Helt, a Novel Basic-Helix-Loop-Helix Transcriptional Repressor Expressed in the Developing Central Nervous System

Abstract

Neuronal differentiation is regulated by many basichelix-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.

Corl1, a Novel Neuronal Lineage-specific Transcriptional Corepressor for the Homeodomain Transcription Factor Lbx1

“…The nucleotide sequence of the Corl1 cDNA was deposited in the DDBJ/EMBL/GenBank TM data base under accession number AB185113. RT-PCR-RT-PCR was performed essentially as described previously (32). ExTaq polymerase (TAKARA) was used for amplification, which was carried out by denaturation at 94°C for 30 s (2 min in the first cycle), annealing at 65°C for 30 s and extension at 72°C for 30 s (2 min in the last cycle).…”

MAGI1 Recruits Dll1 to Cadherin-based Adherens Junctions and Stabilizes It on the Cell Surface

“…The Dll1 WT and Dll1 ⌬C fragments were digested with XhoI/EcoRI and cloned into the corresponding sites in pcDNA-SS-FLAG, pcDNA-SS-HA, and pMX-SS-HA. The MAGI1a fragment was digested with EcoRI and cloned into the corresponding sites in pcDNA-FLAG-NII, pcDNA-HA-NII, and pMX-Myc-NII (34). The Dll1 ICD and Jagged1 ICD fragments were digested with SalI/NotI and cloned into the XhoI/NotI site of pcDNA-FLAG-NII.…”

“…In Situ Hybridization and Immunohistochemistry-In situ hybridization and immunohistochemistry were performed as described previously (34). Polyclonal rabbit anti-MAGI1 and anti-Dll1 antibodies were raised against GST-MAGI1 (aa 379 -454) and GST-Dll1 (aa 646 -722), respectively, and affinity-purified.…”

“…Immunoprecipitation-Immunoprecipitation experiments using 293E cells and detection by Western blotting were performed as described previously (34). Briefly, transfected 293E cells were lysed with a lysis buffer containing 10 mM HEPES (pH 7.6), 250 mM NaCl, 5 mM EDTA, and 1% TX-100 for 1 h at 4°C, and immunoprecipitation was performed with anti-FLAG M2 beads (Sigma).…”

“…Reverse Transcription-PCR-Reverse transcription-PCR was performed as described previously (34). ExTaq polymerase (Takara) was used for amplification, which was carried out by denaturation at 94°C for 30 s (2 min in the first cycle), annealing at 65°C for 30 s, and extension at 72°C for 1 min (3 min in the last cycle).…”