotate mouse, zebrafish and human developmental atlases with nucleolar size information, revealing a conserved coupling between nucleolar activity and proteostasis programs. Our work establishes Visual Cell Sorting as a scalable platform for mapping image-based phenotypes to molecular programs; details the relationship between nuclear compartment phenotypes and development; and provides a transcriptional signature to estimate nucleolar size from existing single-cell datasets.

genomics↗

bioRxiv2026

Cell-type specific allelic dampening of sex-linked genes in sex chromosome aneuploidy

Common sex chromosome aneuploidies (SCAs) often present with cognitive and cardiovascular dysfunction in humans. To address SCA effects on gene expression and DNA methylation in relevant cell types, we differentiated neural precursor cells (NPCs) and cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) with different numbers of sex chromosomes, including isogenic and independent lines. As expected, the expression of genes that escape X inactivation (escapees) mostly increases with the number of inactive X chromosomes (Xi). However, allelic analysis shows dampening of escapees specifically on the Xi in XXY compared to XX in both NPCs and CMs, revealing a novel type of dosage compensation in SCA. In contrast, Y-linked gene expression is higher in XXY versus XY NPCs, but the opposite is observed in CMs. This may explain the greater number of differentially expressed autosomal genes in NPCs versus CMs with an added Y chromosome, while effects of added X chromosomes are similar between cell types. Concordantly, changes in autosomal DNA methylation are mainly driven by the presence of a Y chromosome. These findings highlight the cell-type specificity of sex-linked and autosomal gene regulation in SCA conditions. HighlightsO_LISex chromosome aneuploidy induces cell-type specific changes in gene expression C_LIO_LIDampening of the inactive X chromosome in XXY alleviate X overexpression C_LIO_LIHigh Y-linked gene expression in XXY neuronal precursor cells but not cardiomyocytes C_LIO_LISex chromosome aneuploidy disrupts sex biases in autosomal gene expression C_LI

molecular biology↗

bioRxiv2024

Multi-condition and multi-modal temporal profile inference during mouse embryonic development

The emergence of single-cell time-series datasets enables modeling of changes in various types of cellular profiles over time. However, due to the disruptive nature of single-cell measurements, it is impossible to capture the full temporal trajectory of a particular cell. Furthermore, single-cell profiles can be collected at mismatched time points across different conditions (e.g., sex, batch, disease) and data modalities (e.g., scRNA-seq, scATAC-seq), which makes modeling challenging. Here we propose a joint modeling framework, Sunbear, for integrating multi-condition and multi-modal single-cell profiles across time. Sunbear can be used to impute single-cell temporal profile changes, align multi-dataset and multi-modal profiles across time, and extrapolate single-cell profiles in a missing modality. We applied Sunbear to reveal sex-biased transcription during mouse embryonic development and predict dynamic relationships between epigenetic priming and transcription for cells in which multi-modal profiles are unavailable. Sunbear thus enables the projection of single-cell time-series snapshots to multi-modal and multi-condition views of cellular trajectories.

bioinformatics↗

bioRxiv2023

Pseudotime analysis for time-series single-cell sequencing and imaging data

Many single-cell RNA-sequencing studies have collected time-series data to investigate transcriptional changes concerning various notions of biological time, such as cell differentiation, embryonic development, and response to stimulus. Accordingly, several unsupervised and supervised computational methods have been developed to construct single-cell pseudotime embeddings for extracting the temporal order of transcriptional cell states from these time-series scRNA-seq datasets. However, existing methods, such as psupertime, suffer from low predictive accuracy, and this problem becomes even worse when we try to generalize to other data types such as scATAC-seq or microscopy images. To address this problem, we propose Sceptic, a support vector machine model for supervised pseudotime analysis. Whereas psupertime employs a single joint regression model, Sceptic simultaneously trains multiple classifiers with separate score functions for each time point and also allows for non-linear kernel functions. Sceptic first generates a probability vector for each cell and then aims to predict chronological age via conditional expectation. We demonstrate that Sceptic achieves significantly improved prediction power (accuracy improved by 1.4 - 38.9%) for six publicly available scRNA-seq data sets over state-of-the-art methods, and that Sceptic also works well for single-nucleus image data. Moreover, we observe that the pseudotimes assigned by Sceptic show stronger correlations with nuclear morphology than the observed times, suggesting that these pseudotimes accurately capture the heterogeneity of nuclei derived from a single time point and thus provide more informative time labels than the observed times. Finally, we show that Sceptic accurately captures sex-specific differentiation timing from both scATAC-seq and scRNA-seq data.

genomics↗

bioRxiv2023

Cross-species imputation and comparison of single-cell transcriptomic profiles

Cross-species comparison and prediction of gene expression profiles are important to understand regulatory changes during evolution and to transfer knowledge learned from model organisms to humans. Single-cell RNA-seq (scRNA-seq) profiles enable us to capture gene expression profiles with respect to variations among individual cells; however, cross-species comparison of scRNA-seq profiles is challenging because of data sparsity, batch effects, and the lack of one-to-one cell matching across species. Moreover, single-cell profiles are challenging to obtain in certain biological contexts, limiting the scope of hypothesis generation. Here we developed Icebear, a neural network framework that decomposes single-cell measurements into factors representing cell identity, species, and batch factors. Icebear enables accurate prediction of single-cell gene expression profiles across species, thereby providing high-resolution cell type and disease profiles in under-characterized contexts. Icebear also facilitates direct cross-species comparison of single-cell expression profiles for conserved genes that are located on the X chromosome in eutherian mammals but on autosomes in chicken. This comparison, for the first time, revealed evolutionary and diverse adaptations of X-chromosome upregulation in mammals.

bioinformatics↗

bioRxiv2023

KDM6A facilitates Xist upregulation at the onset of X inactivation

X chromosome inactivation (XCI) is a female-specific process in which one X chromosome is silenced to balance X-linked gene expression between the sexes. XCI is initiated in early development by upregulation of the lncRNA Xist on the future inactive X (Xi). A subset of X-linked genes escape silencing and thus have higher expression in females, suggesting female-specific functions. One of these genes is the highly conserved gene Kdm6a, which encodes a histone demethylase that removes methyl groups at H3K27 to facilitate gene expression. Here, we investigate the role of KDM6A in the regulation of Xist. We observed impaired upregulation of Xist during early stages of differentiation in hybrid mouse ES cells following CRISPR/Cas9 knockout of Kdm6a. This is associated with reduced Xist RNA coating of the Xi, suggesting diminished XCI potency. Indeed, Kdm6a knockout results in aberrant overexpression of genes from the Xi after differentiation. KDM6A binds to the Xist promoter and knockout cells show an increase in H3K27me3 at Xist. These results indicate that KDM6A plays a role in the initiation of XCI through histone demethylase-dependent activation of Xist during early differentiation.

molecular biology↗

bioRxiv2023

CTCF-mediated insulation and chromatin environment modulate Car5b escape from X inactivation

BackgroundThe number and escape levels of genes that escape X chromosome inactivation (XCI) in female somatic cells vary among tissues and cell types, potentially contributing to specific sex differences. Here we investigate the role of CTCF, a master chromatin conformation regulator, in regulating escape from XCI. CTCF binding profiles and epigenetic features were systematically examined at constitutive and facultative escape genes using mouse allelic systems to distinguish the inactive X (Xi) and active X (Xa) chromosomes. ResultsWe found that escape genes are located inside domains flanked by convergent arrays of CTCF binding sites, consistent with the formation of loops. In addition, strong and divergent CTCF binding sites often located at the boundaries between escape genes and adjacent neighbors subject to XCI would help insulate domains. Facultative escapees show clear differences in CTCF binding dependent on their XCI status in specific cell types/tissues. Concordantly, deletion but not inversion of a CTCF binding site at the boundary between the facultative escape gene Car5b and its silent neighbor Siah1b resulted in loss of Car5b escape. Reduced CTCF binding and enrichment of a repressive mark over Car5b in cells with a boundary deletion indicated loss of looping and insulation. In mutant lines in which either the Xi-specific compact structure or its H3K27me3 enrichment was disrupted, escape genes showed an increase in gene expression and associated active marks, supporting the roles of the 3D Xi structure and heterochromatic marks in constraining levels of escape. ConclusionOur findings indicate that escape from XCI is modulated both by looping and insulation of chromatin via convergent arrays of CTCF binding sites and by compaction and epigenetic features of the surrounding heterochromatin.

genomics↗

bioRxiv2023

A single-cell transcriptional timelapse of mouse embryonic development, from gastrula to pup

The house mouse, Mus musculus, is an exceptional model system, combining genetic tractability with close homology to human biology. Gestation in mouse development lasts just under three weeks, a period during which its genome orchestrates the astonishing transformation of a single cell zygote into a free-living pup composed of >500 million cells. Towards a global framework for exploring mammalian development, we applied single cell combinatorial indexing (sci-*) to profile the transcriptional states of 12.4 million nuclei from 83 precisely staged embryos spanning late gastrulation (embryonic day 8 or E8) to birth (postnatal day 0 or P0), with 2-hr temporal resolution during somitogenesis, 6-hr resolution through to birth, and 20-min resolution during the immediate postpartum period. From these data (E8 to P0), we annotate dozens of trajectories and hundreds of cell types and perform deeper analyses of the unfolding of the posterior embryo during somitogenesis as well as the ontogenesis of the kidney, mesenchyme, retina, and early neurons. Finally, we leverage the depth and temporal resolution of these whole embryo snapshots, together with other published data, to construct and curate a rooted tree of cell type relationships that spans mouse development from zygote to pup. Throughout this tree, we systematically nominate sets of transcription factors (TFs) and other genes as candidate drivers of the in vivo differentiation of hundreds of mammalian cell types. Remarkably, the most dramatic shifts in transcriptional state are observed in a restricted set of cell types in the hours immediately following birth, and presumably underlie the massive changes in physiology that must accompany the successful transition of a placental mammal to extrauterine life.

developmental biology↗

bioRxiv2023

Oligonucleotide-directed proximity-interactome mapping (O-MAP): A unified method for discovering RNA-interacting proteins, transcripts and genomic loci in situ.

Throughout biology, RNA molecules form complex networks of molecular interactions that are central to their function, but remain challenging to investigate. Here, we introduce Oligonucleotide-mediated proximity-interactome MAPping (O-MAP), a straightforward method for elucidating the biomolecules near an RNA of interest, within its native cellular context. O-MAP uses programmable oligonucleotide probes to deliver proximity-biotinylating enzymes to a target RNA, enabling nearby molecules to be enriched by streptavidin pulldown. O-MAP induces exceptionally precise RNA-localized in situ biotinylation, and unlike alternative methods it enables straightforward optimization of its targeting accuracy. Using the 47S pre-ribosomal RNA and long noncoding RNA Xist as models, we develop O-MAP workflows for unbiased discovery of RNA-proximal proteins, transcripts, and genomic loci. This revealed unexpected co-compartmentalization of Xist and other chromatin-regulatory RNAs and enabled systematic characterization of nucleolar-chromatin interactions across multiple cell lines. O-MAP is portable to cultured cells, organoids, and tissues, and to RNAs of various lengths, abundances, and sequence composition. And, O-MAP requires no genetic manipulation and uses exclusively off-the-shelf parts. We therefore anticipate its application to a broad array of RNA phenomena.

molecular biology↗

bioRxiv2021

Systematic reconstruction of the cellular trajectories of mammalian embryogenesis

Mammalian embryogenesis is characterized by rapid cellular proliferation and diversification. Within a few weeks, a single cell zygote gives rise to millions of cells expressing a panoply of molecular programs, including much of the diversity that will subsequently be present in adult tissues. Although intensively studied, a comprehensive delineation of the major cellular trajectories that comprise mammalian development in vivo remains elusive. Here we set out to integrate several single cell RNA-seq datasets (scRNA-seq) that collectively span mouse gastrulation and organogenesis. We define cell states at each of 19 successive stages spanning E3.5 to E13.5, heuristically connect them with their pseudo-ancestors and pseudo-descendants, and for a subset of stages, deconvolve their approximate spatial distributions. Despite being constructed through automated procedures, the resulting trajectories of mammalian embryogenesis (TOME) are largely consistent with our contemporary understanding of mammalian development. We leverage TOME to nominate transcription factors (TF) and TF motifs as key regulators of each branch point at which a new cell type emerges. Finally, to facilitate comparisons across vertebrates, we apply the same procedures to single cell datasets of zebrafish and frog embryogenesis, and nominate "cell type homologs" based on shared regulators and transcriptional states.

developmental biology↗

bioRxiv2020

Single-cell landscape of nuclear configuration and gene expression during stem cell differentiation and X inactivation

Mammalian 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 obtained by high-throughput single-cell RNA-seq, ATAC-seq, and Hi-C. In differentiated cells, contact decay profiles, which clearly distinguish the active and inactive X chromosomes, reveal loss of the inactive X-specific structure at mitosis followed by a rapid reappearance, suggesting a bookkeeping mechanism. In differentiating embryonic stem cells, changes in contact decay profiles are detected in parallel on both the X chromosomes and autosomes, suggesting profound simultaneous reorganization. The onset of the inactive X-specific structure in single cells is notably delayed relative to that of gene silencing, consistent with the idea that chromatin compaction is a late event of X inactivation. Novel computational approaches to effectively align single-cell gene expression, chromatin accessibility, and 3D chromosome structure reveal that long-range structural changes to chromosomes appear as discrete events, unlike progressive changes in gene expression and chromatin accessibility.

genomics↗

bioRxiv2019

Architectural RNA is required for heterochromatin organization

In addition to its known roles in protein synthesis and enzyme catalysis, RNA has been proposed to stabilize higher-order chromatin structure. To distinguish presumed architectural roles of RNA from other functions, we applied a ribonuclease digestion strategy to our CUT&RUN in situ chromatin profiling method (CUT&RUN.RNase). We find that depletion of RNA compromises association of the murine nucleolar protein Nucleophosmin with pericentric heterochromatin and alters the chromatin environment of CCCTC-binding factor (CTCF) bound regions. Strikingly, we find that RNA maintains the integrity of both constitutive (H3K9me3 marked) and facultative (H3K27me3 marked) heterochromatic regions as compact domains, but only moderately stabilizes euchromatin. To establish the specificity of heterochromatin stabilization by RNA, we performed CUT&RUN on cells deleted for the Firre long non-coding RNA and observed disruption of H3K27me3 domains on several chromosomes. We conclude that RNA maintains local and global chromatin organization by acting as a structural scaffold for heterochromatic domains.

genomics↗

bioRxiv2019

Trans- and cis-acting effects of the lncRNA Firre on epigenetic and structural features of the inactive X chromosome

Firre encodes a lncRNA involved in nuclear organization in mammals. Here we find that Firre RNA is transcribed from the active X chromosome (Xa) and exerts trans-acting effects on the inactive X chromosome (Xi). Allelic deletion of Firre on the Xa in a mouse hybrid fibroblast cell line results in a dramatic loss of the histone modification H3K27me3 and of components of the PRC2 complex on the Xi as well as the disruption of the perinucleolar location of the Xi. These features are measurably rescued by ectopic expression of a mouse or human Firre/FIRRE cDNA transgene, strongly supporting a conserved trans-acting role of the Firre transcript in maintaining the Xi heterochromatin environment. Surprisingly, CTCF occupancy is decreased on the Xi upon loss of Firre RNA, but is partially recovered by ectopic transgene expression, suggesting a functional link between Firre RNA and CTCF in maintenance of epigenetic features and/or location of the Xi. Loss of Firre RNA results in dysregulation of genes implicated in cell division and development, but not in reactivation of genes on the Xi, which retains its bipartite structure despite some changes in chromatin contact distribution. Allelic deletion or inversion of Firre on the Xi causes localized redistribution of chromatin contacts, apparently dependent on the orientation of CTCF binding sites clustered at the locus. Thus, the Firre locus and its RNA have roles in the maintenance of epigenetic features and structure of the Xi.

genomics↗

bioRxiv2019

Sci-Hi-C: a single-cell Hi-C method for mapping 3D genome organization in large number of single cells

The highly dynamic nature of chromosome conformation and three-dimensional (3D) genome organization leads to cell-to-cell variability in chromatin interactions within a cell population, even if the cells of the population appear to be functionally homogeneous. Hence, although Hi-C is a powerful tool for mapping 3D genome organization, this heterogeneity of chromosome higher order structure among individual cells limits the interpretive power of population based bulk Hi-C assays. Moreover, single-cell studies have the potential to enable the identification and characterization of rare cell populations or cell subtypes in a heterogeneous population. However, it may require surveying relatively large numbers of single cells to achieve statistically meaningful observations in single-cell studies. By applying combinatorial cellular indexing to chromosome conformation capture, we developed single-cell combinatorial indexed Hi-C (sci-Hi-C), a high throughput method that enables mapping chromatin interactomes in large number of single cells. We demonstrated the use of sci-Hi-C data to separate cells by karytoypic and cell-cycle state differences and to identify cellular variability in mammalian chromosomal conformation. Here, we provide a detailed description of method design and step-by-step working protocols for sci-Hi-C.

genomics↗

bioRxiv2018

High-throughput mapping of meiotic crossover and chromosome mis-segregation events in interspecific hybrid mice

We developed \"sci-LIANTI\", a high-throughput, high-coverage single-cell DNA sequencing method that combines single-cell combinatorial indexing (\"sci\") and linear amplification via transposon insertion (\"LIANTI\"). To characterize rare chromosome mis-segregation events in male meiosis and their relationship to the landscape of meiotic crossovers, we applied sci-LIANTI to profile the genomes of 6,928 sperm and sperm precursors from infertile, interspecific F1 male mice. From 1,663 haploid and 292 diploid cells, we mapped 24,672 crossover events and identified genomic and epigenomic contexts that influence crossover hotness. Surprisingly, we observed frequent mitotic chromosome segregation during meiosis. Moreover, segregation during meiosis in individual cells was highly biased towards either mitotic or meiotic events. We anticipate that sci-LIANTI can be applied to fully characterize various recombination landscapes, as well as to other fields requiring high-throughput, high-coverage single-cell genome sequencing.\n\nOne Sentence SummarySingle-cell genome sequencing maps crossover and non-meiotic chromosome segregation during spermatogenesis in interspecific hybrid mice.

genetics↗

bioRxiv2017

Using DNase Hi-C techniques to map global and local three-dimensional genome architecture at high resolution

The folding and three-dimensional (3D) organization of chromatin in the nucleus critically impacts genome function. The past decade has witnessed rapid advances in genomic tools for delineating 3D genome architecture. Among them, chromosome conformation capture (3C)-based methods such as Hi-C are the most widely used techniques for mapping chromatin interactions. However, traditional Hi-C protocols rely on restriction enzymes (REs) to fragment chromatin and are therefore limited in resolution. We recently developed DNase Hi-C for mapping 3D genome organization, which uses DNase I for chromatin fragmentation. DNase Hi-C overcomes RE-related limitations associated with traditional Hi-C methods, leading to improved methodological resolution. Furthermore, combining this method with DNA capture technology provides a high-throughput approach (targeted DNase Hi-C) that allows for mapping fine-scale chromatin architecture at exceptionally high resolution. Hence, targeted DNase Hi-C will be valuable for delineating the physical landscapes of cis-regulatory networks that control gene expression and for characterizing phenotype-associated chromatin 3D signatures. Here, we provide a detailed description of method design and step-by-step working protocols for these two methods.\n\nHighlightsO_LIDNase Hi-C, a method for comprehensive mapping of chromatin contacts on a whole-genome scale, is based on random chromatin fragmentation by DNase I digestion instead of sequence-specific restriction enzyme (RE) digestion.\nC_LIO_LITargeted DNase Hi-C, which combines DNase Hi-C with DNA capture technology, is a high-throughput method for mapping fine-scale chromatin architecture of genomic loci of interest at a resolution comparable to that of genomic annotations of functional elements.\nC_LIO_LIDNase Hi-C and targeted DNase Hi-C provide the first high-throughput way to overcome the RE-digestion-associated resolution limit of 3C-based methods.\nC_LIO_LIStep-by-step whole-genome and targeted DNase Hi-C protocols for mapping global and local 3D genome architecture, respectively, are described.\nC_LI

genomics↗

bioRxiv2017

Orientation-dependent Dxz4 contacts shape the 3D structure of the inactive X chromosome

The mammalian inactive X chromosome (Xi) condenses into a bipartite structure with two superdomains of frequent long-range contacts separated by a boundary or hinge region. Using in situ DNase Hi-C in mouse cells with deletions or inversions within the hinge we show that the conserved repeat locus Dxz4 alone is sufficient to maintain the bipartite structure and that Dxz4 orientation controls the distribution of long-range contacts on the Xi. Frequent long-range contacts between Dxz4 and the telomeric superdomain are either lost after its deletion or shifted to the centromeric superdomain after its inversion. This massive reversal in contact distribution is consistent with the reversal of CTCF motif orientation at Dxz4. De-condensation of the Xi after Dxz4 deletion is associated with partial restoration of TADs normally attenuated on the Xi. There is also an increase in chromatin accessibility and CTCF binding on the Xi after Dxz4 deletion or inversion, but few changes in gene expression, in accordance with multiple epigenetic mechanisms ensuring X silencing. We propose that Dxz4 represents a structural platform for frequent long-range contacts with multiple loci in a direction dictated by the orientation of a bank of CTCF motifs at Dxz4, which may work as a ratchet to form the distinctive bipartite structure of the condensed Xi.

molecular biology↗