Members of the HRT family of basic helix-loop-helix proteins act as transcriptional repressors downstream of Notch signaling

Abstract

The Hairy-related transcription-factor (HRT) genes encode three related basic helix-loop-helix transcription factors that show sequence similarity to the Hairy and Enhancer of split family of transcriptional repressors. HRT proteins are expressed in specific regions of the developing heart, vasculature, pharyngeal arches and somites, and the periodicity of their expression in somitic precursors mirrors that of Notch signaling-related molecules. In the present study, we show that the intracellular domain of the Notch1 receptor (Notch1 IC), which is constitutively active, up-regulates HRT2 expression in 10T(1/2) fibroblasts. Luciferase reporter assays using the regulatory regions of the mouse HRT genes revealed that transcription of all three genes is stimulated by Notch1 IC. The promoters of the HRT genes share homology in a binding site for Suppressor of Hairless [Su(H)], a transcriptional mediator of Notch signaling. A dominant-negative Su(H) mutant abolished Notch-activated HRT2 expression, and mutation of the conserved Su(H) consensus site in the HRT2 promoter attenuated transcriptional activation by Notch. Ectopic expression of HRT proteins also blocked activation of HRT2 expression by Notch1 IC through a mechanism requiring the basic region, but not the conserved carboxyl-terminal YQPW-TEVGAF motif of HRT2. These findings identify HRT genes as downstream targets for Notch signaling and reveal a negative autoregulatory loop whereby HRT proteins repress their own expression through interference with Notch signaling.

Figure 1

Activation of mHRT2 mRNA expression by Notch signaling. 10T½ cells were transfected with a Notch1 IC expression construct or an empty vector (control; 2.7 μg/10-cm dish) and HRT2 transcripts were detected by Northern blot analysis. The blot was exposed to x-ray film for 8 h. Results from three independent transfections with each plasmid are shown. Ethidium bromide staining of 28S rRNA is shown at the bottom and positions of 28S and 18S rRNA are shown on the left.

Figure 2

Genomic structures of mHRT1, mHRT2, and mHRT3 and their transcriptional activation by Notch signaling. (A) Exon–intron structures of the mHRT genes. The filled boxes represent exons for protein-coding regions and open boxes represent exons for 5′ and 3′ noncoding regions. The genomic DNA fragments used for luciferase assays are shown below the gene structure. A schematic of HRT protein functional domains is shown with the positions of introns in the corresponding genomic sequences. Luc, luciferase; b, basic domain; H-L-H, helix–loop–helix motif; Orange, orange domain; YXPW-TEIGAF, carboxyl-terminal YXPW-TE(I/V)GAF motif. (B) Luciferase reporter assays were performed by using the mHRT1, -2, and -3 genomic DNA fragments as shown in A. The indicated cell lines were transfected with luciferase constructs and Notch1 IC expression construct or empty vector (450 ng/35-mm well each). The mHES-1-luciferase construct was used as a positive control. Fold-increase in normalized luciferase activity with Notch1 IC expression is shown.

Figure 3

Notch activation of mHRT2 is mediated by the Su(H) pathway. (A) 10T½ cells were transfected with a Notch1 IC expression plasmid (0.9 μg/10-cm dish) and/or 5.4 μg of dominant-negative RBPJ construct (RBPJR218H), and HRT2 mRNA was detected by Northern blot analysis. Note that less Notch1 IC expression plasmid was used compared with Fig. 1 because of the limitation of total plasmid amount in a transfection, and that the blot was exposed to x-ray film for 24 h. (B) The 10-kb mHRT2-luciferase construct (150 ng/35-mm well) was transfected with or without Notch1 IC and RBPJR218H constructs. (C) A series of 5′-deleted mHRT2-luciferase constructs (450 ng) was transfected with or without 450 ng of Notch1 IC plasmid. (D) Notch responsiveness of the luciferase constructs with a mutation in the proximal Su(H) site was examined as in C. Fold-increase in luciferase activity with Notch1 IC expression is shown in C and D.

Figure 4

Negative autoregulation of mHRT2 gene expression induced by Notch signaling. (A) 10T½ cells were transfected with a Notch1 IC construct (0.9 μg/10-cm dish) and/or 5.4 μg of mHRT2 expression plasmid and HRT2 mRNA was detected by Northern blot analysis. The blot was exposed to x-ray film for 24 h. (B) The 10-kb mHRT2-luciferase construct (150 ng/35-mm well) was transfected with or without Notch1 IC and mHRT2 expression constructs. (C and D) 10T½ cells were transfected with a 10-kb mHRT2-luciferase construct and a Notch1 IC plasmid (150 ng each), and the effects of cotransfection of various HRT constructs were observed. mHRT2 C(−), mHRT2 mutant without carboxyl-terminal YQPW-TEVGAF motif; mHRT2 B(−), mHRT2 mutant lacking basic domain. Luciferase activity without Notch1 IC or HRT cotransfection was given a value of 1 in B–D.

Figure 5

Transcriptional inhibition of mHRT2 independent of the HRT-binding site. (A) Electrophoretic mobility shift assays were performed by using oligonucleotide probes containing a CACGTG motif and in vitro-translated HRT proteins. Specific DNA-protein complexes are indicated by an arrowhead. mHRT2 C(−), mHRT2 mutant without YQPW-TEVGAF motif; mHRT2 B(−), mHRT2 mutant lacking basic domain. (B) 10T½ cells were transfected with 5′-deletion mHRT2-luciferase constructs and Notch1 IC plasmid (150 ng each) and the effects of coexpression of mHRT2 were examined. (C) The 190-bp mHRT2-luciferase construct and Notch1 IC plasmid (150 ng each) were transfected and the effects of cotransfection of various HRT constructs were examined. Luciferase activity without Notch1 IC or HRT cotransfection was given a value of 1 in B and C. (D) The 10-kb mHRT2-luciferase construct (150 ng) and Notch1 IC and/or mHRT2 expression constructs were transfected into 10T½ cells. Trichostatin A (TSA; 300 μM) or vehicle was added to the medium 24 h after transfection and cells were incubated for 20 h. TSA treatment increased luciferase and LacZ activities in control cells with vector transfection, and the luciferase activity of cells with Notch1 IC cotransfection was given a value of 100%.