Basic Helix-Loop-Helix Proteins Bind to TrkB and p21^Cip1 Promoters Linking Differentiation and Cell Cycle Arrest in Neuroblastoma Cells

Abstract

Differentiation of precursor into specialized cells involves an increasing restriction in proliferative capacity, culminating in cell cycle exit. In this report we used a human neuroblastoma cell line to study the molecular mechanisms that coordinate cell cycle arrest and neuronal differentiation. Exposure to retinoic acid (RA), a differentiation agent in many cell types, causes an upregulation of neurotrophin receptor TrkB and the cyclin kinase inhibitor p21^Cip1 at a transcriptional level. Full transcriptional activation of these two genes requires canonical E-box sequences found in the TrkB and p21^Cip1 promoters. As reported for other E-box-regulated promoters, ectopic expression of E47 and E12 basic helix-loop-helix (bHLH) proteins enhances RA-dependent expression of TrkB and p21^Cip1, whereas the inhibitory HLH Id2 exerts opposite effects. In addition, ectopic expression of E47 and NeuroD, a neuronal bHLH protein, is able to activate TrkB transcription in the absence of RA. More importantly, we show that E47 and NeuroD proteins bind the TrkB and p21^Cip1 promoter sequences in vivo. Since they establish a direct transcriptional link between a cell cycle inhibitor, p21^Cip1, and a neurotrophic receptor, TrkB, bHLH proteins would play an important role in coordinating key events of cell cycle arrest and neuronal differentiation.

We gratefully acknowledge Sònia Rius and Lidia Piedrafita for excellent technical assistance. We thank our colleagues from the Molecular Neurobiology group of Lleida and the members of our group, especially Eloi Garí, for many discussions, technical support, and critical reading of the manuscript. We also thank Vicente Andrés for stimulating discussions. We are indebted to M. Nakamura for pCMV-E12 and pCMV-E47 plasmids.

This study was funded by Fundació La Caixa, the Ministerio de Ciencia y Tecnología of Spain, and FEDER.