Biology subjects

Rothenberg, E. V.

Publications and source records attributed to Rothenberg, E. V..

10 recordsLinked to original sources

bioRxiv2026

Bcl11b dose-dependently regulates positive selection of CD8 T cells to the virtual memory fate

Virtual memory T cells are increasingly recognized as a functionally distinct lineage within the CD8 T cell pool, but when and how commitment to the lineage is enforced remain poorly understood. Here we demonstrate that TVM lineage choice is exceptionally sensitive to dosage and repression competence of the key T cell transcription factor Bcl11b. Three different genetic models of slightly reduced Bcl11b each biased CD8 cell development to TVM generation without deregulating effector differentiation. Timed conditional knockouts and adoptive transfers narrowed the developmental window and showed that Bcl11b levels determine diversion to virtual memory fate uniquely during intrathymic positive selection. Whereas total Bcl11b loss disrupts TCR signalling, a <2-fold dose reduction of Bcl11b enhanced selective responses to TCR stimulation. Chromatin accessibility profiling and single cell RNA-seq indicated that Bcl11b dose reduction redirects cells to the TVM fate, from the late cycling fraction of mature CD8SP thymocytes, by a mechanism independent of previously described cytokine-driven pathways.

immunology↗

bioRxiv2025

T cell development from expanded hematopoietic progenitors reveals progression control by Lmo2, Erg, Spi1, Hoxa9, and Meis1

To gain access to the earliest stages of T cell development, we adapted a serum-free culture system that expands hematopoietic stem and progenitor-like cells. These expanded cells efficiently undergo normal T-cell differentiation in vivo and in vitro, verified by early gene expression trajectories from single-cell RNA sequencing, though their absolute differentiation speed is slower than that of fresh progenitors and can be modulated with cytokine priming. Leveraging this expansion system to observe the first T-lineage events, we revealed that initial Notch activation immediately induces chromatin opening and transcriptional activation of the TCR-C{beta} locus. Additionally, acute CRISPR knockouts confirmed T-lineage entry requirements for Ikzf1, Hes1, Gabpa, and Myb while revealing that Lmo2, Erg, Spi1, Hoxa9, and Meis1 retard developmental progression with differing effects on proliferation. Endogenous expression of the stem, progenitor, and leukemia-associated factor Lmo2 markedly restrains initiation of the T cell program, with Lmo2 knockout greatly accelerating germline TCR{beta} locus transcription and expression of Tcf7, Gata3, Runx family, and E protein genes and their targets.

immunology↗

bioRxiv2023

T-cell commitment inheritance - an agent-based multi-scale model

T-cell development provides an excellent model system for studying lineage commitment from a multipotent progenitor. The intrathymic development process has been thoroughly studied. The molecular circuitry controlling it has been dissected and the necessary steps like programmed shut off of progenitor genes and T-cell genes upregulation have been revealed. However, the exact timing between decision-making and commitment stage remains unexplored. To this end, we implemented an agent-based multi-scale model to investigate inheritance in early T-cell development. Treating each cell as an agent provides a powerful tool as it tracks each individual cell of a simulated T-cell colony, enabling the construction of lineage trees. Based on the lineage trees, we introduce the concept of the last common ancestors (LCA) of committed cells and analyse their relations, both at single-cell level and population level. In addition to simulating wild-type development, we also conduct knockdown analysis. Our simulations showed that the commitment is a three-step process over several cell generations where a cell is first prepared by a transcriptional switch. This is followed by the loss of the Bcl11b-opposing function two to three generations later which is when the decision to commit is taken. Finally, after another one to two generations, the cell becomes committed by transitioning to the DN2b state. Our results showed that there is inheritance in the commitment mechanism.

systems biology↗

bioRxiv2023

Multiplex, quantitative, high-resolution imaging of protein:protein complexes via hybridization chain reaction

Signal amplification based on the mechanism of hybridization chain reaction (HCR) facilitates spatial exploration of gene regulatory networks by enabling multiplex, quantitative, high-resolution imaging of RNA and protein targets. Here, we extend these capabilities to the imaging of protein:protein complexes, using proximity-dependent cooperative probes to conditionally generate a single amplified signal if and only if two target proteins are colocalized within the sample. HCR probes and amplifiers combine to provide automatic background suppression throughout the protocol, ensuring that even if reagents bind nonspecifically in the sample, they will not generate amplified background. We demonstrate protein:protein imaging with high signal-to-background in human cells, mouse proT cells, and highly autofluorescent formalin-fixed paraffin-embedded (FFPE) human breast tissue sections. Further, we demonstrate multiplex imaging of 3 different protein:protein complexes simultaneously and validate that HCR enables accurate and precise relative quantitation of protein:protein complexes with subcellular resolution in an anatomical context. Moreover, we establish a unified framework for simultaneous multiplex, quantitative, high-resolution imaging of RNA, protein, and protein:protein targets, with 1-step, isothermal, enzyme-free HCR signal amplification performed for all target classes simultaneously. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/550181v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@dee6f4org.highwire.dtl.DTLVardef@cc6a6org.highwire.dtl.DTLVardef@c5ea79org.highwire.dtl.DTLVardef@1a4edee_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

Runx factors launch T-cell and innate lymphoid programs via direct and gene network-based mechanisms

Runx factors are essential for lineage specification of various hematopoietic cells, including T lymphocytes. However, they regulate context-specific genes and occupy distinct genomic regions in different cell types. Here, we show that dynamic Runx binding shifts in early T-cell development are mostly not restricted by local chromatin state but regulated by Runx dosage and functional partners. Runx co-factors compete to recruit a limited pool of Runx factors in early T-progenitors, and a modest increase in Runx protein availability at pre-commitment stages causes premature Runx occupancy at post-commitment binding sites. This results in striking T-lineage developmental acceleration by selectively activating T-identity and innate lymphoid cell programs. These are collectively regulated by Runx together with other, Runx-induced transcription factors that co-occupy Runx target genes and propagate gene network changes.

immunology↗

bioRxiv2022

Distinct super-enhancer elements differentially control Il2ra gene expression in a cell-type specific fashion

The IL-2 receptor -chain (IL-2R/CD25) is constitutively expressed on DN2/DN3 thymocytes and Treg cells but induced by IL-2 on mature T and NK cells. Il2ra expression is regulated by a super-enhancer extensively bound by STAT5 in mature T cells. Here, we demonstrate that STAT5 cooperates with Notch to induce/maintain Il2ra/CD25 expression in DN2/DN3 cells. Moreover, we systematically investigated CD25 regulation using a series of mice with deletions spanning STAT5 binding elements. Deleting the upstream super-enhancer region mainly affected constitutive CD25 expression on DN2/DN3 thymocytes and Tregs, whereas deleting an intronic region primarily decreased IL-2-induced CD25 on peripheral T and NK cells. Thus, distinct elements preferentially control constitutive versus inducible expression in a cell-type-specific manner, with the MED1 coactivator co-localizing with specific STAT5 binding sites. Moreover, the intronic region was a dominant element whose deletion altered the structure throughout the super-enhancer in mature T cells. These results demonstrate differential functions for distinct super-enhancer elements, thereby indicating ways to manipulate CD25 expression in a cell-type specific fashion.

immunology↗

bioRxiv2021

Single-cell perturbation dissects transcription factor control of progression speed and trajectory choice in early T-cell development

In early T-cell development, single cells dynamically shift expression of multiple transcription factors (TFs) during transition from multipotentiality to T-lineage commitment, but the functional roles of many TFs have been obscure. Here, synchronized in vitro differentiation systems, scRNA-seq with batch indexing, and controlled gene-disruption strategies have unraveled single-cell impacts of perturbing individual TFs at two stages in early T-cell development. Single-cell CRISPR perturbation revealed that early-acting TFs Bcl11a, Erg, Spi1 (PU.1), Gata3, and Tcf7 (TCF1) each play individualized roles promoting or retarding T-lineage progression and suppressing alternative trajectories, collectively determining population dynamics and path topologies. Later, during T-lineage commitment, cells prevented from expressing TF Bcl11b realized this abnormality not with a developmental block, but by shifting into a divergent path via bZIP and Sox TF activation as well as E protein antagonism, finally exiting the T-lineage trajectory. These TFs thus exert a network of impacts to control progression kinetics, trajectories, and differentiation outcomes of early pro-T cells.

immunology↗

bioRxiv2019

Multi-scale dynamical modelling of T-cell development from an early thymic progenitor state to lineage commitment

Thymic development of committed pro-T-cells from multipotent hematopoietic precursors offers a unique opportunity to dissect the molecular circuitry establishing cell identity in response to environmental signals. This transition encompasses programmed shutoff of stem/progenitor genes, upregulation of T-cell specification genes, extensive proliferation, and commitment after a delay. We have incorporated these factors, as well as new single cell gene expression and developmental kinetics data, into a three-level dynamic model of commitment based upon regulation of the commitment gene Bcl11b. The first level is a core gene regulatory network architecture determined by transcription factor perturbation data, the second a stochastically controlled epigenetic gate, and the third a proliferation model validated by growth and commitment kinetics measured at single-cell levels. Using expression values consistent with single molecule RNA-FISH measurements of key transcription factors, this single-cell model exhibits state switching consistent with measured population and clonal proliferation and commitment times. The resulting multi-scale model provides a powerful mechanistic framework for dissecting commitment dynamics.

systems biology↗

bioRxiv2018

Pioneering, chromatin remodeling, and epigenetic constraint in early T-cell gene regulation by PU.1

PU.1 is a dominant but transient regulator in early T-cell precursors and a potent transcriptional controller of developmentally important pro-T cell genes. Before T-lineage commitment, open chromatin is frequently occupied by PU.1, and many PU.1 sites lose accessibility when PU.1 is later downregulated. Pioneering activity of PU.1 was tested in in this developmentally dynamic context, by quantitating the relationships between PU.1 occupancy and site quality and accessibility as PU.1 levels naturally declined in pro-T cell development, and by using stage-specific gain and loss of function perturbations to relate binding to effects on target genes. PU.1 could bind closed genomic sites, but rapidly opened many of them, despite the absence of its frequent collaborators, C/EBP factors. The dynamic properties of PU.1 engagements implied that PU.1 binding affinity and concentration determine its occupancy choices, but with quantitative tradeoffs for occupancy between site sequence quality and stage-dependent site accessibility in chromatin. At non-promoter sites PU.1 binding criteria were more stringent than at promoters, and PU.1 was also much more effective as a transcriptional regulator at non-promoter sites where local chromatin accessibility depended on the presence of PU.1. Runx motifs and Runx1 binding were often linked to PU.1 at open sites, but highly expressed PU.1 could bind its sites without Runx1. Notably, closed chromatin presented a qualitative barrier to occupancy by the PU.1 DNA binding domain alone. Thus, effective pioneering at closed chromatin sites also depends on requirements beyond site recognition served by non-DNA binding domains of PU.1.

immunology↗

bioRxiv2018

A stochastic epigenetic switch controls the dynamics of T-cell lineage commitment

Cell fate decisions occur through the switch-like, irreversible activation of fate-specifying genes. These activation events are often assumed to be tightly-coupled to changes in upstream transcription factors, but could also be constrained by cis-epigenetic mechanisms at individual gene loci. Here, we studied the activation of Bcl11b, which controls T-cell fate commitment. To disentangle cis and trans effects, we generated mice where two Bcl11b copies are tagged with distinguishable fluorescent proteins. Quantitative live microscopy of progenitors from these mice revealed that Bcl11b turned on after a stochastic delay averaging multiple days, which varied not only between cells but also between Bcl11b alleles within the same cell. Genetic perturbations, together with mathematical modeling, showed that a distal enhancer controls the rate of epigenetic activation, while a parallel Notch-dependent trans-acting step stimulates expression from activated loci. These results show that developmental fate transitions can be controlled by stochastic cis-acting events on individual loci.

bioinformatics↗