Biology subjects

Ilia, K.

Publications and source records attributed to Ilia, K..

3 recordsLinked to original sources

bioRxiv2024

Analog epigenetic cell memory by graded DNA methylation

Chemical modifications to histones and DNA play a crucial role in the regulation of transcription and in the maintenance of chromatin states that are not permissive to gene expression [1-3]. However, the landscape of gene expression states that these modifications stably maintain remains uncharted. Here, we show that gene expression can be memorized at a wide range of levels thus implementing analog epigenetic memory. Mechanistically, we find that DNA methylation serves a primary role in maintaining memory across cell divisions while histone modifications only follow DNA methylation to regulate gene expression. Employing targeted epigenetic editing and time-course analysis, we analyzed the temporal stability of gene expression and DNA methylation post removal of epigenetic effectors. We found that the grade of DNA methylation in the genes promoter, defined as the mean fraction of methylated CpGs, remains stable over time and inversely correlates with gene expression level. By contrast, Histone 3 lysine 9 trimethylation (H3K9me3) could not persist after removal of its writer in the absence of DNA methylation. These experimental findings, combined with our chromatin modification model, indicate that the absence of positive feedback mechanisms around DNA methylation - unlike those found in histone modifications - enable the temporal stability of the DNA methylation grade, which leads to analog memory. These results expand current knowledge on how epigenetic memory is achieved in natural systems. Moreover, we anticipate that analog memory through graded DNA methylation will enable to program mammalian cells with fine-grained information storage. This capability will significantly enhance the sophistication of engineered cell functionality in applications including tissue engineering, organoids, and cell therapies.

bioengineering↗

bioRxiv2023

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. One-sentence summaryGenetic controllers and live-cell imaging offer a versatile strategy for probing the role of transcription factor dynamics in cell fate transitions.

synthetic biology↗

bioRxiv2021

Robust and tunable signal processing in mammalian cells via engineered covalent modification cycles

Rewired and synthetic signaling networks can impart cells with new functionalities and enable efforts in engineering cell therapies and directing cell development. However, there is a need for tools to build synthetic signaling networks that are tunable, can precisely regulate target gene expression, and are robust to perturbations within the complex context of mammalian cells. Here, we use proteins derived from bacterial two-component signaling pathways to develop synthetic phosphorylation-based and feedback-controlled devices in mammalian cells with such properties. First, we isolate kinase and phosphatase proteins from the bifunctional histidine kinase EnvZ. We then use these proteins to engineer a synthetic covalent modification cycle, in which the kinase and phosphatase competitively regulate phosphorylation of the cognate response regulator OmpR, enabling analog tuning of OmpR-driven gene expression. Further, we show that the phosphorylation cycle can be extended by connecting phosphatase expression to small molecule and miRNA inputs in the cell, with the latter enabling cell-type specific signaling responses and accurate cell type classification. Finally, we implement a tunable negative feedback controller by co-expressing the kinase-driven output gene with the small molecule-tunable phosphatase. This negative feedback substantially reduces cell-to-cell noise in output expression and mitigates the effects of cell context perturbations due to off-target regulation and resource competition. Our work thus lays the foundation for establishing tunable, precise, and robust control over cell behavior with synthetic signaling networks.

synthetic biology↗