The TALE-homeodomain transcription factor MEIS2 in neuronal differentiation of murine embryonic stem cells
Authors:   , Müller Tamara 
Published: January 2025

This dissertation investigated the role of the transcription factor MEIS2 (myeloid ecotropic viral integration site) in early neuronal differentiation of murine embryonic stem cells (mESCs). MEIS family transcription factors are members of the three amino acid loop extension (TALE) superclass of homeodomain proteins, which exert crucial functions during development. Three Meis genes (Meis1-3) are known in mammals and MEIS1 and MEIS2 have been repeatedly associated with brain development. MEIS2 in particular has been shown to be involved in neurogenesis in the adult subventricular zone (SVZ) and neuronal subtype differentiation. Whereas the functions of MEIS2 in the adult SVZ system are well established, much less is known about a possible contribution of MEIS2 to cell fate decisions during early brain development. A frequently used in vitro system to study early neuronal development is the differentiation of ESCs into neurons. The understanding of the contribution of MEIS2 to this differentiation has the potential to shed light on the molecular basis underlying the phenotypes of patients suffering from the rare neurodevelopmental MEIS2-related disorder. Thus, the presented thesis aimed at filling this knowledge gap and providing first insights into the explanations for phenotypes observed in MEIS2-related disorder patients. The experiments of this study were conducted with distinct murine ESC-lines, including wildtype (WT) as well as Meis1/Meis2 double knockout (dKO) and Meis2 single knockout (sKO) cell lines, which were generated by CRISPR/Cas and were provided for the experiments of the present thesis.
The expression of Meis2 and its homologue Meis1 was induced at early stages after induction of neuronal differentiation in WT cells and mainly increased during the differentiation process. Work from our group demonstrated that the splice isoforms MEIS2A and MEIS2D exhibit opposing functions in cancer, with MEIS2A being able to induce neuronal differentiation. In neuronally differentiated ESCs, expression of Meis2a (together with Meis2b) exceeded that of Meis2c/d throughout the observation period. To examine the consequences of a Meis2 loss, cells deficient of Meis1 and Meis2 were compared with WT cells during early neuronal differentiation. Concomitant deletion of Meis1 and Meis2 was initially chosen to circumvent the well-known compensation between MEIS-family proteins. Whereas no differences between the genotypes could be detected in the pluripotent ESC-stage, the first obvious difference was detected during differentiation when dKO cell cultures exhibited a massive amount of apparent cell death. The point in time of dKO cells showing this phenotype correlated with a decrease of Oct4 (pluripotency marker) and increase of Sox1 (neuroectodermal marker) expression, which indicates that dKO cells were less able to exit pluripotency and commit to the neuronal cell fate. Examining the expression of the neuronal marker Doublecortin (Dcx), resulted in a delay in the onset of Dcx expression in dKO cells and overall lower levels compared to WT cells. This was confirmed on the protein level and in a first approach via three-dimensional Fluorescence in situ hybridization a trend towards more Dcx loci at the repressive nuclear periphery in dKO cells was observed.
After having established that dKO cells exhibited a reduction of the neuronal differentiation potential, the question arose why a small population among the survived cells was still able to differentiate into young neurons albeit the loss of Meis1 and Meis2. To test for a possible compensatory mechanism, a genome-wide expression analysis in WT and dKO cells after four days of neurogenic differentiation was performed. Among the genes whose expression increased in the absence of Meis1 and Meis2 were the forebrain-specific transcription factors Fezf1 and Foxg1. FOXG1 controls the pool of progenitor cells and prevents premature neuronal differentiation. In the present thesis it is therefore suggested that the up-regulation of Foxg1 may serve to slow down differentiation of dKO cells and thereby allow these cells sufficient time to change their transcriptional neuronal differentiation program into one that is independent of MEIS1 and MEIS2.
To understand, how MEIS2 might regulate Foxg1 and other targets, Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) with an antibody against MEIS1 and MEIS2 was performed in WT cells differentiated for three days. This experiment revealed many binding events in distal intergenic regions, which suggested binding to regulatory elements. Indeed, MEIS1/MEIS2 was bound to a considerable number of enhancers. In the context of Foxg1, binding events were detected in a 954 kb large ‘gene desert’ between Foxg1 and its downstream gene Prkd1. Among these, binding to four experimentally verified brain-specific enhancer sequences, of which three were forebrain-specific, were detected. This indicates that MEIS1/MEIS2 may repress Foxg1 via binding to its regulatory sequences. Further data demonstrate that single KO of Meis2 was sufficient to reduce Dcx expression and increase Foxg1 expression, which indicates that MEIS2 rather than MEIS1 was responsible for the observed phenotypes. To investigate whether MEIS2 had the ability to repress Foxg1, lentiviral overexpression of Meis2a was performed. This experiment resulted in a significant decrease of FOXG1-immunoreactivity in transduced cells, which strongly suggests that MEIS2 can act as repressor of Foxg1. The results of this work for the first time describe a repressive function of MEIS2 on Foxg1 and provide a profound basis for further research on the molecular mechanisms underlying the phenotypes observed in patients suffering from the MEIS2-related disorder.