Transcriptional corepressors of the Groucho (Gro)/TLE family play important roles during a variety of developmental pathways, including neuronal differentiation. In particular, they act as negative regulators of neurogenesis, together with Hairy/Enhancer of split (Hes) DNA-binding proteins. The interaction with Hes1 leads to Gro/TLE hyperphosphorylation and increased transcription repression activity in mammalian cells, but the underlying molecular mechanisms are poorly characterized. We now show that Gro/TLE1 is phosphorylated in vivo by protein kinase CK2. This phosphorylation occurs at serine 239 within the conserved CcN domain present in all Gro/TLE family members. Mutation of serine 239 into alanine decreases Hes1-induced hyperphosphorylation of Gro/TLE1 and also reduces its nuclear association and transcription repression activity. We demonstrate further that Gro/TLE1 inhibits the transition of cortical neural progenitors into neurons and that its antineurogenic activity is inhibited by a serine-239-alanine mutation but not by a serine-239-glutamate mutation. These results suggest that CK2 phosphorylation of serine 239 of Gro/TLE1 is important for its function during neuronal differentiation.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene exhibits a tightly regulated pattern of expression in human epithelial cells. The mechanism of this regulation is complex and is likely to involve a number of genetic elements that effect temporal and spatial expression. To date none of the elements that have been identified in the CFTR promoter regulate tissue-specific expression. We have identified a putative regulatory element within the first intron of the CFTR gene at 181+10kb. The region containing this element was first identified as a DNase I hypersensitive site that was present in cells that express the CFTR gene but absent from cells not transcribing CFTR. In vitro analysis of binding of proteins to this region of DNA sequence by gel mobility shift assays and DNase I footprinting revealed that some proteins that are only present in CFTR-expressing cells bound to specific elements, and other proteins that bound to adjacent elements were present in all epithelial cells irrespective of their CFTR expression status. When assayed in transient expression systems in a cell line expressing CFTR endogenously, this DNA sequence augmented reporter gene expression through activation of the CFTR promoter but had no effect in nonexpressing cells.

Neurogenesis requires factors that regulate the decision of dividing progenitors to leave the cell cycle and activate the neuronal differentiation program. It is shown here that the murine runt-related gene Runx1 is expressed in proliferating cells on the basal side of the olfactory epithelium. These include both Mash1ϩ olfactory receptor neuron (ORN) progenitors and NeuroDϩ ORN precursors. Disruption of Runx1 function in vivo does not cause a change in Mash1 expression but leads to a decrease in the number of NeuroDϩ neuronal precursors and an increase in differentiated ORNs. These effects result in premature and ectopic ORN differentiation. It is shown further that exogenous Runx1 expression in cultured olfactory neural progenitors causes an expansion of the mitotic cell population. In agreement with these findings, exogenous Runx1 expression also promotes cortical neural progenitor cell proliferation without inhibiting neuronal differentiation. These effects are phenocopied by a chimeric protein containing ETO, the eight twenty one transcriptional repressor, fused to the Runx1 DNA-binding domain, which suggests the involvement of transcription repression mechanisms. Consistent with this possibility, Runx1 represses transcription driven by the promoter of the cell cycle inhibitor p21Cip 1 in cortical progenitors. Together, these findings suggest a previously unrecognized role for Runx1 in coordinating the proliferation and neuronal differentiation of selected populations of neural progenitors.

Transcriptional corepressors of the Groucho/transducin-like Enhancer of split (Gro/TLE) family regulate a number of developmental pathways in both invertebrates and vertebrates. They form transcription repression complexes with members of several DNA-binding protein families and participate in the regulation of the expression of numerous genes. Despite their pleiotropic roles, little is known about the mechanisms that regulate the functions of Gro/TLE proteins. It is shown here that Gro/TLEs become hyperphosphorylated in response to neural cell differentiation and interaction with the DNA-binding cofactor Hairy/Enhancer of split 1 (Hes1). Hyperphosphorylation of Gro/TLEs is correlated with a tight association with the nuclear compartment through interaction with chromatin, suggesting that hyperphosphorylated Gro/TLEs may mediate transcriptional repression via chromatin remodeling mechanisms. Pharmacological inhibition of protein kinase CK2 reduces the Hes1-induced hyperphosphorylation of Gro/TLEs and causes a decrease in the chromatin association of the latter. Moreover, the transcription repression activity of Gro/TLEs is reduced by protein kinase CK2 inhibition. Consistent with these observations, Gro/TLEs are phosphorylated in vitro by purified protein kinase CK2. Taken together, these results implicate protein kinase CK2 in Gro/TLE functions. They suggest further that this kinase is involved in a hyperphosphorylation mechanism activated by Hes1 that promotes the transcription repression functions of Hes1-Gro/TLE protein complexes.