Siyuan (Steven) Wang, PhD

Image-based 3D genomics and spatial multi-omics

At Harvard: Wang’s research interest is to understand the spatiotemporal complexity of molecular and cellular systems, and how the complexity affects biological functions. Especially, he aims to understand the spatial organization of mammalian chromatin – the complex of genomic DNA and associated proteins. The spatial organization of chromatin in the nucleus is of critical importance to many essential genomic functions, from the regulation of gene expression to the replication of the genome. Unfortunately, relatively little is known about the three-dimensional (3D) organization of chromatin beyond the length scale of the nucleosomes, in large part due to the lack of tools that allow direct visualization of the 3D folding of chromatin in individual chromosomes. To address this need, his main postdoctoral work (Science 2016) involved the development of a first-in-kind image-based 3D genomics method termed “chromatin tracing“, via multiplexed DNA fluorescence in situ hybridization (multiplexed DNA FISH). This novel method enabled direct spatial tracing of numerous genomic regions in individual chromosomes in single cells, offering a powerful tool to study the 3D folding of chromatin. As the first application of this method, he studied the spatial organization of topologically associating domains (TADs), also termed contact domains, by tracing the 3D positions of TADs in individual chromosomes in interphase human cells, and revealed a series of unexpected structural features. This work opened up many opportunities to study the spatial organization of chromatin at different length scales in a variety of important biological processes and in diseases. He also co-developed a highly-multiplexed RNA FISH technique termed “MERFISH” that enabled localized detection and quantification of 1000 different RNA species in a single cell (Science 2015). In comparison to single-cell RNA sequencing, this multiplexed FISH method easily retains the spatial information of all the probed transcripts, and is highly sensitive for counting low-copy-number transcripts. Additionally, he led the development of a new photoactivatable fluorescent protein (PAFP), named mMaple3 (PNAS 2014), that outperforms previously existing PAFPs in single-molecule-based superresolution imaging (STORM/PALM) and has been adopted by hundreds of research labs around the world, and an RNA-aptamer-based two-color CRISPR labeling system for studying chromatin dynamics (Scientific Reports 2016).

At Yale: Wang’s independent lab at Yale is devoted to understand mammalian genome architectures and spatial transcriptome in health and disease. In the past few years, the lab introduced a new integrative technique, termed Multiplexed Imaging of Nucleome Architectures (MINA) – the first comprehensive 3D nucleomic imaging technique (bioRxiv 2019; Nat Comm 2020; Nat Protoc 2021). This method enabled multi-omic and multiscale visualization of single-cell nucleome architectures and gene expression to functionally define promoter-enhancer interactions, chromatin domains, compartments, territories, and chromatin interactions with nuclear landmarks in the same single cells of complex mammalian tissues, generating true 3D maps of all major chromatin architectures reported by various sequencing methods (P-E loops, TADs, LADs, NADs, A-B compartments, and chromosome territories). In applying the technique to mouse fetal liver, the team discovered cell-type-specific chromatin architectures associated with gene expression as well as chromatin organization principles independent of cell type (bioRxiv 2019; Nat Comm 2020). They also applied chromatin tracing to elucidate novel architectures and their regulation in the folding conformations of the two copies of X chromosomes in female human cells (Genome Biology 2021; Science Advances 2023). Overall, the chromatin tracing and MINA technologies have revolutionized 3D genomics and multi-omics studies (Trends in Cell Biology “Best of 2021”, Nature Reviews Methods Primers 2024).

Using genome-wide chromatin tracing, the team recently resolved the first single-cell cancer 3D genome atlas of any cancer using any technique. The resulting data afford an unprecedented view of single-cell cancer 3D genome organization during lung and pancreatic cancer progression in vivo, revealing: 1) Stage-specific alterations in global 3D genome folding heterogeneity, compactness, and compartmentalization as cancers progress from normal to preinvasive to invasive tumors, elucidating a potential structural bottleneck during early cancer progression. 2) 3D genome organization reflects histologic cancer cell states at the single cell level with high precision. 3) The utility of 3D genome mapping in discovering prognostic and predictive biomarkers – uncovering novel cancer genes associated with patient survival and dependency in cancer cells. 4) New 3D genome function, as our data reveal that cancer genes associated with 3D compartment changes are more stably regulated than non-compartment-regulated genes. 5) A novel ubiquitin ligase-independent role for polycomb-group protein Rnf2 in 3D genome regulation, as knockdown or rapid targeted degradation partially reverses the 3D genome reorganization associated with lung cancer progression. Overall, this work charts a comprehensive blueprint of 3D genome alterations during cancer progression in the native tissue context and systematically demonstrates how this rich resource can provide novel biological insights and potential diagnostic, prognostic, and therapeutic biomarkers (bioRxiv 2023, Nature Genetics 2025).

Despite the fundamental importance of chromatin organization, relatively little is known regarding the molecular mechanisms that control chromatin organization, due to a lack of tools to efficiently screen for 3D genome regulators. To address this need, the team developed a high-content pooled CRISPR screen paired with chromatin tracing to efficiently identify new regulators of 3D genome architectures across multiple length scales (Nature Methods 2025). This technology, termed Perturb-tracing, laid the foundation for a complete mapping of the “3D genomic regulatome”, which may lead to a new avenue of therapeutics by halting or reversing the deleterious 3D genome reorganization in aging and diseases. To study the functional relationship between the 3D genome and epigenome, the team recently introduced Epigenetic Proximity Hybridization Reaction (Epi-PHR), the first non-disruptive, high-genomic-resolution, image-based epigenetic profiling method. Epi-PHR enables the first phased single-cell, single-copy examination of histone mark deposition and 3D genome co-profiling at an imprinted locus by any technique (bioRxiv 2025).

The lab is also interested in the spatial organization of transcriptome at both the subcellular and cell-to-tissue scales (Cell Discovery 2021, Neuron 2023, Cell Reports 2025). Most recently, the team invented Reverse-padlock Amplicon Encoding Fluorescence In Situ Hybridization (RAEFISH), a new flagship image-based spatial transcriptomics technology that addresses the major compromises of previous image-based and sequencing-based spatial transcriptomics techniques by simultaneously enabling single-molecule spatial resolution and whole-genome level coverage of long and short, endogenous and engineered RNA species in cell cultures and intact tissues, setting a new standard in spatial transcriptomics (bioRXiv 2025b, Cell 2025). This work represents the first time that transcripts from more than 20,000 genes were directly probed and imaged in situ with any technology, and the first time numerous different gRNAs were directly probed and distinguished by imaging in a high-content CRISPR screen.

Earlier research

As a graduate student at Princeton University, Wang studied bacterial cell mechanics, especially how the bacterial cytoskeleton coordinates cell wall synthesis. The first project (PNAS 2010) in his dissertation showed that the bacterial actin homologue MreB contributes nearly as much to the rigidity of an E. coli cell as the peptidoglycan cell wall. This conclusion provided the premise for several theoretical works that assumed MreB applies force to the cell wall during growth, and suggested an evolutionary origin of cytoskeleton-governed cell rigidity. His second project (PNAS 2011) dealt with the discovery of the motion of E. coli MreB linked to cell wall synthesis. This was the first observation of a cell-wall assembly driven molecular motor in bacteria. (Simultaneously with the work, Garner et al and Dominguez-Escobar et al discovered the same phenomenon in B. subtilis.) His third project (PNAS 2012) elucidated that both cell wall synthesis and the peptidoglycan network have a chiral ordering, which is established by MreB. This work linked the molecular structures of the cytoskeleton and of the cell wall with organismal-scale behavior. His fourth project (Biophysical Journal 2013) developed a generic, quantitative model to explain the various spatial patterns adopted by bacterial cytoskeletal proteins. The model set up a new theoretical framework for the study of membrane-polymer interaction, and is useful for the exploration of the physical limits of cytoskeleton organization.