a-web-based-workflow-for-selecting-gene-and-tissue-specific-enhancers

A Web-Based Workflow for Selecting Gene- and Tissue-Specific Enhancers

Enhancers are DNA regions that regulate gene expression. Mutations within enhancers can result in abnormal gene regulation leading to disease. Therefore, ...

University of Melbourne

Monash University

The Royal Children's Hospital

Murdoch Children'S Research Institute

A strong focus of the post-genomic era is mining of the non-coding regulatory genome in order to unravel the function of regulatory elements that coordinate gene expression (Nat 489:57-74, 2012; Nat 507:462-70, 2014; Nat 507:455-61, 2014; Nat 518:317-30, 2015). Whole-genome approaches based on next-generation sequencing (NGS) have provided insight into the genomic location of regulatory elements throughout different cell types, organs and organisms. These technologies are now widespread and commonly used in laboratories from various fields of research. This highlights the need for fast and user-friendly software tools dedicated to extracting cis-regulatory information contained in these regulatory regions; for instance transcription factor binding site (TFBS) composition. Ideally, such tools should not require prior programming knowledge to ensure they are accessible for all users.

TrawlerWeb: an online de novo motif discovery tool for next-generation sequencing datasets.

The growth and progression of solid tumors involves dynamic cross-talk between cancer epithelium and the surrounding microenvironment. To date, molecular profiling has largely been restricted to the epithelial component of tumors; therefore, features underpinning the persistent protumorigenic phenotype of the tumor microenvironment are unknown. Using whole-genome bisulfite sequencing, we show for the first time that cancer-associated fibroblasts (CAFs) from localized prostate cancer display remarkably distinct and enduring genome-wide changes in DNA methylation, significantly at enhancers and promoters, compared to nonmalignant prostate fibroblasts (NPFs). Differentially methylated regions associated with changes in gene expression have cancer-related functions and accurately distinguish CAFs from NPFs. Remarkably, a subset of changes is shared with prostate cancer epithelial cells, revealing the new concept of tumor-specific epigenome modifications in the tumor and its microenvironment. The distinct methylome of CAFs provides a novel epigenetic hallmark of the cancer microenvironment and promises new biomarkers to improve interpretation of diagnostic samples.

Enduring epigenetic landmarks define the cancer microenvironment.

Spatially adjacent habitats on coral reefs can represent highly distinct environments, often harbouring different coral communities. Yet, certain coral species thrive across divergent environments. It is unknown whether the forces of selection are sufficiently strong to overcome the counteracting effects of the typically high gene flow over short distances, and for local adaptation to occur. We screened the coral genome (using restriction site-associated sequencing) and characterized both the dinoflagellate photosymbiont- and tissue-associated prokaryote microbiomes (using metabarcoding) of a reef flat and slope population of the reef-building coral, Pocillopora damicornis, at two locations on Heron Island in the southern Great Barrier Reef. Reef flat and slope populations were separated by <100 m horizontally and ~5 m vertically, and the two study locations were separated by ~1 km. For the coral host, genetic divergence between habitats was much greater than between locations, suggesting limited gene flow between the flat and slope populations. Consistent with environmental selection, outlier loci primarily belonged to the conserved, minimal cellular stress response, likely reflecting adaptation to the different temperature and irradiance regimes on the reef flat and slope. The prokaryote community differed across both habitat and, to a lesser extent, location, whereas the dinoflagellate photosymbionts differed by habitat but not location. The observed intraspecific diversity associated with divergent habitats supports that environmental adaptation involves multiple members of the coral holobiont. Adaptive alleles or microbial associations present in coral populations from the environmentally variable reef flat may provide a source of adaptive variation for assisted evolution approaches, through assisted gene flow, artificial cross-breeding or probiotic inoculations, with the aim to increase climate resilience in the slope populations.

Adaptation to reef habitats through selection on the coral animal and its associated microbiome.

Disease severity in SLE is an important concept related to disease activity, treatment burden and prognosis. We set out to evaluate if high disease activity status (HDAS), based on ever attainment of a Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) disease activity score of ≥10, is an indicator for disease severity in SLE.

High disease activity status suggests more severe disease and damage accrual in systemic lupus erythematosus.

Understanding the spatial gene expression and regulation in the heart is key to uncovering its developmental and physiological processes, during homeostasis and disease. Numerous techniques exist to gain gene expression and regulation information in organs such as the heart, but few utilize intuitive true-to-life three-dimensional representations to analyze and visualise results. Here we combined transcriptomics with 3D-modelling to interrogate spatial gene expression in the mammalian heart. For this, we microdissected and sequenced transcriptome-wide 18 anatomical sections of the adult mouse heart. Our study has unveiled known and novel genes that display complex spatial expression in the heart sub-compartments. We have also created 3D-cardiomics, an interface for spatial transcriptome analysis and visualization that allows the easy exploration of these data in a 3D model of the heart. 3D-cardiomics is accessible from http://3d-cardiomics.erc.monash.edu/.

3D-cardiomics: A spatial transcriptional atlas of the mammalian heart.

Congenital heart diseases are the major cause of death in newborns, but the genetic etiology of this developmental disorder is not fully known. The conventional approach to identify the disease-causing genes focuses on screening genes that display heart-specific expression during development. However, this approach would have discounted genes that are expressed widely in other tissues but may play critical roles in heart development.

A cis-regulatory-directed pipeline for the identification of genes involved in cardiac development and disease.

Organ fibroblasts are essential components of homeostatic and diseased tissues. They participate in sculpting the extracellular matrix, sensing the microenvironment, and communicating with other resident cells. Recent studies have revealed transcriptomic heterogeneity among fibroblasts within and between organs. To dissect the basis of interorgan heterogeneity, we compare the gene expression of murine fibroblasts from different tissues (tail, skin, lung, liver, heart, kidney, and gonads) and show that they display distinct positional and organ-specific transcriptome signatures that reflect their embryonic origins. We demonstrate that expression of genes typically attributed to the surrounding parenchyma by fibroblasts is established in embryonic development and largely maintained in culture, bioengineered tissues and ectopic transplants. Targeted knockdown of key organ-specific transcription factors affects fibroblast functions, in particular genes involved in the modulation of fibrosis and inflammation. In conclusion, our data reveal that adult fibroblasts maintain an embryonic gene expression signature inherited from their organ of origin, thereby increasing our understanding of adult fibroblast heterogeneity. The knowledge of this tissue-specific gene signature may assist in targeting fibrotic diseases in a more precise, organ-specific manner.

Adult mouse fibroblasts retain organ-specific transcriptomic identity.

Thymic epithelium is critical for the structural integrity of the thymus and for T cell development. Within the fully formed thymus, large numbers of hematopoietic cells shape the thymic epithelium into a scaffold-like structure which bears little similarity to classical epithelial layers, such as those observed in the skin, intestine or pancreas. Here, we show that human thymic epithelial cells (TECs) possess an epithelial identity that also incorporates the expression of mesenchymal cell associated genes, whose expression levels vary between medullary and cortical TECs (m/cTECs). Using pluripotent stem cell (PSC) differentiation systems, we identified a unique population of cells that co-expressed the master TEC transcription factor , as well as the epithelial associated marker EPCAM and the mesenchymal associated gene CD90. Using the same serum free culture conditions, we also observed co-expression of EPCAM and CD90 on cultured TECs derived from neonatal human thymus . Single cell RNA-sequencing revealed these cultured TECs possessed an immature mTEC phenotype and expressed epithelial and mesenchymal associated genes, such as , ,  and . Importantly, flow cytometry and single cell RNA-sequencing analysis further confirmed the presence of an EPCAM+CD90+ population in the CD45- fraction of neonatal human thymic stromal cells . Using the human thymus cell atlas, we found that cTECs displayed more pronounced mesenchymal characteristics than mTECs during embryonic development. Collectively, these results suggest human TECs possess a hybrid gene expression program comprising both epithelial and mesenchymal elements, and provide a basis for the further exploration of thymus development from primary tissues and from the  differentiation of PSCs.

CD90 Marks a Mesenchymal Program in Human Thymic Epithelial Cells  and .

Human macrophages are a natural host of many mycobacterium species, including Mycobacterium abscessus (M. abscessus), an emerging pathogen affecting immunocompromised and cystic fibrosis patients with few available treatments. The search for an effective treatment is hindered by the lack of a tractable in vitro intracellular infection model. Here, we established a reliable model for M. abscessus infection using human pluripotent stem cell-derived macrophages (hPSC-macrophages). hPSC differentiation permitted reproducible generation of functional macrophages that were highly susceptible to M. abscessus infection. Electron microscopy demonstrated that M. abscessus was present in the hPSC-macrophage vacuoles. RNA sequencing analysis revealed a time-dependent host cell response, with differing gene and protein expression patterns post-infection. Engineered tdTOMATO-expressing hPSC-macrophages with GFP-expressing mycobacteria enabled rapid image-based high-throughput analysis of intracellular infection and quantitative assessment of antibiotic efficacy. Our study describes the first to our knowledge hPSC-based model for M. abscessus infection, representing a novel and accessible system for studying pathogen-host interaction and drug discovery.

Human pluripotent stem cell-derived macrophages host Mycobacterium abscessus infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily infects the respiratory tract, but pulmonary and cardiac complications occur in severe coronavirus disease 2019 (COVID-19). To elucidate molecular mechanisms in the lung and heart, we conducted paired experiments in human stem cell-derived lung alveolar type II (AT2) epithelial cell and cardiac cultures infected with SARS-CoV-2. With CRISPR-Cas9-mediated knockout of ACE2, we demonstrated that angiotensin-converting enzyme 2 (ACE2) was essential for SARS-CoV-2 infection of both cell types but that further processing in lung cells required TMPRSS2, while cardiac cells required the endosomal pathway. Host responses were significantly different; transcriptome profiling and phosphoproteomics responses depended strongly on the cell type. We identified several antiviral compounds with distinct antiviral and toxicity profiles in lung AT2 and cardiac cells, highlighting the importance of using several relevant cell types for evaluation of antiviral drugs. Our data provide new insights into rational drug combinations for effective treatment of a virus that affects multiple organ systems.

Parallel use of human stem cell lung and heart models provide insights for SARS-CoV-2 treatment.

Treat-to-target strategies have improved outcomes in single-organ diseases with simple clinical or laboratory endpoints. A lack of validated endpoints has prevented adoption of treat to target for complex multiorgan conditions, such as systemic lupus erythematosus (SLE). We report the first prospective study undertaken to specifically validate a treat-to-target endpoint for SLE.

Lupus low disease activity state as a treatment endpoint for systemic lupus erythematosus: a prospective validation study.

Cardiac function requires appropriate proteins in each chamber. Atria requires slow myosin to act as reservoirs, while ventricles demand fast myosin for swift pumping. Myosins are thus under chamber-biased cis-regulation, with myosin gene expression imbalances leading to congenital heart dysfunction. To identify regulatory inputs leading to cardiac chamber-biased expression, we computationally and molecularly dissected the quail Slow Myosin Heavy Chain III (SMyHC III) promoter that drives preferential expression to the atria. We show that SMyHC III gene states are orchestrated by a complex Nuclear Receptor Element (cNRE) of 32 base pairs. Using transgenesis in zebrafish and mice, we demonstrate that preferential atrial expression is achieved by a combinatorial regulatory input composed of atrial activation motifs and ventricular repression motifs. Using comparative genomics, we show that the cNRE might have emerged from an endogenous viral element through infection of an ancestral host germline, revealing an evolutionary pathway to cardiac chamber-specific expression.

Unraveling the evolutionary origin of the complex Nuclear Receptor Element (cNRE), a cis-regulatory module required for preferential expression in the atrial chamber.

Hematopoietic stem cells (HSCs) derived from human induced pluripotent stem cells (iPS cells) have important biomedical applications. We identified differentiation conditions that generate HSCs defined by robust long-term multilineage engraftment in immune-deficient NOD,B6.Prkdc Il2rg Kit mice. We guided differentiating iPS cells, as embryoid bodies in a defined culture medium supplemented with retinyl acetate, through HOXA-patterned mesoderm to hemogenic endothelium specified by bone morphogenetic protein 4 and vascular endothelial growth factor (VEGF). Removal of VEGF facilitated an efficient endothelial-to-hematopoietic transition, evidenced by release into the culture medium of CD34 blood cells, which were cryopreserved. Intravenous transplantation of two million thawed CD34 cells differentiated from four independent iPS cell lines produced multilineage bone marrow engraftment in 25-50% of immune-deficient recipient mice. These functionally defined, multipotent CD34 hematopoietic cells, designated iPS cell-derived HSCs (iHSCs), produced levels of engraftment similar to those achieved following umbilical cord blood transplantation. Our study provides a step toward the goal of generating HSCs for clinical translation.

Long-term engrafting multilineage hematopoietic cells differentiated from human induced pluripotent stem cells.

Membrane contact sites between organelles are critical for the transfer of biomolecules. Lipid droplets store fatty acids and form contacts with mitochondria, which regulate fatty acid oxidation and adenosine triphosphate production. Protein compartmentalization at lipid droplet-mitochondria contact sites and their effects on biological processes are poorly described. Using proximity-dependent biotinylation methods, we identify 71 proteins at lipid droplet-mitochondria contact sites, including a multimeric complex containing extended synaptotagmin (ESYT) 1, ESYT2, and VAMP Associated Protein B and C (VAPB). High resolution imaging confirms localization of this complex at the interface of lipid droplet-mitochondria-endoplasmic reticulum where it likely transfers fatty acids to enable β-oxidation. Deletion of ESYT1, ESYT2 or VAPB limits lipid droplet-derived fatty acid oxidation, resulting in depletion of tricarboxylic acid cycle metabolites, remodeling of the cellular lipidome, and induction of lipotoxic stress. These findings were recapitulated in Esyt1 and Esyt2 deficient mice. Our study uncovers a fundamental mechanism that is required for lipid droplet-derived fatty acid oxidation and cellular lipid homeostasis, with implications for metabolic diseases and survival.

Proximity proteomics reveals a mechanism of fatty acid transfer at lipid droplet-mitochondria- endoplasmic reticulum contact sites.

Craniofacial malformations arise from developmental defects in the head, face, and neck with phenotypes such as 22q11.2 deletion syndrome illustrating a developmental link between cardiovascular and craniofacial morphogenesis. NKX2-5 is a key cardiac transcription factor associated with congenital heart disease and mouse models of Nkx2-5 deficiency highlight roles in cardiac development. In zebrafish, nkx2.5 and nkx2.7 are paralogues in the NK4 family expressed in cardiomyocytes and pharyngeal arches. Despite shared cellular origins of cardiac and craniofacial tissues, the function of NK4 factors in head and neck patterning has not been elucidated. Molecular evolutionary analysis of NK4 genes shows that nkx2.5 and nkx2.7 are ohnologs resulting from whole genome duplication events. Nkx2.7 serves as a previously unappreciated regulator of branchiomeric muscle and cartilage formation for which nkx2.5 cannot fully compensate. Mechanistically, our results highlight that Nkx2.7 patterns the cranial neural crest and functions upstream of Endothelin1 to inhibit Notch signals. Together, our studies shed light on an evolutionarily conserved Nkx transcription factor with unique functions in vertebrate craniofacial development, advancing our understanding of congenital head and neck deformities.

Nkx2.7 is a conserved regulator of craniofacial development.

The field of spatial transcriptomics is rapidly evolving, with increasing sample complexity, resolution, and tissue size. Yet the field lacks comprehensive and intuitive solutions for automated integration and analysis of multi-slice data in either co-planar (2D) or stacked (3D) formation. To address this, we develop VR-Omics, a free, platform-agnostic software that provides end-to-end automated processing of multi-slice data through a biologist-friendly interface. Benchmarking against existing methods demonstrates VR-Omics' unique strengths to perform comprehensive end-to-end analysis of multi-slice stacked data. Through co-planar slice analysis, VR-Omics uncovers previously undetected, dysregulated metabolic networks within rare pediatric cardiac rhabdomyomas, demonstrating its potential for biological discoveries.

Article	Year
TrawlerWeb: an online de novo motif discovery tool for next-generation sequencing datasets. BMC genomics\| PubMed ID: 29621972	2018
Enduring epigenetic landmarks define the cancer microenvironment. Genome research\| PubMed ID: 29650553	2018
Adaptation to reef habitats through selection on the coral animal and its associated microbiome. Molecular ecology\| PubMed ID: 29900626	2018
High disease activity status suggests more severe disease and damage accrual in systemic lupus erythematosus. Lupus science & medicine\| PubMed ID: 32467293	2020
3D-cardiomics: A spatial transcriptional atlas of the mammalian heart. Journal of molecular and cellular cardiology\| PubMed ID: 34624332	2022
A cis-regulatory-directed pipeline for the identification of genes involved in cardiac development and disease. Genome biology\| PubMed ID: 34906219	2021
Adult mouse fibroblasts retain organ-specific transcriptomic identity. eLife\| PubMed ID: 35293863	2022
CD90 Marks a Mesenchymal Program in Human Thymic Epithelial Cells and . Frontiers in immunology\| PubMed ID: 35371075	2022
Human pluripotent stem cell-derived macrophages host Mycobacterium abscessus infection. Stem cell reports\| PubMed ID: 35985333	2022
Parallel use of human stem cell lung and heart models provide insights for SARS-CoV-2 treatment. Stem cell reports\| PubMed ID: 37315523	2023
Lupus low disease activity state as a treatment endpoint for systemic lupus erythematosus: a prospective validation study. The Lancet. Rheumatology\| PubMed ID: 38229349	2019
Unraveling the evolutionary origin of the complex Nuclear Receptor Element (cNRE), a cis-regulatory module required for preferential expression in the atrial chamber. Communications biology\| PubMed ID: 38575811	2024
Long-term engrafting multilineage hematopoietic cells differentiated from human induced pluripotent stem cells. Nature biotechnology\| PubMed ID: 39223325	2024
Proximity proteomics reveals a mechanism of fatty acid transfer at lipid droplet-mitochondria- endoplasmic reticulum contact sites. Nature communications\| PubMed ID: 40032835	2025
Nkx2.7 is a conserved regulator of craniofacial development. Nature communications\| PubMed ID: 40268889	2025
Automated integration of multi-slice spatial transcriptomics data in 2D and 3D using VR-Omics. Genome biology\| PubMed ID: 40598307	2025

Hieu T. Nim

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