Undifferentiated Cell Transcription Research Articles

Transcription of the chicken Grin1 gene is regulated by the activity of SP3 and NRSF in undifferentiated cells and neurons

The NMDA (N-methyl-D-aspartate) receptors are important in the regulation of neuronal development, synaptic plasticity, learning and memory, and are involved in several brain pathologies. The NR1 subunit is essential for the assembly of functional receptors, as it forms the calcium-permeable ion channel and contains the obligatory co-agonist binding site. Previous studies have shown that NR1 gene (Grin1) expression is up-regulated during neuronal differentiation and its expression is widespread in the central nervous system. We have previously cloned the chicken Grin1 gene and 1.9 kb of the 5'-regulatory region. In the present study, we analysed the molecular mechanisms that regulate chicken Grin1 gene transcription in undifferentiated cells and neurons. By functional analysis of chicken Grin1-luciferase gene 5'-regulatory region constructs, we demonstrate that the basal promoter is delimited within 210 bp upstream from the main transcription initiation site. DNA-protein binding and functional assays revealed that the 5'-UTR (untranslated region) has one consensus NRSE (neuron-restrictive silencing element) that binds NRSF (neuron-restrictive silencing factor), and one SP (stimulating protein transcription factor) element that binds SP3, both repressing Grin1 gene transcription in undifferentiated P19 cells (embryonic terato-carcinoma cells) and PC12 cells (phaeochromocytoma cells). The promoter region lacks a consensus TATA box, but contains one GSG/SP (GSG-like box near a SP-consensus site) that binds SP3 and up-regulates gene transcription in embryonic chicken cortical neurons. Taken together, these results demonstrate a dual role of SP3 in regulating the expression of the Grin1 gene, by repressing transcription in the 5'-UTR in undifferentiated cells as well as acting as a transcription factor, increasing Grin1 gene transcription in neurons.

HoxA9 Activates Transcription of the Gene Encoding Gp91phox, a Respiratory Burst Oxidase Protein, during Myeloid Differentiation.

Transcription of the CYBB gene, which encodes the respiratory burst oxidase protein, gp91phox, is restricted to phagocytic cells, differentiated beyond the promyelocyte stage. Regulation of CYBB transcription involves several repressor cis elements in the promoter. These repressor elements are homologous to the derived consensus sequence for DNA-binding of HoxA10 as a hetero-dimer with Pbx1a. The HoxA10/Pbx1 complex recruits histone deacetylase 2 and represses CYBB transcription in undifferentiated cells. During myeloid differentiation, HoxA10 is tyrosine phosphorylated, which decreases DNA-binding affinity for the CYBB repressor element. In our previous investigations, we found this decreased binding affinity requires phosphorylation of two tyrosine residues in the DNA-binding homeodomain. These residues are highly conserved with other HoxA proteins. However, we also found decreased DNA-binding requires interaction of these homeodomain tyrosines with a non-conserved domain, amino terminal in HoxA10. In previous investigations, we determined these CYBB repressor cis elements function as positive cis elements in differentiated myeloid cells, and interact with an unidentified protein complex. In these studies, we investigate the proteins involved in transcriptional activation of the CYBB gene, via these promoter sequences. We find this cis element is activated by HoxA9 and Pbx1a, in differentiating myeloid cells. Consistent with this, we find increased binding of HoxA9 to this CYBB cis element, during myeloid differentiation, in vitro and in vivo. Consistent with our results with HoxA10, we find HoxA9 is tyrosine phosphorylated during myeloid differentiation. However, in contrast to HoxA10, we find tyrosine phosphorylation of the conserved residues in the HoxA9 DNA-binding homeodomain increases binding to the CYBB cis element. Therefore, we conclude HoxA10 and HoxA9 have opposing functions, for myeloid specific gene transcription, during differentiation. We also find these functions are regulated by post translational modification of conserved tyrosine residues in the homeodomain regions of these proteins. Additionally, our results suggest differences in the effect of phosphorylation of these residues on DNA-binding is likely due to interaction with non-conserved domains in HoxA9 and HoxA10.