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.

A single-cell time-lapse of mouse prenatal development from gastrula to birth.

The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data4-8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb.

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.

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.

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

Background: The 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. Results: We 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. Conclusion: Our 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.

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.

Phenotypes and Genotypes in Patients with SMC1A-Related Developmental and Epileptic Encephalopathy.

The X-linked SMC1A gene encodes a core subunit of the cohesin complex that plays a pivotal role in genome organization and gene regulation. Pathogenic variants in SMC1A are often dominant-negative and cause Cornelia de Lange syndrome (CdLS) with growth retardation and typical facial features; however, rare SMC1A variants cause a developmental and epileptic encephalopathy (DEE) with intractable early-onset epilepsy that is absent in CdLS. Unlike the male-to-female ratio of 1:2 in those with CdLS associated with dominant-negative SMC1A variants, SMC1A-DEE loss-of-function (LOF) variants are found exclusively in females due to presumed lethality in males. It is unclear how different SMC1A variants cause CdLS or DEE. Here, we report on phenotypes and genotypes of three females with DEE and de novo SMC1A variants, including a novel splice-site variant. We also summarize 41 known SMC1A-DEE variants to characterize common and patient-specific features. Interestingly, compared to 33 LOFs detected throughout the gene, 7/8 non-LOFs are specifically located in the N/C-terminal ATPase head or the central hinge domain, both of which are predicted to affect cohesin assembly, thus mimicking LOFs. Along with the characterization of X-chromosome inactivation (XCI) and SMC1A transcription, these variants strongly suggest that a differential SMC1A dosage effect of SMC1A-DEE variants is closely associated with the manifestation of DEE phenotypes.

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.

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

BACKGROUND: 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 from these three modalities obtained by high-throughput single-cell RNA-seq, ATAC-seq, and Hi-C. RESULTS: Allele-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. CONCLUSIONS: Based 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.

X-factors in human disease: impact of gene content and dosage regulation.

The gene content of the X and Y chromosomes has dramatically diverged during evolution. The ensuing dosage imbalance within the genome of males and females has led to unique chromosome-wide regulatory mechanisms with significant and sex-specific impacts on X-linked gene expression. X inactivation or silencing of most genes on one X chromosome chosen at random in females profoundly affects the manifestation of X-linked diseases, as males inherit a single maternal allele, while females express maternal and paternal alleles in a mosaic manner. An additional complication is the existence of genes that escape X inactivation and thus are ubiquitously expressed from both alleles in females. The mosaic nature of X-linked gene expression and the potential for escape can vary between individuals, tissues, cell types and stages of life. Our understanding of the specialized nature of X-linked genes and of the multilayer epigenetic regulation that influence their expression throughout the organism has been helped by molecular studies conducted by tissue-specific and single-cell-specific approaches. In turn, the definition of molecular events that control X silencing has helped develop new approaches for the treatment of some X-linked disorders. This review focuses on the peculiarities of the X chromosome genetic content and epigenetic regulation in shaping the manifestation of congenital and acquired X-linked disorders in a sex-specific manner.

Deletion of FUNDC2 and CMC4 on Chromosome Xq28 Is Sufficient to Cause Hypergonadotropic Hypogonadism in Men.

BACKGROUND: Hypergonadotropic hypogonadism (HH) is characterized by low sex steroid levels and secondarily elevated gonadotropin levels with either congenital or acquired etiology. Genetic factors leading to HH have yet to be fully elucidated. METHODS: Here, we report on genome and transcriptome data analyses from a male patient with HH and history of growth delay who has an inherited deletion of chromosome Xq28. Expression analyses were done for this patient and his unaffected family members and compared to normal controls to identify dysregulated genes due to this deletion. RESULTS: Our patient's Xq28 deletion is 44,806 bp and contains only two genes, FUNDC2 and CMC4. Expression of both FUNDC2 and CMC4 are completely abolished in the patient. Gene ontology analyses of differentially expressed genes (DEGs) in the patient in comparison to controls show that significantly up-regulated genes in the patient are enriched in Sertoli cell barrier (SCB) regulation, apoptosis, inflammatory response, and gonadotropin-releasing regulation. Indeed, our patient has an elevated follicle stimulating hormone (FSH) level, which regulates Sertoli cell proliferation and spermatogenesis. In his mother and sister, who are heterozygous for this deletion, X-chromosome inactivation (XCI) is skewed toward the deleted X, suggesting a mechanism to avoid FSH dysregulation. CONCLUSION: Compared to the previously reported men with variable sized Xq28 deletions, our study suggests that loss of function of FUNDC2 and CMC4 results in dysregulation of apoptosis, inflammation, and FSH, and is sufficient to cause Xq28-associated HH.

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

Firre encodes a lncRNA involved in nuclear organization. Here, we show that Firre RNA expressed from the active X chromosome maintains histone H3K27me3 enrichment on the inactive X chromosome (Xi) in somatic cells. This trans-acting effect involves SUZ12, reflecting interactions between Firre RNA and components of the Polycomb repressive complexes. Without Firre RNA, H3K27me3 decreases on the Xi and the Xi-perinucleolar location is disrupted, possibly due to decreased CTCF binding on the Xi. We also observe widespread gene dysregulation, but not on the Xi. These effects are measurably rescued by ectopic expression of mouse or human Firre/FIRRE transgenes, supporting conserved trans-acting roles. We also find that the compact 3D structure of the Xi partly depends on the Firre locus and its RNA. In common lymphoid progenitors and T-cells Firre exerts a cis-acting effect on maintenance of H3K27me3 in a 26 Mb region around the locus, demonstrating cell type-specific trans- and cis-acting roles of this lncRNA.

Structural optimization of pyrazolo[1,5-a]pyrimidine derivatives as potent and highly selective DPP-4 inhibitors.

Our previous discovery of pyrazolo [1,5-a]pyrimidin-7(4H)-one scaffold-based DPP-4 inhibitors yielded two potent compounds b2 (IC50 = 79 nM) and d1 (IC50 = 49 nM) but characterized by cytotoxicity. Herein, with scaffold hopping and fragment-based drug design strategies, highly potent and selective pyrazolo [1,5-a]pyrimidine DPP-4 inhibitors were found featured by reduced or diminished cytotoxicity. Specifically, c24 (IC50 = 2 nM) exhibits a 25 to 40-fold increase of inhibitory activity respect to those of b2 and d1, respectively, 2-fold from Alogliptin (IC50 = 4 nM), and remarkable selectivity over DPP-8 and DPP-9 (>2000 fold). Further docking studies confirmed that the pyrazolo [1,5-a]pyrimidine core interacts with the S1 pocket whereas its substituted aromatic ring interacts with the sub-S1 pocket. The interactive mode in this case resembles that of Alogliptin and Trelagliptin. Further in vivo IPGTT assays in diabetic mice demonstrated that c24 effectively reduces glucose excursion by 48% at the dose of 10 mg/kg, suggesting that c24 is worthy of further development as a potent anti-diabetes agent.

Drug discovery approaches targeting the incretin pathway.

Incretin pathway plays an important role in the development of diabetes medications. Interventions in DPP-4 and GLP-1 receptor have shown remarkable efficacy in experimental and clinical studies and imperatively become one of the most promising therapeutic approaches in the T2DM drug discovery pipeline. Herein, we analyzed the actionmechanismsof DPP-4 and GLP-1 receptor targeting the incretin pathway in T2DM treatment. We gave an insight into the structural requirements for the potent DPP-4 inhibitors and revealed a classification of DPP-4 inhibitors by stressing on the binding modes of these ligands to the enzyme. We then reviewed the drug discovery strategies for the development of peptide and non-peptide GLP-1 receptor agonists (GLP-1 RAs). Furthermore, the drug design strategies for DPP-4 inhibitors and GLP-1R agonists were detailed accurately. This review might provide an efficient evidence for the highly potent and selective DPP-4 inhibitors and the GLP-1 RAs, as novel medicines for patients suffering from T2DM.

Moderation of mitochondrial respiration mitigates metabolic syndrome of aging.

Deregulation of mitochondrial dynamics leads to the accumulation of oxidative stress and unhealthy mitochondria; consequently, this accumulation contributes to premature aging and alterations in mitochondria linked to metabolic complications. We postulate that restrained mitochondrial ATP synthesis might alleviate age-associated disorders and extend healthspan in mammals. Herein, we prepared a previously discovered mitochondrial complex IV moderate inhibitor in drinking water and orally administered to standard-diet-fed, wild-type C57BL/6J mice every day for up to 16 mo. No manifestation of any apparent toxicity or deleterious effect on studied mouse models was observed. The impacts of an added inhibitor on a variety of mitochondrial functions were analyzed, such as respiratory activity, mitochondrial bioenergetics, and biogenesis, and a few age-associated comorbidities, including reactive oxygen species (ROS) production, glucose abnormalities, and obesity in mice. It was found that mitochondrial quality, dynamics, and oxidative metabolism were greatly improved, resulting in lean mice with a specific reduction in visceral fat plus superb energy and glucose homeostasis during their aging period compared to the control group. These results strongly suggest that a mild interference in ATP synthesis through moderation of mitochondrial activity could effectively up-regulate mitogenesis, reduce ROS production, and preserve mitochondrial integrity, thereby impeding the onset of metabolic syndrome. We conclude that this inhibitory intervention in mitochondrial respiration rectified the age-related physiological breakdown in mice by protecting mitochondrial function and markedly mitigated certain undesired primary outcomes of metabolic syndrome, such as obesity and type 2 diabetes. This intervention warrants further research on the treatment of metabolic syndrome of aging in humans.

Unsupervised manifold alignment for single-cell multi-omics data.

Integrating single-cell measurements that capture different properties of the genome is vital to extending our understanding of genome biology. This task is challenging due to the lack of a shared axis across datasets obtained from different types of single-cell experiments. For most such datasets, we lack corresponding information among the cells (samples) and the measurements (features). In this scenario, unsupervised algorithms that are capable of aligning single-cell experiments are critical to learning an in silico co-assay that can help draw correspondences among the cells. Maximum mean discrepancy-based manifold alignment (MMD-MA) is such an unsupervised algorithm. Without requiring correspondence information, it can align single-cell datasets from different modalities in a common shared latent space, showing promising results on simulations and a small-scale single-cell experiment with 61 cells. However, it is essential to explore the applicability of this method to larger single-cell experiments with thousands of cells so that it can be of practical interest to the community. In this paper, we apply MMD-MA to two recent datasets that measure transcriptome and chromatin accessibility in ~2000 single cells. To scale the runtime of MMD-MA to a more substantial number of cells, we extend the original implementation to run on GPUs. We also introduce a method to automatically select one of the user-defined parameters, thus reducing the hyperparameter search space. We demonstrate that the proposed extensions allow MMD-MA to accurately align state-of-the-art single-cell experiments.

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.

Anti-diabetic potential of Pueraria lobata root extract through promoting insulin signaling by PTP1B inhibition.

Type 2 diabetes mellitus is a fast-growing epidemic affecting people globally. We initiated the project by searching the possible target of the Pueraria lobata root extract (P. lobata). We conducted the IC50 assays of P. lobata on the four diabetes-related proteins: PTP1B, TCPTP, SHP-2 and DPP-4. Results indicated that P. lobata exhibited high PTP1B inhibitory activity with IC50 of 0.043 mg/ml. Treated insulin-resistant HepG2 cells with 0.0115 mg/ml of P. lobata increased the glucose uptake by two times compared with the negative control. Further, we performed OGTT test on the diabetic C57BL/6 male mice. 20% decreased blood glucose (AUC) was obtained with a dose of 1 g/kg P. lobata compared with the negative control. Herein, we were able to demonstrate the antidiabetic effects of P. lobata might be related to the inhibition of PTP1B and therefore, bettering the insulin signaling pathway.

X-Chromosome Inactivation and Escape from X Inactivation in Mouse.

X chromosome inactivation silences one X chromosome in female mammals. However, this silencing is incomplete, and some genes escape X inactivation. We describe methods to determine the chromosome-wide X inactivation status of genes in tissues or cell lines derived from mice using a combination of skewing of X inactivation and allele-specific analyses of gene expression based on RNA-seq.