Matrin3 mediates differentiation through stabilizing chromatin loop-domain interactions and YY1 mediated enhancer-promoter interactions.

Although emerging evidence indicates that alterations in proteins within nuclear compartments elicit changes in chromosomal architecture and differentiation, the underlying mechanisms are not well understood. Here we investigate the direct role of the abundant nuclear complex protein Matrin3 (Matr3) in chromatin architecture and development in the context of myogenesis. Using an acute targeted protein degradation platform (dTAG-Matr3), we reveal the dynamics of development-related chromatin reorganization. High-throughput chromosome conformation capture (Hi-C) experiments revealed substantial chromatin loop rearrangements soon after Matr3 depletion. Notably, YY1 binding was detected, accompanied by the emergence of novel YY1-mediated enhancer-promoter loops, which occurred concurrently with changes in histone modifications and chromatin-level binding patterns. Changes in chromatin occupancy by Matr3 also correlated with these alterations. Overall, our results suggest that Matr3 mediates differentiation through stabilizing chromatin accessibility and chromatin loop-domain interactions, and highlight a conserved and direct role for Matr3 in maintenance of chromosomal architecture.

Synthetic genetic circuits to uncover the OCT4 trajectories of successful reprogramming of human fibroblasts.

Reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs) is inefficient, with heterogeneity among transcription factor (TF) trajectories driving divergent cell states. Nevertheless, the impact of TF dynamics on reprogramming efficiency remains uncharted. We develop a system that accurately reports OCT4 protein levels in live cells and use it to reveal the trajectories of OCT4 in successful reprogramming. Our system comprises a synthetic genetic circuit that leverages noise to generate a wide range of OCT4 trajectories and a microRNA targeting endogenous OCT4 to set total cellular OCT4 protein levels. By fusing OCT4 to a fluorescent protein, we are able to track OCT4 trajectories with clonal resolution via live-cell imaging. We discover that a supraphysiological, stable OCT4 level is required, but not sufficient, for efficient iPSC colony formation. Our synthetic genetic circuit design and high-throughput live-imaging pipeline are generalizable for investigating TF dynamics for other cell fate programming applications.

Synthetic genetic circuits to uncover and enforce the OCT4 trajectories of successful reprogramming of human fibroblasts.

Reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs) is inefficient, with heterogeneity among transcription factor (TF) trajectories driving divergent cell states. Nevertheless, the impact of TF dynamics on reprogramming efficiency remains uncharted. Here, we identify the successful reprogramming trajectories of the core pluripotency TF, OCT4, and design a genetic controller that enforces such trajectories with high precision. By combining a genetic circuit that generates a wide range of OCT4 trajectories with live-cell imaging, we track OCT4 trajectories with clonal resolution and find that a distinct constant OCT4 trajectory is required for colony formation. We then develop a synthetic genetic circuit that yields a tight OCT4 distribution around the identified trajectory and outperforms in terms of reprogramming efficiency other circuits that less accurately regulate OCT4. Our synthetic biology approach is generalizable for identifying and enforcing TF dynamics for cell fate programming applications.

EZH1 repression generates mature iPSC-derived CAR T cells with enhanced antitumor activity.

Human induced pluripotent stem cells (iPSCs) provide a potentially unlimited resource for cell therapies, but the derivation of mature cell types remains challenging. The histone methyltransferase EZH1 is a negative regulator of lymphoid potential during embryonic hematopoiesis. Here, we demonstrate that EZH1 repression facilitates in vitro differentiation and maturation of T cells from iPSCs. Coupling a stroma-free T cell differentiation system with EZH1-knockdown-mediated epigenetic reprogramming, we generated iPSC-derived T cells, termed EZ-T cells, which display a highly diverse T cell receptor (TCR) repertoire and mature molecular signatures similar to those of TCRαß T cells from peripheral blood. Upon activation, EZ-T cells give rise to effector and memory T cell subsets. When transduced with chimeric antigen receptors (CARs), EZ-T cells exhibit potent antitumor activities in vitro and in xenograft models. Epigenetic remodeling via EZH1 repression allows efficient production of developmentally mature T cells from iPSCs for applications in adoptive cell therapy.

Technical note: Evaluation of a silicone-based custom bolus for radiation therapy of a superficial pelvic tumor.

PURPOSE: Use of standard-of-care radiation therapy boluses may result in air-gaps between the target surface and bolus, as they may not adequately conform to each patient's unique topography. Such air-gaps can be particularly problematic in cases of superficial pelvic tumor radiation, as the density variation may result in the radiation delivered to the target site being inconsistent with the prescribed dose. To increase bolus fit and thereby dose predictability and homogeneity, we designed and produced a custom silicone bolus for evaluation against the clinical standard. METHODS: A custom bolus was created for the pelvic regions of both an anthropomorphic phantom and a pelvic patient with squamous cell carcinoma of the penile shaft. Molds were designed using computed tomography (CT) scans, then 3D-printed and cast with silicone rubber to yield the boluses. Air-gap measurements were performed on custom and standard-of-care Superflab gel sheet boluses by analyzing total volume between the bolus and target surface, as measured from CT scans. Therapeutic doses of radiation were delivered to both boluses. Radiation dose was measured and compared to the expected dose using nine optically stimulated luminescent dosimeters (OSLDs) placed on the phantom. RESULTS: Mean air-gap volume between the bolus and phantom was decreased from 314 ± 141 cm3 with the standard bolus to 4.56 ± 1.59 cm3 using the custom device. In the case of the on-treatment patient, air-gap volume was reduced from 169 cm3 with the standard bolus to 46.1 cm3 with the custom. Dosimetry testing revealed that the mean absolute difference between expected and received doses was 5.69%±4.56% (15.1% maximum) for the standard bolus and 1.91%±1.31% (3.51% maximum) for the custom device. Areas of greater dose difference corresponded to areas of larger air-gap. CONCLUSIONS: The custom bolus reduced air-gap and increased predictability of radiation dose delivered compared to the standard bolus. The custom bolus could increase the certainty of prescribed dose-delivery of radiation therapy for superficial tumors.