PhD Scientific Days 2025

Poster Session I. - A: Molecular Medicine

Investigating the Role of De Novo Mutations in the POU Neuronal Gene Family

Name of the presenter

Molnár Krisztina

Institute/workplace of the presenter

Semmelweis University – Institute of Biochemistry and Molecular Biology, Department of Molecular Biology

Authors

Krisztina Molnár¹, Réka Kovács-Nagy¹

1: Semmelweis University – Institute of Biochemistry and Molecular Biology, Department of Molecular Biology

Text of the abstract

Introduction
POU3F2 and POU3F3 proteins are essential for neuronal differentiation. Exome sequencing of two patients revealed two gene variants, causing amino acid changes in the conserved DNA-binding regions of these proteins, potentially contributing to neuronal developmental disorders.
Aims
Our study focused on the functional analysis and characterization of the two identified mutations in the POU3F2 (E271V) and POU3F3 (K406R) genes to provide insights into molecular diagnosis and improve understanding of the underlying pathomechanism.
Methods
Coding sequences of wild-type POU transcription factors were cloned into pSF-CMV-Puro-NH2-HA expression vector. Mutant variant coding vectors were obtained by site-directed mutagenesis. Besides that, we created an artificial mutant POU3F2 (E271Q). The promoter region of ZEB2 gene, with a putative POU binding site, was cloned into pGL3B luciferase reporter vector. HEK293 and SK-N-FI cell lines were used for transient transfection. The impact of the transcription factor mutations was quantified by measuring relative luciferase activity. Western blot analysis was used for the detection of POU transcription factors. For mRNA level measurement, RT-qPCR was used. The results were statistically analyzed using t-tests.
Results
Based on in silico analysis, we selected ZEB2 from the previously identified target genes for further studies. Functional analysis on HEK293 cell line showed a decreased luciferase activity with the mutant variants both for POU3F2 and POU3F3 proteins (p=0.0431; p=0.0492). The artificial mutant of POU3F2 did not show a significantly lowered luciferase activity (p=0.3415). Baseline was measured using an empty expression vector. The presence of the transcription factor coding insert gave a significantly higher luciferase signal both for POU3F2 and F3. mRNA levels showed no significant change in HEK293 cell lines after transfection.
Conclusion
Our measurements suggest that the mutations do not change the stability but the function of the POU3F2 and POU3F3 proteins, thus possibly play a role in the neurodevelopmental delay of the two patients.
Funding
Students in Research Scholarship, 2022 – Richter Gedeon Centenáriumi Alapítvány
SE 250+ Excellence PhD Scholarship, 2024, 2025 – Semmelweis University
Institutional Grant Baron Münchausen, 2021, 2022 – SE, Dept. of Molecular Biology