β-Catenin Up-regulates Atoh1 Expression in Neural Progenitor Cells by Interaction with an Atoh1 3′ Enhancer

Fuxin Shi,Yen-Fu Cheng,Xiaohui L Wang,Albert S.B Edge

doi:10.1074/jbc.m109.059055

Abstract

Atoh1, a basic helix-loop-helix transcription factor, plays a critical role in the differentiation of several epithelial and neural cell types. We found that beta-catenin, the key mediator of the canonical Wnt pathway, increased expression of Atoh1 in mouse neuroblastoma cells and neural progenitor cells, and baseline Atoh1 expression was decreased by siRNA directed at beta-catenin. The up-regulation of Atoh1 was caused by an interaction of beta-catenin with the Atoh1 enhancer that could be demonstrated by chromatin immunoprecipitation. We found that two putative Tcf-Lef sites in the 3' enhancer of the Atoh1 gene displayed an affinity for beta-catenin and were critical for the activation of Atoh1 transcription because mutation of either site decreased expression of a reporter gene downstream of the enhancer. Tcf-Lef co-activators were found in the complex that bound to these sites in the DNA together with beta-catenin. Inhibition of Notch signaling, which has previously been shown to induce bHLH transcription factor expression, increased beta-catenin expression in progenitor cells of the nervous system. Because this could be a mechanism for up-regulation of Atoh1 after inhibition of Notch, we tested whether siRNA to beta-catenin prevented the increase in Atoh1 and found that beta-catenin expression was required for increased expression of Atoh1 after Notch inhibition.

Highlights

Grants R01 DC007174 and P30 DC05209 from the NIDCD and by the Hamilton H
The Wnt pathway plays a key role in early development of several of these tissues, including the intestinal epithelium and the inner ear (6 –11), and is a potential candidate for upstream signaling leading to Atoh1 expression
Overexpression of ␤-Catenin Up-regulates Atoh1 Expression— The effect of ␤-catenin overexpression and silencing on Atoh1 levels was measured in two neural cell types, a neuroblastoma cell line and neural progenitors derived from embryonic stem cells

Summary

MATERIALS AND METHODS

Cell Culture—Neuro2a cells were grown in DMEM supplemented with 10% heat-inactivated fetal calf serum, 2 mM Glutamax, and penicillin (100 units/ml)/streptomycin (100 ␮g/ml). Wnt3a-conditioned medium was harvested from L-Wnt3a cells (from ATCC, CRL-2647), which were stably transfected with a Wnt3a expression vector and secrete biologically active Wnt3a protein. Cells to be used for the measurement of gene expression were seeded onto 10-cm dishes and transfected with Atoh1 [18], GFP, control pcDNA3 vector, Notch intracellular domain (NICD) [20], ␤-catenin [21], or dominant-negative Tcf expression vector [22] using 5 ␮g of DNA per 15 ␮l of Lipofectamine 2000 (Invitrogen) in 5 ml of opti-MEM for 106 cells seeded. Precipitated proteins were washed 5ϫ with binding buffer and boiled in 50 ␮l of 2ϫ sample buffer, and the supernatant was collected for Western blotting with anti-␤-catenin antibody and anti-Lef-1-Tcf antibody. Cells were lysed after 48 h, and luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega) in a TD-20/20 Luminometer (Turner Designs)

RESULTS

To determine the precise sequences that had an affinity for

DISCUSSION

Notch is a negative regulator of