Biology subjects

Yagci, Z. B.

Publications and source records attributed to Yagci, Z. B..

3 recordsLinked to original sources

bioRxiv2025

A Human Angelman Syndrome Class II Pluripotent Stem Cell line with Fluorescent Paternal UBE3A Reporter

IntroductionAngelman Syndrome (AS) is characterized in large part by the loss of functional UBE3A protein in mature neurons. A majority of AS etiologies is linked to deletion of the maternal copy of the UBE3A gene and epigenetic silencing of the paternal copy. A common therapeutic strategy is to unsilence the intact paternal copy thereby restoring UBE3A levels. Identifying novel therapies has been aided by a UBE3A-YFP reporter mouse model. This study presents an analogous fluorescent UBE3A reporter system in human cells. MethodsPreviously derived induced Pluripotent Stem Cells (iPSCs) with a Class II large deletion at the UBE3A locus are used in this study. mGL and eGFP are integrated downstream of the endogenous UBE3A using CRISPR/Cas9. These reporter iPSCs are differentiated into 2D and 3D neural cultures to monitor long-term neuronal maturation. Green fluorescence dynamics are analyzed by immunostaining and flow cytometry. ResultsThe reporter is successfully integrated into the genome and reports paternal UBE3A expression. Fluorescence expression gradually reduces with UBE3A silencing in neurons as they mature. Expression patterns also reflect expected responses to molecules known to reactivate paternal UBE3A. DiscussionThis human-cell-based model can be used to screen novel therapeutic candidates, facilitate tracking of UBE3A expression in time and space, and study human-specific responses. However, its ability to restore UBE3A function cannot be studied using this model. Further research in human cells is needed to engineer systems with functional UBE3A to fully capture the therapeutic capabilities of novel candidates.

cell biology↗

bioRxiv2025

A High Sensitivity Assay of UBE3A Ubiquitin Ligase Activity

UBE3A is an E3 ubiquitin ligase associated with several neurodevelopmental disorders. The development of several preclinical therapeutic approaches involving UBE3A, such as gene therapy, enzyme replacement therapy, and epigenetic reactivation, require the detection of its ubiquitin ligase activity. Prior commercial assays leveraged Western Blotting to detect shifts in substrate size due to ubiquitination, but these suffered from long assay times and have also been discontinued. Here we develop a new assay that quantifies UBE3A activity. It measures the fluorescence intensity of ubiquitinated p53 substrates with a microplate reader, eliminating the need for Western Blot antibodies and instruments, and enables detection in just 1 hour. The assay is fast, cost-effective, low noise, and uses components with long shelf lives. Importantly, it is also highly sensitive, detecting UBE3A levels as low as 1nM, similar to that observed in human and mouse cerebrospinal fluid. It also differentiates the activity of wild-type UBE3A and catalytic mutants. We also design a p53 substrate with a triple-epitope tag HIS-HA-CMYC on the N terminus, which allows for versatile detection of UBE3A activity from diverse natural and engineered sources. This new assay provides a timely solution for growing needs in preclinical validation, quality control, endpoint measurements for clinical trials, and downstream manufacturing testing and validation.

biochemistry↗

bioRxiv2024

Loss of UBE3A impacts both neuronal and non-neuronal cells in human cerebral organoids

Angelman syndrome is a neurodevelopmental disorder caused by (epi)genetic lesions of maternal UBE3A. Research has focused largely on the role of UBE3A in neurons due to its imprinting in that cell type. Yet, evidence suggests there may be broader neurodevelopmental impacts of UBE3A dysregulation. Human cerebral organoids might reveal these understudied aspects of UBE3A as they recapitulate diverse cell types of the developing human brain. We performed scRNAseq on organoids to reveal the effects of UBE3A disruption on cell type-specific compositions and transcriptomic alterations. In the absence of UBE3A, progenitor proliferation and structures were disrupted while organoid composition shifted away from proliferative cell types. We observed impacts on non-neuronal cells, including choroid plexus enrichment. Furthermore, EMX1+ cortical progenitors were negatively impacted, disrupting corticogenesis, and potentially delaying excitatory neuron maturation. This work reveals novel impacts of UBE3A on understudied cell types and related neurodevelopmental processes and elucidates potential new therapeutic targets. TeaserHuman cerebral organoids exhibit compositional and transcriptomic alterations in both neuronal and non-neuronal cells in the absence of UBE3A.

developmental biology↗