YAP induces a neonatal-like pro-renewal niche in the adult heart.

After myocardial infarction (MI), mammalian hearts do not regenerate, and the microenvironment is disrupted. Hippo signaling loss of function with activation of transcriptional co-factor YAP induces heart renewal and rebuilds the post-MI microenvironment. In this study, we investigated adult renewal-competent mouse hearts expressing an active version of YAP, called YAP5SA, in cardiomyocytes (CMs). Spatial transcriptomics and single-cell RNA sequencing revealed a conserved, renewal-competent CM cell state called adult (a)CM2 with high YAP activity. aCM2 co-localized with cardiac fibroblasts (CFs) expressing complement pathway component C3 and macrophages (MPs) expressing C3ar1 receptor to form a cellular triad in YAP5SA hearts and renewal-competent neonatal hearts. Although aCM2 was detected in adult mouse and human hearts, the cellular triad failed to co-localize in these non-renewing hearts. C3 and C3ar1 loss-of-function experiments indicated that C3a signaling between MPs and CFs was required to assemble the pro-renewal aCM2, C3+ CF and C3ar1+ MP cellular triad.

scGIST: gene panel design for spatial transcriptomics with prioritized gene sets.

A critical challenge of single-cell spatial transcriptomics (sc-ST) technologies is their panel size. Being based on fluorescence in situ hybridization, they are typically limited to panels of about a thousand genes. This constrains researchers to build panels from only the marker genes of different cell types and forgo other genes of interest, e.g., genes encoding ligand-receptor complexes or those in specific pathways. We propose scGIST, a constrained feature selection tool that designs sc-ST panels prioritizing user-specified genes without compromising cell type detection accuracy. We demonstrate scGIST's efficacy in diverse use cases, highlighting it as a valuable addition to sc-ST's algorithmic toolbox.

Increased iron uptake by splenic hematopoietic stem cells promotes TET2-dependent erythroid regeneration.

Hematopoietic stem cells (HSCs) are capable of regenerating the blood system, but the instructive cues that direct HSCs to regenerate particular lineages lost to the injury remain elusive. Here, we show that iron is increasingly taken up by HSCs during anemia and induces erythroid gene expression and regeneration in a Tet2-dependent manner. Lineage tracing of HSCs reveals that HSCs respond to hemolytic anemia by increasing erythroid output. The number of HSCs in the spleen, but not bone marrow, increases upon anemia and these HSCs exhibit enhanced proliferation, erythroid differentiation, iron uptake, and TET2 protein expression. Increased iron in HSCs promotes DNA demethylation and expression of erythroid genes. Suppressing iron uptake or TET2 expression impairs erythroid genes expression and erythroid differentiation of HSCs; iron supplementation, however, augments these processes. These results establish that the physiological level of iron taken up by HSCs has an instructive role in promoting erythroid-biased differentiation of HSCs.

Endothelial cells adopt a pro-reparative immune responsive signature during cardiac injury.

Modulation of the heart's immune microenvironment is crucial for recovery after ischemic events such as myocardial infarction (MI). Endothelial cells (ECs) can have immune regulatory functions; however, interactions between ECs and the immune environment in the heart after MI remain poorly understood. We identified an EC-specific IFN responsive and immune regulatory gene signature in adult and pediatric heart failure (HF) tissues. Single-cell transcriptomic analysis of murine hearts subjected to MI uncovered an EC population (IFN-ECs) with immunologic gene signatures similar to those in human HF. IFN-ECs were enriched in regenerative-stage mouse hearts and expressed genes encoding immune responsive transcription factors (Irf7, Batf2, and Stat1). Single-cell chromatin accessibility studies revealed an enrichment of these TF motifs at IFN-EC signature genes. Expression of immune regulatory ligand genes by IFN-ECs suggests bidirectional signaling between IFN-ECs and macrophages in regenerative-stage hearts. Our data suggest that ECs may adopt immune regulatory signatures after cardiac injury to accompany the reparative response. The presence of these signatures in human HF and murine MI models suggests a potential role for EC-mediated immune regulation in responding to stress induced by acute injury in MI and chronic adverse remodeling in HF.

ScribbleDom: using scribble-annotated histology images to identify domains in spatial transcriptomics data.

MOTIVATION: Spatial domain identification is a very important problem in the field of spatial transcriptomics. The state-of-the-art solutions to this problem focus on unsupervised methods, as there is lack of data for a supervised learning formulation. The results obtained from these methods highlight significant opportunities for improvement. RESULTS: In this article, we propose a potential avenue for enhancement through the development of a semi-supervised convolutional neural network based approach. Named "ScribbleDom", our method leverages human expert's input as a form of semi-supervision, thereby seamlessly combines the cognitive abilities of human experts with the computational power of machines. ScribbleDom incorporates a loss function that integrates two crucial components: similarity in gene expression profiles and adherence to the valuable input of a human annotator through scribbles on histology images, providing prior knowledge about spot labels. The spatial continuity of the tissue domains is taken into account by extracting information on the spot microenvironment through convolution filters of varying sizes, in the form of "Inception" blocks. By leveraging this semi-supervised approach, ScribbleDom significantly improves the quality of spatial domains, yielding superior results both quantitatively and qualitatively. Our experiments on several benchmark datasets demonstrate the clear edge of ScribbleDom over state-of-the-art methods-between 1.82% to 169.38% improvements in adjusted Rand index for 9 of the 12 human dorsolateral prefrontal cortex samples, and 15.54% improvement in the melanoma cancer dataset. Notably, when the expert input is absent, ScribbleDom can still operate, in a fully unsupervised manner like the state-of-the-art methods, and produces results that remain competitive. AVAILABILITY AND IMPLEMENTATION: Source code is available at Github (https://github.com/1alnoman/ScribbleDom) and Zenodo (https://zenodo.org/badge/latestdoi/681572669).

Structural underpinnings of mutation rate variations in the human genome.

Single nucleotide mutation rates have critical implications for human evolution and genetic diseases. Importantly, the rates vary substantially across the genome and the principles underlying such variations remain poorly understood. A recent model explained much of this variation by considering higher-order nucleotide interactions in the 7-mer sequence context around mutated nucleotides. This model's success implicates a connection between DNA shape and mutation rates. DNA shape, i.e. structural properties like helical twist and tilt, is known to capture interactions between nucleotides within a local context. Thus, we hypothesized that changes in DNA shape features at and around mutated positions can explain mutation rate variations in the human genome. Indeed, DNA shape-based models of mutation rates showed similar or improved performance over current nucleotide sequence-based models. These models accurately characterized mutation hotspots in the human genome and revealed the shape features whose interactions underlie mutation rate variations. DNA shape also impacts mutation rates within putative functional regions like transcription factor binding sites where we find a strong association between DNA shape and position-specific mutation rates. This work demonstrates the structural underpinnings of nucleotide mutations in the human genome and lays the groundwork for future models of genetic variations to incorporate DNA shape.

Noncanonical binding of transcription factors: time to revisit specificity?

Transcription factors (TFs) are one of the most studied classes of DNA-binding proteins that have a direct functional impact on gene transcription and thus, on human physiology and disease. The mechanisms that TFs use for recognizing target DNA binding sites have been studied for nearly five decades, yet they remain poorly understood. It is classically assumed that a TF recognizes a specific sequence pattern, or motif, as its binding sites. However, recent studies are consistently finding examples of noncanonical binding, that is, TFs binding at sites that do not resemble their sequence motifs. Here we review the current literature on four major types of noncanonical TF binding, namely binding based on DNA shape readout, at Guanine-quadruplex structures, at repeat sequences, and bispecific binding. These examples point to a critical need for studies to unify our current observations, many of which are at odds with the "one TF, one motif" view, into a more comprehensive definition of the DNA-binding specificity of TFs.

NoVaTeST: identifying genes with location-dependent noise variance in spatial transcriptomics data.

MOTIVATION: Spatial transcriptomics (ST) can reveal the existence and extent of spatial variation of gene expression in complex tissues. Such analyses could help identify spatially localized processes underlying a tissue's function. Existing tools to detect spatially variable genes assume a constant noise variance across spatial locations. This assumption might miss important biological signals when the variance can change across locations. RESULTS: In this article, we propose NoVaTeST, a framework to identify genes with location-dependent noise variance in ST data. NoVaTeST models gene expression as a function of spatial location and allows the noise to vary spatially. NoVaTeST then statistically compares this model to one with constant noise and detects genes showing significant spatial noise variation. We refer to these genes as "noisy genes." In tumor samples, the noisy genes detected by NoVaTeST are largely independent of the spatially variable genes detected by existing tools that assume constant noise, and provide important biological insights into tumor microenvironments. AVAILABILITY AND IMPLEMENTATION: An implementation of the NoVaTeST framework in Python along with instructions for running the pipeline is available at https://github.com/abidabrar-bracu/NoVaTeST.

DeepBend: An interpretable model of DNA bendability.

The bendability of genomic DNA impacts chromatin packaging and protein-DNA binding. However, we do not have a comprehensive understanding of the motifs influencing DNA bendability. Recent high-throughput technologies such as Loop-Seq offer an opportunity to address this gap but the lack of accurate and interpretable machine learning models still remains. Here we introduce DeepBend, a convolutional neural network model with convolutions designed to directly capture the motifs underlying DNA bendability and their periodic occurrences or relative arrangements that modulate bendability. DeepBend consistently performs on par with alternative models while giving an extra edge through mechanistic interpretations. Besides confirming the known motifs of DNA bendability, DeepBend also revealed several novel motifs and showed how the spatial patterns of motif occurrences influence bendability. DeepBend's genome-wide prediction of bendability further showed how bendability is linked to chromatin conformation and revealed the motifs controlling the bendability of topologically associated domains and their boundaries.

Outersphere Approach to Increasing the Persistance of Oxygen-Sensitive Europium(II)-Containing Contrast Agents for Magnetic Resonance Imaging with Perfluorocarbon Nanoemulsions toward Imaging of Hypoxia.

Radiographic mapping of hypoxia is needed to study a wide range of diseases. Complexes of Eu(II) are a promising class of molecules to fit this need, but they are generally limited by their rapid oxidation rates in vivo. Here, a perfluorocarbon-nanoemulsion perfused with N2 , forms an interface with aqueous layers to hinder oxidation of a new perfluorocarbon-soluble complex of Eu(II). Conversion of the perfluorocarbon solution of Eu(II) into nanoemulsions results in observable differences between reduced and oxidized forms by magnetic resonance imaging both in vitro and in vivo. Oxidation in vivo occurrs over a period of ≈30 min compared to <5 min for a comparable Eu(II)-containing complex without nanoparticle interfaces. These results represent a critical step toward delivery of Eu(II)-containing complexes in vivo for the study of hypoxia.

Assigning pathogenicity for TAB2 variants using a novel scalable functional assay and expanding TAB2 disease spectrum.

Haploinsufficiency of TGF-beta-activated kinase 1 (MAP3K7) binding protein 2 (TAB2) has been associated with congenital heart disease and more recently multiorgan structural abnormalities. Missense variant represents a major proportion of non-synonymous TAB2 variants reported in gnomAD (295/576) and Clinvar (16/73), most of which are variants of uncertain significance (VUSs). However, interpretation of TAB2 missense variants remains challenging because of lack of functional assays. To address this issue, we established a cell-based luciferase assay that enables high-throughput screening of TAB2 variants to assess the functional consequence for predicting variant pathogenicity. Using this platform, we screened 47 TAB2 variants including five pathogenic controls and one benign control, and the results showed that the transcriptional activity of activator protein 1 (AP-1) but not nuclear factor kappa B predicts the TAB2 variant pathogenicity. This assay provides accurate functional readout for both loss-of-function (LOF) and gain-of-function variants, which are associated with distinct phenotypes. In all, 22 out of 32 tested VUSs were reclassified. Genotype-Phenotype association showed that most patients with partial LOF variants do not exhibit congenital heart disease but high frequency of developmental delay, hypotonia and dysmorphic features, which suggests that genetic testing for TAB2 is needed for a broader spectrum of patients with more diverse phenotypes. Molecular modeling with Npl4 zinc finger (NZF) domain variants revealed that the stability of the NZF domain in TAB2 protein is crucial for AP-1 activation. In conclusion, we developed a highly effective functional assay for TAB2 variant prediction and interpretation.

Hippo-deficient cardiac fibroblasts differentiate into osteochondroprogenitors.

Cardiac fibrosis, a common pathophysiology associated with various heart diseases, occurs from the excess deposition of extracellular matrix (ECM) 1 . Cardiac fibroblasts (CFs) are the primary cells that produce, degrade, and remodel ECM during homeostasis and tissue repair 2 . Upon injury, CFs gain plasticity to differentiate into myofibroblasts 3 and adipocyte-like 4,5 and osteoblast-like 6 cells, promoting fibrosis and impairing heart function 7 . How CFs maintain their cell state during homeostasis and adapt plasticity upon injury are not well defined. Recent studies have shown that Hippo signalling in CFs regulates cardiac fibrosis and inflammation 8-11 . Here, we used single-nucleus RNA sequencing (snRNA-seq) and spatially resolved transcriptomic profiling (ST) to investigate how the cell state was altered in the absence of Hippo signaling and how Hippo-deficient CFs interact with macrophages during cardiac fibrosis. We found that Hippo-deficient CFs differentiate into osteochondroprogenitors (OCPs), suggesting that Hippo restricts CF plasticity. Furthermore, Hippo-deficient CFs colocalized with macrophages, suggesting their intercellular communications. Indeed, we identified several ligand-receptor pairs between the Hippo-deficient CFs and macrophages. Blocking the Hippo-deficient CF-induced CSF1 signaling abolished macrophage expansion. Interestingly, blocking macrophage expansion also reduced OCP differentiation of Hippo-deficient CFs, indicating that macrophages promote CF plasticity.

Multinomial Convolutions for Joint Modeling of Regulatory Motifs and Sequence Activity Readouts.

A common goal in the convolutional neural network (CNN) modeling of genomic data is to discover specific sequence motifs. Post hoc analysis methods aid in this task but are dependent on parameters whose optimal values are unclear and applying the discovered motifs to new genomic data is not straightforward. As an alternative, we propose to learn convolutions as multinomial distributions, thus streamlining interpretable motif discovery with CNN model fitting. We developed MuSeAM (Multinomial CNNs for Sequence Activity Modeling) by implementing multinomial convolutions in a CNN model. Through benchmarking, we demonstrate the efficacy of MuSeAM in accurately modeling genomic data while fitting multinomial convolutions that recapitulate known transcription factor motifs.

Dual-Mode Tumor Imaging Using Probes That Are Responsive to Hypoxia-Induced Pathological Conditions.

Hypoxia in solid tumors is associated with poor prognosis, increased aggressiveness, and strong resistance to therapeutics, making accurate monitoring of hypoxia important. Several imaging modalities have been used to study hypoxia, but each modality has inherent limitations. The use of a second modality can compensate for the limitations and validate the results of any single imaging modality. In this review, we describe dual-mode imaging systems for the detection of hypoxia that have been reported since the start of the 21st century. First, we provide a brief overview of the hallmarks of hypoxia used for imaging and the imaging modalities used to detect hypoxia, including optical imaging, ultrasound imaging, photoacoustic imaging, single-photon emission tomography, X-ray computed tomography, positron emission tomography, Cerenkov radiation energy transfer imaging, magnetic resonance imaging, electron paramagnetic resonance imaging, magnetic particle imaging, and surface-enhanced Raman spectroscopy, and mass spectrometric imaging. These overviews are followed by examples of hypoxia-relevant imaging using a mixture of probes for complementary single-mode imaging techniques. Then, we describe dual-mode molecular switches that are responsive in multiple imaging modalities to at least one hypoxia-induced pathological change. Finally, we offer future perspectives toward dual-mode imaging of hypoxia and hypoxia-induced pathophysiological changes in tumor microenvironments.

Approaching deconvolution with Fermi's mindset.

Spatial transcriptomics (ST) can catalyze discoveries linking tissue function with spatial organization of cell types. However, technical variations and subtle differences between cell subtypes make this a challenging goal. Recent algorithms that carefully incorporate the details of the ST data generation process show the promise to overcome these challenges.

Decoding the PITX2-controlled genetic network in atrial fibrillation.

Atrial fibrillation (AF), the most common sustained cardiac arrhythmia and a major risk factor for stroke, often arises through ectopic electrical impulses derived from the pulmonary veins (PVs). Sequence variants in enhancers controlling expression of the transcription factor PITX2, which is expressed in the cardiomyocytes (CMs) of the PV and left atrium (LA), have been implicated in AF predisposition. Single nuclei multiomic profiling of RNA and analysis of chromatin accessibility combined with spectral clustering uncovered distinct PV- and LA-enriched CM cell states. Pitx2-mutant PV and LA CMs exhibited gene expression changes consistent with cardiac dysfunction through cell type-distinct, PITX2-directed, cis-regulatory grammars controlling target gene expression. The perturbed network targets in each CM were enriched in distinct human AF predisposition genes, suggesting combinatorial risk for AF genesis. Our data further reveal that PV and LA Pitx2-mutant CMs signal to endothelial and endocardial cells through BMP10 signaling with pathogenic potential. This work provides a multiomic framework for interrogating the basis of AF predisposition in the PVs of humans.

Cell-type modeling in spatial transcriptomics data elucidates spatially variable colocalization and communication between cell-types in mouse brain.

Single-cell spatial transcriptomics (sc-ST) holds the promise to elucidate architectural aspects of complex tissues. Such analyses require modeling cell types in sc-ST datasets through their integration with single-cell RNA-seq datasets. However, this integration, is nontrivial since the two technologies differ widely in the number of profiled genes, and the datasets often do not share many marker genes for given cell types. We developed a neural network model, spatial transcriptomics cell-types assignment using neural networks (STANN), to overcome these challenges. Analysis of STANN's predicted cell types in mouse olfactory bulb (MOB) sc-ST data delineated MOB architecture beyond its morphological layer-based conventional description. We find that cell-type proportions remain consistent within individual morphological layers but vary significantly between layers. Notably, even within a layer, cellular colocalization patterns and intercellular communication mechanisms show high spatial variations. These observations imply a refinement of major cell types into subtypes characterized by spatially localized gene regulatory networks and receptor-ligand usage.

Systematic identification of non-canonical transcription factor motifs.

Sequence-specific transcription factors (TFs) recognize motifs of related nucleotide sequences at their DNA binding sites. Upon binding at these sites, TFs regulate critical molecular processes such as gene expression. It is widely assumed that a TF recognizes a single "canonical" motif, although recent studies have identified additional "non-canonical" motifs for some TFs. A comprehensive approach to identify non-canonical DNA binding motifs and the functional importance of those motifs' matches in the human genome is necessary for fully understanding the mechanisms of TF-regulated molecular processes in human cells. To address this need, we developed a statistical pipeline for in vitro HT-SELEX data that identifies and characterizes the distributions of non-canonical TF motifs in a stringent manner. Analyzing ~170 human TFs' HT-SELEX data, we found non-canonical motifs for 19 TFs (11%). These non-canonical motifs occur independently of the TFs' canonical motifs. Non-canonical motif occurrences in the human genome show similar evolutionary conservation to canonical motif occurrences, explain TF binding in locations without canonical motifs, and occur within gene promoters and epigenetically marked regulatory sequences in human cell lines and tissues. Our approach and collection of non-canonical motifs expand current understanding of functionally relevant DNA binding sites for human TFs.

A De Novo Shape Motif Discovery Algorithm Reveals Preferences of Transcription Factors for DNA Shape Beyond Sequence Motifs.

DNA shape adds specificity to sequence motifs but has not been explored systematically outside this context. We hypothesized that DNA-binding proteins (DBPs) preferentially occupy DNA with specific structures ("shape motifs") regardless of whether or not these correspond to high information content sequence motifs. We present ShapeMF, a Gibbs sampling algorithm that identifies de novo shape motifs. Using binding data from hundreds of in vivo and in vitro experiments, we show that most DBPs have shape motifs and can occupy these in the absence of sequence motifs. This "shape-only binding" is common for many DBPs and in regions co-bound by multiple DBPs. When shape and sequence motifs co-occur, they can be overlapping, flanking, or separated by consistent spacing. Finally, DBPs within the same protein family have different shape motifs, explaining their distinct genome-wide occupancy despite having similar sequence motifs. These results suggest that shape motifs not only complement sequence motifs but also facilitate recognition of DNA beyond conventionally defined sequence motifs.

Quantitative Measurement and Thermodynamic Modeling of Fused Enhancers Support a Two-Tiered Mechanism for Interpreting Regulatory DNA.

Computational models of enhancer function generally assume that transcription factors (TFs) exert their regulatory effects independently, modeling an enhancer as a "bag of sites." These models fail on endogenous loci that harbor multiple enhancers, and a "two-tier" model appears better suited: in each enhancer TFs work independently, and the total expression is a weighted sum of their expression readouts. Here, we test these two opposing views on how cis-regulatory information is integrated. We fused two Drosophila blastoderm enhancers, measured their readouts, and applied the above two models to these data. The two-tier mechanism better fits these readouts, suggesting that these fused enhancers comprise multiple independent modules, despite having sequence characteristics typical of single enhancers. We show that short-range TF-TF interactions are not sufficient to designate such modules, suggesting unknown underlying mechanisms. Our results underscore that mechanisms of how modules are defined and how their outputs are combined remain to be elucidated.