PhD Scientific Days 2024

Budapest, 9-10 July 2024

Poster Session E - Molecular Medicine 2.

Functional Analysis of De Novo Variants in the POU Neuronal Gene Family

Author(s)

Dr. Krisztina Molnár1
1: Semmelweis University – Institute of Biochemistry and Molecular Biology, Department of Molecular Biology

Text of the abstract

Introduction
Pou3f2 and Pou3f3 proteins play a crucial role in neuronal differentiation. Two gene variants in two patients were identified by exome sequencing, resulting in amino acid changes in the conserved DNA-binding region of the proteins, potentially leading to neuronal developmental disorders.
Aims
Our research aimed to conduct the functional analysis and the description of the two identified mutations in POU3F2 and POU3F3 genes for a potential molecular diagnosis and a better understanding of the pathomechanism.
Methods
We cloned the promoter regions of two previously chosen target genes (ZEB2 and H2AC6) into pGL3B luciferase vectors. The cloned promoter regions contained one and two putative binding sites for the Pou transcription factors in the ZEB2 and H2AC6 5’ regions, respectively.
We designed protein-expressing constructs using a pSF-CMV-Puro-NH2-HA vector containing the wild-type genes of POU3F2 and POU3F3. DNA constructs coding the mutant variants of the proteins were created by site-directed mutagenesis. HEK293 cells were used for transient transfection. The efficacy of the transfection and the endogenous levels of the Pou3-proteins were determined by Western blot analysis. The impact of the transcription factors was quantified by measuring relative luciferase activity. The results of the luciferase assays were statistically analyzed using t-tests.
Results
Pou3f2 and Pou3f3 target genes and their putative binding sequences were previously identified. Based on in silico analysis, we selected H2AC6 and ZEB2 genes for further studies. We carried out the functional analysis on HEK293 cell line, which showed that the mutant variants of Pou3f2 and Pou3f3 proteins could not exert their function to the extent as the wild types of the transcription factors could. This significantly lowered the relative luciferase activity for POU3F2 with the ZEB2 promoter (p = 0,00068) and for POU3F3 with the H2AC6 promoter (p = 0,017).
Conclusion
The results of our measurements suggest that the mutations change the function of the Pou3f2 and Pou3f3 proteins, and thus, they could cause the neurodevelopmental delay of the two patients.
Funding
Students in Research Scholarship, 2022 – Richter Gedeon Centenáriumi Alapítvány
SE 250+ Excellence PhD Scholarship, 2024 – Semmelweis University
Institutional Grant Baron Münchausen, 2021, 2022 – SE, Dept. of Molecular Biology