Single-cell sequencing reveals lineage-specific dynamic genetic regulation of gene expression during human cardiomyocyte differentiation.

Dynamic and temporally specific gene regulatory changes may underlie unexplained genetic associations with complex disease. During a dynamic process such as cellular differentiation, the overall cell type composition of a tissue (or an in vitro culture) and the gene regulatory profile of each cell can both experience significant changes over time. To identify these dynamic effects in high resolution, we collected single-cell RNA-sequencing data over a differentiation time course from induced pluripotent stem cells to cardiomyocytes, sampled at 7 unique time points in 19 human cell lines. We employed a flexible approach to map dynamic eQTLs whose effects vary significantly over the course of bifurcating differentiation trajectories, including many whose effects are specific to one of these two lineages. Our study design allowed us to distinguish true dynamic eQTLs affecting a specific cell lineage from expression changes driven by potentially non-genetic differences between cell lines such as cell composition. Additionally, we used the cell type profiles learned from single-cell data to deconvolve and re-analyze data from matched bulk RNA-seq samples. Using this approach, we were able to identify a large number of novel dynamic eQTLs in single cell data while also attributing dynamic effects in bulk to a particular lineage. Overall, we found that using single cell data to uncover dynamic eQTLs can provide new insight into the gene regulatory changes that occur among heterogeneous cell types during cardiomyocyte differentiation.

Elevated and sustained expression of the transcription factors Egr1 and Egr2 controls NKT lineage differentiation in response to TCR signaling.

Interactions driven by the T cell antigen receptor (TCR) determine the lineage fate of CD4(+)CD8(+) thymocytes, but the molecular mechanisms that induce the lineage-determining transcription factors are unknown. Here we found that TCR-induced transcription factors Egr2 and Egr1 had higher and more-prolonged expression in precursors of the natural killer T (NKT) than in cells of conventional lineages. Chromatin immunoprecipitation followed by deep sequencing showed that Egr2 directly bound and activated the promoter of Zbtb16, which encodes the NKT lineage-specific transcription factor PLZF. Egr2 also bound the promoter of Il2rb, which encodes the interleukin 2 (IL-2) receptor ß-chain, and controlled the responsiveness to IL-15, which signals the terminal differentiation of the NKT lineage. Thus, we propose that persistent higher expression of Egr2 specifies the early and late stages of NKT lineage differentiation, providing a discriminating mechanism that enables TCR signaling to 'instruct' a thymic lineage.

Characterizing and inferring quantitative cell cycle phase in single-cell RNA-seq data analysis.

Cellular heterogeneity in gene expression is driven by cellular processes, such as cell cycle and cell-type identity, and cellular environment such as spatial location. The cell cycle, in particular, is thought to be a key driver of cell-to-cell heterogeneity in gene expression, even in otherwise homogeneous cell populations. Recent advances in single-cell RNA-sequencing (scRNA-seq) facilitate detailed characterization of gene expression heterogeneity and can thus shed new light on the processes driving heterogeneity. Here, we combined fluorescence imaging with scRNA-seq to measure cell cycle phase and gene expression levels in human induced pluripotent stem cells (iPSCs). By using these data, we developed a novel approach to characterize cell cycle progression. Although standard methods assign cells to discrete cell cycle stages, our method goes beyond this and quantifies cell cycle progression on a continuum. We found that, on average, scRNA-seq data from only five genes predicted a cell's position on the cell cycle continuum to within 14% of the entire cycle and that using more genes did not improve this accuracy. Our data and predictor of cell cycle phase can directly help future studies to account for cell cycle-related heterogeneity in iPSCs. Our results and methods also provide a foundation for future work to characterize the effects of the cell cycle on expression heterogeneity in other cell types.

Fully interpretable deep learning model of transcriptional control.

MOTIVATION: The universal expressibility assumption of Deep Neural Networks (DNNs) is the key motivation behind recent worksin the systems biology community to employDNNs to solve important problems in functional genomics and moleculargenetics. Typically, such investigations have taken a 'black box' approach in which the internal structure of themodel used is set purely by machine learning considerations with little consideration of representing the internalstructure of the biological system by the mathematical structure of the DNN. DNNs have not yet been applied to thedetailed modeling of transcriptional control in which mRNA production is controlled by the binding of specific transcriptionfactors to DNA, in part because such models are in part formulated in terms of specific chemical equationsthat appear different in form from those used in neural networks. RESULTS: In this paper, we give an example of a DNN whichcan model the detailed control of transcription in a precise and predictive manner. Its internal structure is fully interpretableand is faithful to underlying chemistry of transcription factor binding to DNA. We derive our DNN from asystems biology model that was not previously recognized as having a DNN structure. Although we apply our DNNto data from the early embryo of the fruit fly Drosophila, this system serves as a test bed for analysis of much larger datasets obtained by systems biology studies on a genomic scale. . AVAILABILITY AND IMPLEMENTATION: The implementation and data for the models used in this paper are in a zip file in the supplementary material. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

An in silico analysis of robust but fragile gene regulation links enhancer length to robustness.

Organisms must ensure that expression of genes is directed to the appropriate tissues at the correct times, while simultaneously ensuring that these gene regulatory systems are robust to perturbation. This idea is captured by a mathematical concept called r-robustness, which says that a system is robust to a perturbation in up to r - 1 randomly chosen parameters. r-robustness implies that the biological system has a small number of sensitive parameters and that this number can be used as a robustness measure. In this work we use this idea to investigate the robustness of gene regulation using a sequence level model of the Drosophila melanogaster gene even-skipped. We consider robustness with respect to mutations of the enhancer sequence and with respect to changes of the transcription factor concentrations. We find that gene regulation is r-robust with respect to mutations in the enhancer sequence and identify a number of sensitive nucleotides. In both natural and in silico predicted enhancers, the number of nucleotides that are sensitive to mutation correlates negatively with the length of the sequence, meaning that longer sequences are more robust. The exact degree of robustness obtained is dependent not only on DNA sequence, but also on the local concentration of regulatory factors. We find that gene regulation can be remarkably sensitive to changes in transcription factor concentrations at the boundaries of expression features, while it is robust to perturbation elsewhere.

A synthetic biology approach to the development of transcriptional regulatory models and custom enhancer design.

Synthetic biology offers novel opportunities for elucidating transcriptional regulatory mechanisms and enhancer logic. Complex cis-regulatory sequences--like the ones driving expression of the Drosophila even-skipped gene--have proven difficult to design from existing knowledge, presumably due to the large number of protein-protein interactions needed to drive the correct expression patterns of genes in multicellular organisms. This work discusses two novel computational methods for the custom design of enhancers that employ a sophisticated, empirically validated transcriptional model, optimization algorithms, and synthetic biology. These synthetic elements have both utilitarian and academic value, including improving existing regulatory models as well as evolutionary questions. The first method involves the use of simulated annealing to explore the sequence space for synthetic enhancers whose expression output fit a given search criterion. The second method uses a novel optimization algorithm to find functionally accessible pathways between two enhancer sequences. These paths describe a set of mutations wherein the predicted expression pattern does not significantly vary at any point along the path. Both methods rely on a predictive mathematical framework that maps the enhancer sequence space to functional output.

Guided Differentiation of Pluripotent Stem Cells for Cardiac Cell Diversity.

We have developed a guided differentiation protocol for induced pluripotent stem cells (iPSCs) that rapidly generates a temporally and functionally diverse set of cardiac-relevant cell types. By leveraging techniques used in embryoid body and cardiac organoid generation, we produce both progenitor and terminal cardiac cell types concomitantly in just 10 days. Our results show that guided differentiation generates functionally relevant cardiac cell types that closely align with the transcriptional profiles of cells from differentiation time-course collections, mature cardiac organoids, and in vivo heart tissue. Guided differentiation prioritizes simplicity by minimizing the number of reagents and steps required, thereby enabling rapid and cost-effective experimental throughput. We expect this approach will provide a scalable cardiac model for population-level studies of gene regulatory variation and gene-by-environment interactions.

Cell-type and dynamic state govern genetic regulation of gene expression in heterogeneous differentiating cultures.

Identifying the molecular effects of human genetic variation across cellular contexts is crucial for understanding the mechanisms underlying disease-associated loci, yet many cell-types and developmental stages remain underexplored. Here we harnessed the potential of heterogeneous differentiating cultures ( HDCs ), an in vitro system in which pluripotent cells asynchronously differentiate into a broad spectrum of cell-types. We generated HDCs for 53 human donors and collected single-cell RNA-sequencing data from over 900,000 cells. We identified expression quantitative trait loci in 29 cell-types and characterized regulatory dynamics across diverse differentiation trajectories. This revealed novel regulatory variants for genes involved in key developmental and disease-related processes while replicating known effects from primary tissues, and dynamic regulatory effects associated with a range of complex traits.

The relationship between regulatory changes in cis and trans and the evolution of gene expression in humans and chimpanzees.

BACKGROUND: Comparative gene expression studies in apes are fundamentally limited by the challenges associated with sampling across different tissues. Here, we used single-cell RNA sequencing of embryoid bodies to collect transcriptomic data from over 70 cell types in three humans and three chimpanzees. RESULTS: We find hundreds of genes whose regulation is conserved across cell types, as well as genes whose regulation likely evolves under directional selection in one or a handful of cell types. Using embryoid bodies from a human-chimpanzee fused cell line, we also infer the proportion of inter-species regulatory differences due to changes in cis and trans elements between the species. Using the cis/trans inference and an analysis of transcription factor binding sites, we identify dozens of transcription factors whose inter-species differences in expression are affecting expression differences between humans and chimpanzees in hundreds of target genes. CONCLUSIONS: Here, we present the most comprehensive dataset of comparative gene expression from humans and chimpanzees to date, including a catalog of regulatory mechanisms associated with inter-species differences.

FK506-binding protein (FKBP) partitions a modified HIV protease inhibitor into blood cells and prolongs its lifetime in vivo.

HIV protease inhibitors are a key component of anti-retroviral therapy, but their susceptibility to cytochrome P(450) metabolism reduces their systemic availability and necessitates repetitive dosing. Importantly, failure to maintain adequate inhibitor levels is believed to provide an opportunity for resistance to emerge; thus, new strategies to prolong the lifetime of these drugs are needed. Toward this goal, numerous prodrug approaches have been developed, but these methods involve creating inactive precursors that require enzymatic processing. Using an alternative strategy inspired by the natural product FK506, we have synthetically modified an HIV protease inhibitor such that it acquires high affinity for the abundant, cytoplasmic chaperone, FK506-binding protein (FKBP). This modified protease inhibitor maintains activity against HIV-1 protease (IC(50) = 19 nM) and, additionally, it is partitioned into the cellular component of whole blood via binding to FKBP. Interestingly, redistribution into this protected niche reduces metabolism and improves its half-life in mice by almost 20-fold compared with the unmodified compound. Based on these findings, we propose that addition of FKBP-binding groups might partially overcome the poor pharmacokinetic properties of existing HIV protease inhibitors and, potentially, other drug classes.

Human embryoid bodies as a novel system for genomic studies of functionally diverse cell types.

Practically all studies of gene expression in humans to date have been performed in a relatively small number of adult tissues. Gene regulation is highly dynamic and context-dependent. In order to better understand the connection between gene regulation and complex phenotypes, including disease, we need to be able to study gene expression in more cell types, tissues, and states that are relevant to human phenotypes. In particular, we need to characterize gene expression in early development cell types, as mutations that affect developmental processes may be of particular relevance to complex traits. To address this challenge, we propose to use embryoid bodies (EBs), which are organoids that contain a multitude of cell types in dynamic states. EBs provide a system in which one can study dynamic regulatory processes at an unprecedentedly high resolution. To explore the utility of EBs, we systematically explored cellular and gene expression heterogeneity in EBs from multiple individuals. We characterized the various cell types that arise from EBs, the extent to which they recapitulate gene expression in vivo, and the relative contribution of technical and biological factors to variability in gene expression, cell composition, and differentiation efficiency. Our results highlight the utility of EBs as a new model system for mapping dynamic inter-individual regulatory differences in a large variety of cell types.

One major goal of human genetics is to understand how changes in the way genes are regulated affect human traits, including disease susceptibility. To date, most studies of gene regulation have been performed in adult tissues, such as liver or kidney tissue, that were collected at a single time point. Yet, gene regulation is highly dynamic and context-dependent, meaning that it is important to gather data from a greater variety of cell types at different stages of their development. Additionally, observing which genes switch on and off in response to external treatments can shed light on how genetic variation can drive errors in gene regulation and cause diseases. Stem cells can produce more cells like themselves or differentiate ­ acquire the characteristics ­ of many cell types. These cells have been used in the laboratory to research gene regulation. Unfortunately, these studies often fail to capture the complex spatial and temporal dynamics of stem cell differentiation; in particular, these studies are unable to observe gene regulation in the transient cell types that appear early in embryonic development. To overcome these limitations, scientists developed systems such as embryoid bodies: three-dimensional aggregates of stem cells that, when grown under certain conditions, spontaneously develop into a variety of cell types. Rhodes, Barr et al. wanted to assess the utility of embryoid bodies as a model to study how genes are dynamically regulated in different cell types, by different individuals who have distinct genetic makeups. To do this, they grew embryoid bodies made from human stem cells from different individuals to examine which genes switched on and off as the stem cells that formed the embryoid bodies differentiated into different types of cells. The results showed that it was possible to grow embryoid bodies derived from genetically distinct individuals that consistently produce diverse cell types, similar to those found during human fetal development. Rhodes, Barr et al.'s findings suggest that embryoid bodies are a useful model to study gene regulation across individuals with different genetic backgrounds. This could accelerate research into how genetics are associated with disease by capturing gene regulatory dynamics at an unprecedentedly high spatial and temporal resolution. Additionally, embryoid bodies could be used to explore how exposure to different environmental factors during early development affect disease-related outcomes in adulthood in different individuals.

Structure-activity relationship (SAR) of the α-amino acid residue of potent tetrahydroisoquinoline (THIQ)-derived LFA-1/ICAM-1 antagonists.

This letter describes the structure-activity relationship (SAR) of the 'right-wing' α-amino acid residue of potent tetrahydroisoquinoline (THIQ)-derived LFA-1/ICAM-1 antagonists. Novel (S)-substituted heteroaryl-bearing α-amino acids have been identified as replacements of the 'right-wing' (S)-2,3-diaminopropanoic acid (DAP) moiety. Improvement of potency in the Hut-78 assay in the presence of 10% human serum has also been achieved.

Divergence in alternative polyadenylation contributes to gene regulatory differences between humans and chimpanzees.

While comparative functional genomic studies have shown that inter-species differences in gene expression can be explained by corresponding inter-species differences in genetic and epigenetic regulatory mechanisms, co-transcriptional mechanisms, such as alternative polyadenylation (APA), have received little attention. We characterized APA in lymphoblastoid cell lines from six humans and six chimpanzees by identifying and estimating the usage for 44,432 polyadenylation sites (PAS) in 9518 genes. Although APA is largely conserved, 1705 genes showed significantly different PAS usage (FDR 0.05) between species. Genes with divergent APA also tend to be differentially expressed, are enriched among genes showing differences in protein translation, and can explain a subset of observed inter-species protein expression differences that do not differ at the transcript level. Finally, we found that genes with a dominant PAS, which is used more often than other PAS, are particularly enriched for differentially expressed genes.

Discovery of tetrahydroisoquinoline (THIQ) derivatives as potent and orally bioavailable LFA-1/ICAM-1 antagonists.

This letter describes the discovery of a novel series of tetrahydroisoquinoline (THIQ)-derived small molecules that potently inhibit both human T-cell migration and super-antigen induced T-cell activation through disruption of the binding of integrin LFA-1 to its receptor, ICAM-1. In addition to excellent in vitro potency, 6q shows good pharmacokinetic properties and its ethyl ester (6t) demonstrates good oral bioavailability in both mouse and rat. Either intravenous administration of 6q or oral administration of its ethyl ester (6t) produced a significant reduction of neutrophil migration in a thioglycollate-induced murine peritonitis model.

Structure-Based Design and Identification of FT-2102 (Olutasidenib), a Potent Mutant-Selective IDH1 Inhibitor.

Inhibition of mutant IDH1 is being evaluated clinically as a treatment option for oncology. Here we describe the structure-based design and optimization of quinoline lead compounds to identify FT-2102, a potent, orally bioavailable, brain penetrant, and selective mIDH1 inhibitor. FT-2102 has excellent ADME/PK properties and reduces 2-hydroxyglutarate levels in an mIDH1 xenograft tumor model. This compound has been selected as a candidate for clinical development in hematologic malignancies, solid tumors, and gliomas with mIDH1.

Discovery of N-(Indazol-3-yl)piperidine-4-carboxylic Acids as RORγt Allosteric Inhibitors for Autoimmune Diseases.

The clinical success of anti-IL-17 monoclonal antibodies (i.e., Cosentyx and Taltz) has validated Th17 pathway modulation for the treatment of autoimmune diseases. The nuclear hormone receptor RORγt is a master regulator of Th17 cells and affects the production of a host of cytokines, including IL-17A, IL-17F, IL-22, IL-26, and GM-CSF. Substantial interest has been spurred across both academia and industry to seek small molecules suitable for RORγt inhibition. A variety of RORγt inhibitors have been reported in the past few years, the majority of which are orthosteric binders. Here we disclose the discovery and optimization of a class of inhibitors, which bind differently to an allosteric binding pocket. Starting from a weakly active hit 1, a tool compound 14 was quickly identified that demonstrated superior potency, selectivity, and off-target profile. Further optimization focused on improving metabolic stability. Replacing the benzoic acid moiety with piperidinyl carboxylate, modifying the 4-aza-indazole core in 14 to 4-F-indazole, and incorporating a key hydroxyl group led to the discovery of 25, which possesses exquisite potency and selectivity, as well as an improved pharmacokinetic profile suitable for oral dosing.

Allosteric inhibition of protein tyrosine phosphatase 1B.

Obesity and type II diabetes are closely linked metabolic syndromes that afflict >100 million people worldwide. Although protein tyrosine phosphatase 1B (PTP1B) has emerged as a promising target for the treatment of both syndromes, the discovery of pharmaceutically acceptable inhibitors that bind at the active site remains a substantial challenge. Here we describe the discovery of an allosteric site in PTP1B. Crystal structures of PTP1B in complex with allosteric inhibitors reveal a novel site located approximately 20 A from the catalytic site. We show that allosteric inhibitors prevent formation of the active form of the enzyme by blocking mobility of the catalytic loop, thereby exploiting a general mechanism used by tyrosine phosphatases. Notably, these inhibitors exhibit selectivity for PTP1B and enhance insulin signaling in cells. Allosteric inhibition is a promising strategy for targeting PTP1B and constitutes a mechanism that may be applicable to other tyrosine phosphatases.

Discovery and Optimization of Quinolinone Derivatives as Potent, Selective, and Orally Bioavailable Mutant Isocitrate Dehydrogenase 1 (mIDH1) Inhibitors.

Mutations at the arginine residue (R132) in isocitrate dehydrogenase 1 (IDH1) are frequently identified in various human cancers. Inhibition of mutant IDH1 (mIDH1) with small molecules has been clinically validated as a promising therapeutic treatment for acute myeloid leukemia and multiple solid tumors. Herein, we report the discovery and optimization of a series of quinolinones to provide potent and orally bioavailable mIDH1 inhibitors with selectivity over wild-type IDH1. The X-ray structure of an early lead 24 in complex with mIDH1-R132H shows that the inhibitor unexpectedly binds to an allosteric site. Efforts to improve the in vitro and in vivo absorption, distribution, metabolism, and excretion (ADME) properties of 24 yielded a preclinical candidate 63. The detailed preclinical ADME and pharmacology studies of 63 support further development of quinolinone-based mIDH1 inhibitors as therapeutic agents in human trials.

Design and synthesis of 2-amino-pyrazolopyridines as Polo-like kinase 1 inhibitors.

A series of 2-amino-pyrazolopyridines was designed and synthesized as Polo-like kinase (Plk) inhibitors based on a low micromolar hit. The SAR was developed to provide compounds exhibiting low nanomolar inhibitory activity of Plk1; the phenotype of treated cells is consistent with Plk1 inhibition. A co-crystal structure of one of these compounds with zPlk1 confirms an ATP-competitive binding mode.

Structure of the Brachydanio rerio Polo-like kinase 1 (Plk1) catalytic domain in complex with an extended inhibitor targeting the adaptive pocket of the enzyme.

Polo-like kinase 1 (Plk1) is a member of the Polo-like kinase family of serine/threonine kinases involved in the regulation of cell-cycle progression and cytokinesis and is an attractive target for the development of anticancer therapeutics. The catalytic domain of this enzyme shares significant primary amino-acid homology and structural similarity with another mitotic kinase, Aurora A. While screening an Aurora A library of ATP-competitive compounds, a urea-containing inhibitor with low affinity for mouse Aurora A but with submicromolar potency for human and zebrafish Plk1 (hPlk1 and zPlk1, respectively) was identified. A crystal structure of the zebrafish Plk1 kinase domain-inhibitor complex reveals that the small molecule occupies the purine pocket and extends past the catalytic lysine into the adaptive region of the active site. Analysis of the structures of this protein-inhibitor complex and of similar small molecules cocrystallized with other kinases facilitates understanding of the specificity of the inhibitor for Plk1 and documents for the first time that Plk1 can accommodate extended ATP-competitive compounds that project toward the adaptive pocket and help the enzyme order its activation segment.