Abstract
Runx3, a Runt domain transcription factor, determines neurotrophin receptor phenotype in dorsal root ganglion (DRG) neurons. Molecular mechanisms by which Runx3 controls distinct neurotrophin receptors are largely unknown. Here, we show that RUNX3 abolished mRNA induction of TRKB expression, and concomitantly altered the neurotrophin response in a differentiating neuroblastoma cell line. In contrast, RUNX3 did not play a significant role in TRKC regulation even under the relevant BMP signaling pathway. We identified putative regulatory elements of Ntrk2/NTRK2 (a gene that codes for TrkB) using an unbiased computational approach. One of these elements was a highly conserved intronic sequence that contains a cluster of Runx binding sites. In a primary culture of DRG neurons, endogenous Runx3 bound to the consensus cluster, which had repressor activity against the Ntrk2 promoter under the control of NT-3 signaling. Consistent with these findings, Runx3-deficient embryos showed an increased number of trkB+ DRG neurons and failed to maintain trkC expression. Taken together, Runx3 determines TrkC positive sensory neuron identities through the transcriptional repression of TrkB when Trk-BTrkC double positive neurons differentiate into TrkC single positive neurons.
Highlights
Genetic evidence clearly showed that Runx3 contributes to selective repression of TrkB synthesis within TrkC-positive sensory neurons [20]
In the mock control, brain-derived neurotrophic factor (BDNF) supported the survival of Retinoic acid (RA)-treated cells, which was consistent with the high level of induced TRKB expression
We observed that NT-3 was sufficient to protect the mock transfectant from cell death (Fig. 1D), presumably because NT-3 binds to TrkB, albeit with less affinity compared with BDNF [2]
Summary
Cell Culture—DRGs were dissected from rat embryos at E15.5 (corresponds to mouse E13.5). 0.125% trypsin/versene was added to the ganglions and incubated at 37 °C for 8 min. Cell Culture—DRGs were dissected from rat embryos at E15.5 (corresponds to mouse E13.5). A point mutation (R178Q) was introduced by site-directed mutagenesis according to the instructions in the QuikChange XL manual (Stratagene) This mutation has not been found in human disease, the corresponding point mutation R174Q in the conserved Runt domain was reported for both RUNX1 and RUNX2 in patients with leukemia [24] and cleidocranial dysplasia [25], respectively. Reporter Assay—Dissociated rat DRG cells from E15.5 embryos were transfected by electroporation with 50 ng of phRL-SV40 (Promega) luciferase, as an internal control, and 5 g of each of the designated Firefly reporter plasmid containing the sequence of interest. A rabbit antibody raised against mouse ER81 (1:8000, from Dr Jessell, Columbia University) was used and visualized with a Cy3-conjugated anti-rabbit IgG antibody (1:400, Chemicon)
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