Biology subjects

Salibi, E.

Publications and source records attributed to Salibi, E..

4 recordsLinked to original sources

bioRxiv2025

Reversing transgene silencing via targeted chromatin editing

Mammalian cell engineering offers the opportunity to uncover biological principles and develop next-generation biotechnologies. However, epigenetic silencing of transgenes hinders the control of gene expression in mammalian cells. Here, we use chromatin editing of an integrated reporter in CHO-K1 and human induced pluripotent stem cells to study the molecular interactions driving silencing and its reversal. After transient induction of either DNA methylation or H3K9me3, stable silencing was exclusively observed with both marks. Due to the positive feedback between DNA methylation and H3K9me3 and the relative low stability of H3K9me3, our model predicts that removing DNA methylation is sufficient for transgene reactivation. Accordingly, targeted DNA demethylation reactivated the reporter irrespective of whether silencing was achieved by inducing DNA methylation, H3K9me3, or by the endogenous cellular machinery. These results shed light on molecular mechanisms at play during silencing and provide engineering tools for potent and specific transgene reactivation in mammalian cells.

bioengineering↗

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↗

bioRxiv2022

Ribozyme-phenotype coupling in peptide-based coacervate protocells

Condensed coacervate phases are now understood to be important features of modern cell biology, as well as valuable protocellular models in origin of life studies and synthetic biology. In each of these fields, the development of model systems with varied and tuneable material properties is of great importance for replicating properties of life. Here, we develop a ligase ribozyme system capable of concatenating short RNA fragments into extremely long chains. Our results show that formation of coacervate microdroplets with the ligase ribozyme and poly(L-lysine) enhances ribozyme rate and yield, which in turn increases the length of the anionic polymer component of the system and imparts specific physical properties to the droplets. Droplets containing active ribozyme sequences resist growth, do not wet or spread on unpassivated surfaces, and exhibit reduced transfer of RNA between droplets when compared to controls containing inactive sequences. These altered behaviours, which stem from RNA sequence and catalytic activity, constitute a specific phenotype and potential fitness advantage, opening the door to selection and evolution experiments based on a genotype - phenotype linkage.

synthetic biology↗

bioRxiv2020

CRISPR interference as low burden logic inverters in synthetic circuits: characterization and tuning

The rational design of complex biological systems through the interconnection of single functional building blocks is hampered by many unpredictability sources; this is mainly due to the tangled context-dependency behavior of those parts once placed into an intrinsically complex living system. Among others, the finite amount of translational resources in prokaryotic cells leads to load effects in heterologous protein expression. As a result, hidden interactions among protein synthesis rates arise, leading to unexpected and counterintuitive behaviors. To face this issue in rational design of synthetic circuits in bacterial cells, CRISPR interference is here evaluated as genetic logic inverters with low translational resource usage, compared with traditional transcriptional regulators. This system has been studied and characterized in several circuit configurations. Each module composing the circuit architecture has been optimized in order to meet the desired specifications, and its reduced metabolic load has been eventually demonstrated via in-vivo assays.

synthetic biology↗