Abstract
Neuronal differentiation is regulated by many basic-helix-loop-helix (bHLH) family transcriptional activators and repressors, and the balance of activity between these factors is important for the differentiation process. Here, we report the identification of a novel transcriptional repressor, designated Helt. Helt encoded a Hey-related bHLH protein containing the bHLH and Orange domains. Helt could homodimerize, and heterodimerize with Hes5 or Hey2. Both the bHLH and Orange domains were involved in the homodimerization. In contrast, only the bHLH domain was required for the heterodimerization with Hey2, whereas only the Orange domain mediated the interaction between Helt and Hes5. Thus, Helt has two dimerization domains, and these domains independently select a partner. Identification of preferred recognition sequences by CASTing experiments revealed that Helt bound to the E box, which was distinct from the Hes1 optimal sequence around the E box core. Not only the core sequence but also sequences flanking the E box were essential for the recognition by Helt and Hes1. Furthermore, Helt repressed transcription from an artificial promoter through binding to the optimal E box elements, as well as transcription from its own promoter. Using in situ hybridization and immunohistochemistry, Helt expression in embryos was investigated. Helt was mainly expressed in undifferentiated neural progenitors in some of the developing brain regions, including the mesencephalon and diencephalon, at the neurogenesis stage. These results suggest that Helt acts as a transcriptional repressor to regulate neuronal differentiation and/or identity.
Highlights
Transcription factors of the basic-helix-loop-helix1 family play important roles in lineage restriction and differentiation in many developmental processes
Neuronal differentiation is regulated by many basichelix-loop-helix family transcriptional activators and repressors, and the balance of activity between these factors is important for the differentiation process
Computer data base searches revealed that Helt had a bHLH domain and an Orange domain, which are characteristic motifs among the bHLH-O family transcriptional repressors (Fig. 1A)
Summary
Isolation of Helt—Brain regions including the mesencephalon and metencephalon were dissected from E10.5 mouse embryos and divided into two portions, the ventral and dorsal regions. Amplified DNA was purified using a Qiaquick PCR purification kit (Qiagen), and 50 ng of degenerate oligonucleotides was incubated with 25 l of cell lysates of transfected 293E cells in an EMSA reaction buffer. The sections were washed three times with buffer 1 (50% formamide, 5ϫ SSC, 1% SDS) at 68 °C for 20 min and treated with 0.05 g/ml RNase A in 1ϫ RNase buffer (0.5 M NaCl, 10 mM Tris-HCl, 1 mM EDTA) at room temperature for 5 min followed by three 5-min incubations in 1ϫ RNase buffer at room temperature. For immunological detection of DIG-labeled hybrids, the sections were first blocked in 1ϫ blocking reagent (Roche Applied Science) at room temperature for 1 h and incubated overnight at 4 °C in a solution containing alkaline phosphatase-conjugated rabbit anti-DIG Fab fragments (DAKO) diluted 1:80 in 1ϫ TBS-T.
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