Biology subjects

Szu-Tu, C.

Publications and source records attributed to Szu-Tu, C..

4 recordsLinked to original sources

bioRxiv2026

Lentiviral single-cell MPRA of synthetic enhancers reveals motif affinity-based encoding of cell type specificity

Cell-state-specific gene expression programs emerge from the interplay between cis-regulatory elements (CREs), such as enhancers, and transcription factors (TFs). Massively parallel reporter assays (MPRAs) have enabled large-scale dissection of CRE design principles, but bulk approaches cannot resolve cell state-specific regulatory logic on continuous trajectories of cellular differentiation, and existing single-cell MPRAs are not readily applicable to primary cell differentiation models. Here, we developed a single-cell lentiviral Massively Parallel Reporter Assay (sc-lentiMPRA) that overcomes these limitations and enables parallel quantification of enhancer activity and cellular transcriptome. Applying sc-lentiMPRA in blood stem differentiation, we profiled the activity and specificity of ~160 fully synthetic enhancers with controlled motif composition and affinities across ~190,000 single cells. Focusing on Trp53 and Cebpa, we show that enhancers with high and low affinity motifs differ qualitatively and quantitatively in their responses to TF expression gradients. For Trp53, low-affinity motifs exhibited near-linear correlation with TF expression, whereas high-affinity motifs showed reduced sensitivity to TF levels and a potential contribution of cofactor availability. In contrast, Cebpa-associated enhancers displayed nonlinear behaviors. Together, sc-lentiMPRA establishes a powerful framework for systematically relating enhancer architecture and TF expression to regulatory output at single-cell resolution during cellular differentiation.

genomics↗

bioRxiv2024

Synthetic enhancers reveal design principles of cell state specific regulatory elements in hematopoiesis

During cellular differentiation, enhancers transform overlapping gradients of transcription factors (TFs) to highly specific gene expression patterns. However, the vast complexity of regulatory DNA impedes the identification of the underlying cis-regulatory rules. Here, we have characterized 62,126 fully synthetic DNA sequences to bottom-up dissect design principles of cell-state specific enhancers in the context of the differentiation of blood stem cells to seven myeloid lineages. Focusing on binding sites for 38 TFs and their pairwise interactions, we found that identical sites displayed both repressive and activating function, as a consequence of cellular context, site combinatorics, or simply predicted occupancy of a TF on an enhancer. Surprisingly, we found that combinations of activating sites frequently neutralized each other or even gained repressive function. These negative synergies convert quantitative imbalances in transcription factor expression into binary downstream activity patterns, a principle that can be exploited to build differentiation-state specific enhancers from scratch.

molecular biology↗

bioRxiv2024

Somatic epimutations enable single-cell lineage tracing in native hematopoiesis across the murine and human lifespan

Current approaches to lineage tracing of stem cell clones require genetic engineering or rely on sparse somatic DNA variants, which are difficult to capture at single-cell resolution. Here, we show that targeted single-cell measurements of DNA methylation at single-CpG resolution deliver joint information about cellular differentiation state and clonal identities. We develop EPI-clone, a droplet-based method for transgene-free lineage tracing, and apply it to study hematopoiesis, capturing hundreds of clonal trajectories across almost 100,000 single-cells. Using ground-truth genetic barcodes, we demonstrate that EPI-clone accurately identifies clonal lineages throughout hematopoietic differentiation. Applied to unperturbed hematopoiesis, we describe an overall decline of clonal complexity during murine ageing and the expansion of rare low-output stem cell clones. In aged human donors, we identified expanded hematopoietic clones with and without genetic lesions, and various degrees of clonal complexity. Taken together, EPI-clone enables accurate and transgene-free single-cell lineage tracing at scale.

genomics↗

bioRxiv2022

Clonally resolved single-cell multi-omics identifies routes of cellular differentiation in acute myeloid leukemia

Inter-patient variability and the similarity of healthy and leukemic stem cells have impeded the characterization of leukemic stem cells (LSCs) in acute myeloid leukemia (AML), and their differentiation landscape. Here, we introduce CloneTracer, a novel method that adds clonal resolution to single-cell RNA-seq datasets. Applied to samples from 19 AML patients, CloneTracer revealed routes of leukemic differentiation. While residual healthy cells dominated the dormant stem cell compartment, active leukemic stem cells resembled their healthy counterpart and retained erythroid capacity. By contrast, downstream myeloid progenitors were highly aberrant and constituted the disease-defining compartment: Their gene expression and differentiation state determined both chemotherapy response and the leukemias ability to differentiate to transcriptomically normal monocytes. Finally, we demonstrated the potential of CloneTracer to identify surface markers mis-regulated specifically in leukemic cells by intra-patient comparisons. Taken together, CloneTracer revealed a differentiation landscape that mimics its healthy counterpart and determines biology and therapy response in AML.

cancer biology↗