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Background & Aims: Metabolic dysfunction-associated steatotic liver disease ranges from metabolic dysfunction-associated steatotic liver (MASL) to metabolic dysfunction-associated steatohepatitis (MASH) with fibrosis. Transdifferentiation of hepatic stellate cells (HSCs) into fibrogenic myofibroblasts plays a critical role in the pathogenesis of MASH liver fibrosis. We compared transcriptome and chromatin accessibility of human HSCs from NORMAL, MASL, and MASH livers at single-cell resolution. We aimed to identify genes that are upregulated in activated HSCs and to determine which of these genes are key in the pathogenesis of MASH fibrosis., Methods: Eighteen human livers were profiled using single-nucleus (sn)RNA-seq and snATAC-seq. High priority targets were identified, then tested in 2D human HSC cultures, 3D human liver spheroids, and HSC-specific gene knockout mice., Results: MASH-enriched activated HSC subclusters are the major source of extracellular matrix proteins. We identified a set of concurrently upregulated and more accessible core genes (GAS7, SPON1, SERPINE1, LTBP2, KLF9, EFEMP1) that drive activation of HSCs. Expression of these genes was regulated via crosstalk between lineage-specific (JUNB/AP1), cluster-specific (RUNX1/2) and signal-specific (FOXA1/2) transcription factors. The pathological relevance of the selected targets, such as SERPINE1 (PAI-1), was demonstrated using dsiRNA-based HSC-specific gene knockdown or pharmacological inhibition of PAI-1 in 3D human MASH liver spheroids, and HSC-specific Serpine1 knockout mice., Conclusion: This study identified novel gene targets and regulatory mechanisms underlying activation of MASH fibrogenic HSCs and demonstrated that genetic or pharmacological inhibition of select genes suppressed liver fibrosis., Impact and Implications: Herein, we present the results of a multi-modal sequencing analysis of human hepatic stellate cells (HSCs) from NORMAL, MASL (metabolic dysfunction-associated steatotic liver), and metabolic dysfunction-associated steatohepatitis (MASH) livers. We identified additional subclusters that were not detected by previous studies and characterized the mechanism by which HSCs are activated in MASH livers, including the transcriptional machinery that induces the transdifferentiation of HSCs into myofibroblasts. For the first time, we described the pathogenic role of activated HSC-derived PAI-1 (a product of the SERPINE1 gene) in the development of MASH liver fibrosis. Targeting the RUNX1/2-SERPINE1 axis could be a novel strategy for the treatment of liver fibrosis in patients., Competing Interests: Conflict of interest The authors of this study declare that they do not have any conflict of interest. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Enhlink is a computational tool for scATAC-seq data analysis, facilitating precise interrogation of enhancer function at the single-cell level. It employs an ensemble approach incorporating technical and biological covariates to infer condition-specific regulatory DNA linkages. Enhlink can integrate multi-omic data for enhanced specificity, when available. Evaluation with simulated and real data, including multi-omic datasets from the mouse striatum and novel promoter capture Hi-C data, demonstrate that Enhlink outperfoms alternative methods. Coupled with eQTL analysis, it identified a putative super-enhancer in striatal neurons. Overall, Enhlink offers accuracy, power, and potential for revealing novel biological insights in gene regulation., (© 2024. The Author(s).)

Single-cell sequencing could help to solve the fundamental challenge of linking millions of cell-type-specific enhancers with their target genes. However, this task is confounded by patterns of gene co-expression in much the same way that genetic correlation due to linkage disequilibrium confounds fine-mapping in genome-wide association studies (GWAS). We developed a non-parametric permutation-based procedure to establish stringent statistical criteria to control the risk of false-positive associations in enhancer-gene association studies (EGAS). We applied our procedure to large-scale transcriptome and epigenome data from multiple tissues and species, including the mouse and human brain, to predict enhancer-gene associations genome wide. We tested the functional validity of our predictions by comparing them with chromatin conformation data and causal enhancer perturbation experiments. Our study shows how controlling for gene co-expression enables robust enhancer-gene linkage using single-cell sequencing data., Competing Interests: J.R.E. serves on the scientific advisory board of Zymo Research, Inc. B.R. is a shareholder of Arima Genomics, Inc. and Epigenome Technologies, Inc. H.Z. is on the scientific advisory board of MapLight Therapeutics, Inc., (© 2023 The Authors.)

Enhancers play a crucial role in regulating gene expression and their functional status can be queried with cell type precision using using single-cell (sc)ATAC-seq. To facilitate analysis of such data, we developed Enhlink, a novel computational approach that leverages single-cell signals to infer linkages between regulatory DNA sequences, such as enhancers and promoters. Enhlink uses an ensemble strategy that integrates cell-level technical covariates to control for batch effects and biological covariates to infer robust condition-specific links and their associated p -values. It can integrate simultaneous gene expression and chromatin accessibility measurements of individual cells profiled by multi-omic experiments for increased specificity. We evaluated Enhlink using simulated and real scATAC-seq data, including those paired with physical enhancer-promoter links enumerated by promoter capture Hi-C and with multi-omic scATAC-/RNA-seq data we generated from the mouse striatum. These examples demonstrated that our method outperforms popular alternative strategies. In conjunction with eQTL analysis, Enhlink revealed a putative super-enhancer regulating key cell type-specific markers of striatal neurons. Taken together, our analyses demonstrate that Enhlink is accurate, powerful, and provides features that can lead to novel biological insights., Competing Interests: Competing interests The author(s) declare(s) that they have no competing interests.

The heart, a vital organ which is first to develop, has adapted its size, structure and function in order to accommodate the circulatory demands for a broad range of animals. Although heart development is controlled by a relatively conserved network of transcriptional/chromatin regulators, how the human heart has evolved species-specific features to maintain adequate cardiac output and function remains to be defined. Here, we show through comparative epigenomic analysis the identification of enhancers and promoters that have gained activity in humans during cardiogenesis. These cis-regulatory elements (CREs) are associated with genes involved in heart development and function, and may account for species-specific differences between human and mouse hearts. Supporting these findings, genetic variants that are associated with human cardiac phenotypic/disease traits, particularly those differing between human and mouse, are enriched in human-gained CREs. During early stages of human cardiogenesis, these CREs are also gained within genomic loci of transcriptional regulators, potentially expanding their role in human heart development. In particular, we discovered that gained enhancers in the locus of the early human developmental regulator ZIC3 are selectively accessible within a subpopulation of mesoderm cells which exhibits cardiogenic potential, thus possibly extending the function of ZIC3 beyond its conserved left-right asymmetry role. Genetic deletion of these enhancers identified a human gained enhancer that was required for not only ZIC3 and early cardiac gene expression at the mesoderm stage but also cardiomyocyte differentiation. Overall, our results illuminate how human gained CREs may contribute to human-specific cardiac attributes, and provide insight into how transcriptional regulators may gain cardiac developmental roles through the evolutionary acquisition of enhancers., Competing Interests: Ethics declarations The authors declare no competing interests.