Expression of Zfhep/deltaEF1 protein in palate, neural progenitors, and differentiated neurons.

Zfhep/deltaEF1 is essential for embryonic development. We have investigated the expression pattern of Zfhep protein during mouse embryogenesis. We show expression of Zfhep in the mesenchyme of the palatal shelves, establishing concordance of expression with the reported cleft palate of the deltaEF1-null mice. Zfhep protein is strongly expressed in proliferating progenitors of the nervous system. In most regions of the brain, post-mitotic cells stop expressing Zfhep when they migrate out of the ventricular zone (VZ) and differentiate. However, in the hindbrain, Zfhep protein is also highly expressed in post-mitotic migratory neuronal cells of the precerebellar extramural stream that arise from the neuroepithelium adjacent to the lower rhombic lip. Also, Zfhep is expressed as cells migrate from a narrow region of the pons VZ towards the trigeminal nucleus. Co-expression with Islet1 shows that Zfhep is expressed in motor neurons of the trigeminal nucleus of the pons, but not in the inferior olive motor neurons at E12.5. Therefore, Zfhep is strongly expressed in a tightly regulated pattern in proliferating neural stem cells and a subset of neurons. Zfhep protein is also strongly expressed in trigeminal ganglia, and is moderately expressed in other cranial ganglia. In vitro studies have implicated Zfhep as a repressor of myogenesis, however, we find that Zfhep protein expression increases during muscle differentiation.

Cell-specific phosphorylation of Zfhep transcription factor.

Zinc finger homeodomain enhancer-binding protein (Zfhep/Zfhx1a) is a transcription factor essential for immune system development, skeletal patterning, and life. Regulation of the interleukin-2 gene in T cells has been suggested to depend on post-translational processing of Zfhep, however, no modifications of Zfhep are known. Here we demonstrate that Zfhep is present in both hyperphosphorylated and hypophosphorylated forms. Western blot analysis demonstrates two forms of Zfhep with different mobilities. Differences in phosphorylation are sufficient to explain the difference in mobilities. Zfhep is primarily phosphorylated on Ser and Thr residues since PP2A dephosphorylates the slower mobility band. Treatment of nuclear extract with O-GlcNAcase did not detect O-linked sugar. Importantly, post-translational processing is cell-specific. Doublets of Zfhep were detected in five cell lines, whereas 6 cell lines contain only, or predominantly, non-phosphorylated Zfhep, and Saos-2 cells contain predominantly the phosphorylated form. These data provide the first demonstration that Zfhep is post-translationally modified.