Lengthening of the oscillatory period of the <i>Hes7</i> promoter activity by LiCl.

<p>(A,B) Timelapse imaging of E10.5 <i>Hes7</i> promoter luciferase reporter embryos in the presence of 20 and 40 mM LiCl and period quantification. The control sample shows the oscillatory promoter activity bands (White arrows), while the 20 and 40 mM LiCl-treated samples show an abnormal posterior band (Red arrows) (A). Quantification shows an increase of the oscillation period in a dose-dependent manner for the control (n = 11), 20 mM LiCl (n = 12) and 40 mM LiCl (n = 4) of 2.5 h, 2.9 h and 3.6 h (B).</p

<i>Hes7</i> promoter analysis with lacZ reporter transgenic embryos.

<p>The left side is a scheme of the <i>Hes7</i> promoter constructs. From top to bottom: H7p2.6wt, H7p2.6dR, H7p1.9dR, H7p1.4dR and H7p1.0dR. The promoter size relative to the transcription start is shown. The red boxes show conserved sequences according to the Vista browser <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053323#pone.0053323-Dubchak1" target="_blank">[33]</a>. The yellow and green boxes show Rbpj and T-box binding sites, respectively, while the gray boxes stand for mutated sites. The two numbers show the number of X-gal positive embryos and transgene positive embryos, respectively.</p

Role of Tbx6 and Wnt pathway on <i>Hes7</i> regulation.

<p>Tbx6 binding sites and the Wnt pathway are required for normal <i>Hes7</i> expression in the PSM. Furthermore, the Gsk3 inhibitor LiCl activates the Wnt pathway and lengthens the oscillatory period of <i>Hes7</i> promoter activity.</p

Activation of <i>Hes7</i> expression by the Wnt pathway.

<p>(A,B,C) <i>Hes7</i> promoter luciferase reporter assay of the H7p2.6dR promoter reporter after co-transfection of constitutively active <i>Ctnnb1</i>, <i>Lef1</i>, <i>T</i> and <i>Tbx6</i> expression plasmids. (A,B) The H7p2.6dR promoter reporter is activated by constitutively active <i>Ctnnb1</i> and <i>Lef1</i> expression plasmids. (C) The Ctnnb1 and Lef1 mediated activation of the H7p2.6dR promoter reporter is synergistically enhanced by co-transfection of both T and Tbx6 expression plasmids. (D) <i>Hes7</i> intronic expression in <i>Wnt3a</i> hypomorph E10.5 mutant embryos. In the mutant, <i>Hes7</i> intronic expression (n = 7) is downregulated compared to the control (n = 9).</p

Requirement of Tbx6 for <i>Hes7</i> expression.

<p>(A) Alignment of mouse (−1350 to −1291, Top) and human (Bottom) <i>Hes7</i> promoter sequences with two T-box binding sites (T-1306 and T-1350). (B) Luciferase assay of the H7p2.6wt promoter after transfection of NICD and Tbx6 expression plasmids. The <i>Hes7</i> promoter is activated by NICD and further enhanced by Tbx6. (C) Electromobility gel shift assay (EMSA) of the T-box containing oligos and Tbx6. Tbx6 binds to T-box containing oligos. Conditions: control lysate (Lanes 1,8), low (Lanes 2,9) and high Tbx6 lysate concentration (Lanes 3–7,10–14), supershift for Tbx6 antibody (Lanes 4,11), wild-type (WT) (Lanes 2–4,6,7,9–11,13,14) and mutant labeled oligonucleotides (Lanes 5,12) and competition with unlabeled wild-type (Lanes 6,13) and mutant (Lanes 7,14) oligonucleotides. (D,E) X-gal staining of transgenic embryos with the H7p1.4dR (WT T-box sites; n = 8) and H7p1.4dRdT (mutated T-box sites; n = 2) reporter constructs and vibratome sections of control and H7p1.4dRdT reporters. Mutated T-box binding sites of the <i>Hes7</i> promoter reporter prevent the WT X-gal staining (D). Vibratome sections of control reporter constructs with WT T-box binding sites show that all positive X-gal staining embryos also present staining in the paraxial mesoderm (E top, n = 13). By contrast, reporter constructs with mutated T-box binding sites prevent paraxial mesoderm staining and induce lateral plate and ventral mesoderm staining (E bottom, n = 2). NT, neural tube; PSM, presomitic mesoderm; HG, hindgut; LPM, lateral plate mesoderm. (F) Double immunofluorescence detection of Hes7 (magenta) and Tbx6 (green) (Phase I, n = 3; Phase II, n = 2). The anterior limit of Hes7 protein coincides or slightly exceeds the Tbx6 protein domain suggesting that the <i>Hes7</i> mRNA domain is always included in the Tbx6 protein domain.</p

Reconstitution of an Ultradian Oscillator in Mammalian Cells by a Synthetic Biology Approach

The Notch effector gene <i>Hes1</i> is an ultradian clock exhibiting cyclic gene expression in several progenitor cells, with a period of a few hours. Because of the complexity of studying Hes1 in the endogenous setting, and the difficulty of imaging these fast oscillations <i>in vivo</i>, the mechanism driving oscillations has never been proven. Here, we applied a “build it to understand it” synthetic biology approach to construct simplified “hybrid” versions of the Hes1 ultradian oscillator combining synthetic and natural parts. We successfully constructed a simplified synthetic version of the <i>Hes1</i> promoter matching the endogenous regulation logic. By mathematical modeling and single-cell real-time imaging, we were able to demonstrate that Hes1 is indeed able to generate stable oscillations by a delayed negative feedback loop. Moreover, we proved that introns in <i>Hes1</i> contribute to the transcriptional delay but may not be strictly necessary for oscillations to occur. We also developed a novel reporter of endogenous Hes1 oscillations able to amplify the bioluminescence signal 5-fold. Our results have implications also for other ultradian oscillators

Identification of HERC2 and NEURL4 as LRRK2-binding proteins.

<p>(A) Silver-stained gel image for LRRK2-binding proteins. LRRK2 and representative co-purified proteins are indicated. (B) Domain structures of HERC2, NEURL4 and their related proteins Neuralized and Mindbomb homolog 1 (MIB1). RCC1-like, Regulator of chromosome condensation 1-like domain; Cyt-b5, cytochrome b5-like motif; MIB-HERC2, MIB/HERC2 domain; NHR, Neuralized homology repeat domain. (C) HERC2 and NEURL4 specifically bind to LRRK2 in HEK293T cells. Lysates expressing FLAG-tagged LRRK2 or FLAG-tagged LKB1 were subjected to immunoprecipitation with anti-FLAG antibody (FLAG-IP) and were detected by Western blotting with anti-FLAG, anti-HERC2 and anti-NEURL4 antibodies. (D) Endogenous associations between LRRK2, NEURL4 and HERC2. Mouse brain lysates were subjected to immunoprecipitation with anti-LRRK2 (LRRK2-IP) or anti-dFoxO (Control-IP) antibodies as a control and analyzed by Western blotting with the indicated antibodies. The results of two independent experiments are shown. (E) LRRK2, NEURL4 and HERC2 are partially colocalized as vesicular signals in the cytosol. HeLa cells transfected with LRRK2 were visualized with anti-NEURL4 (green) and anti-LRRK2 (red) antibodies, and the nuclei were counterstained with DAPI (blue). Colocalization of the proteins appears as yellow. Insets show higher-magnification images of the boxed regions and the values ± SE in (E, F) indicates colocalized green signals with red (n = 4). Scale bar, 10 μm. (F) HeLa cells transfected with HERC2 were visualized with anti-NEURL4 (green) and anti-HERC2 (red) antibodies as in (E). (G) LRRK2 binds to HERC2 via NEURL4. HEK293T cell lysates transfected with FLAG-LRRK2, Myc-NEURL4, an siRNA duplex against NEURL4 and/or an empty plasmid were subjected to immunoprecipitation with an anti-FLAG antibody and detected by Western blotting with antibodies against the indicated proteins. (H) LRRK2 GTPase mutations (TN, T1348N; RL, R1398L; RG, R1441G) modulate the affinity to NEURL4 and HERC2. Note that LRRK2^TN consistently exhibited lower expression levels, suggesting instability. The graph indicates the relative levels of endogenous proteins co-precipitated with FLAG-LRRK2. The data are shown as the mean ± SE from five repeated experiments (**, <i>p</i> < 0.01; *, <i>p</i> < 0.05 by one-way ANOVA).</p

LRRK2, NEURL4 and HERC2 modulate Notch signal intensity in cultured cells.

<p>(A) Schematic of non-cell-autonomous Notch signaling and ‘<i>cis</i>-inhibition’ reconstituted in cultured cells. (B) Notch signal intensity assessed by the Hes1 promoter activity. SH-SY5Y cells were transfected with Hes1 reporter and Notch1 plasmids and control LacZ or Dll1-∆ICD plasmids. CHO cells stably expressing Dll1 (D) and parental CHO (P) cells were co-cultured as signal-sending and mock cells, respectively. (C) LRRK2 and HERC2 suppress Notch signal intensity. (D) LRRK2 ROC mutations affect the suppressive potency of Notch signaling. (E, F) ‘Weak <i>cis</i>-inhibition’ condition accentuates the apparent suppressive effects of LRRK2, NEURL4 and HERC2 on Notch signal intensity. (G) LRRK2 knockdown activates the Notch signal. (C, E, F) ***, <i>p</i> < 0.001; **, <i>p</i> < 0.01; N.S., not significant <i>vs</i>. LacZ and (D) **, <i>p</i> < 0.01; *, <i>p</i> < 0.05 <i>vs</i>. LRRK2 WT by one-way ANOVA. (G) *, <i>p</i> < 0.05 by Student’s <i>t</i>-test. The data represent the mean ± SE at least from three experiments performed in triplicate. KD, kinase-dead.</p

LRRK2 complex modulates Dll1 turnover in the endosomal pathway.

<p>(A) Co-expression of LRRK2, NEURL4 and HERC2 stabilizes Dll1. HeLa cells stably expressing Dll1-HA were transfected with a mixture of plasmids for LRRK2/NEURL4/HERC2 or with LacZ as a control. At 24 h after transfection, the cells were then treated with cycloheximide (CHX, 100 μg ml^-1) for the indicated times, and Western blotting assays were performed. (B) The level of Dll1 remaining at different time points was plotted as the percentage of the initial Dll1 level (0 h of CHX treatment). The data are shown as the mean ± SE from four repeated experiments (*, <i>p</i> < 0.05 by Student’s <i>t</i>-test). (C) Dynamics of cell surface Dll1. HeLa cells stably expressing Dll1-SNAP were transfected as in (A). Cell surface Dll1 was labeled with SNAP-Surface Alexa Fluor 647 for 20 min at 37°C. Representative grayscale images of Dll1 labeled with Alexa Fluor 647 0–6 h after washout of the tracking dye (t = 0) are shown. (D) LRRK2 complex increases the amount of cell-surface Dll1. The data are presented as the mean ± SE for six independent experiments, with 13–26 cells counted per sample. **, <i>p</i> < 0.01 <i>vs</i>. Mock at 6 h determined by Student’s <i>t</i>-test. (E) LRRK2 stimulates the recycling of Dll1 via the endosomes. <i>LRRK2</i>-deficient mouse embryonic fibroblasts stably expressing Dll1-SNAP along with EGFP-Rab5, EGFP-Rab7 or EGFP-Rab11 were transfected with LacZ or LRRK2 and were labeled with SNAP-Surface Alexa Fluor 647 as in (C). Graph showing that Dll1 colocalized with the indicated Rab proteins (mean ± SE for 3–7 independent experiments, with 5–7 cells counted per sample). ** <i>p</i> < 0.01, * <i>p</i> < 0.05 by Student’s <i>t</i>-test.</p

LRRK2, NEURL4 and HERC2 bind to the Notch ligand Dll1.

<p>(A) NEURL4 binds to Dll1. HEK293T cell lysate transfected with the indicated plasmids was subjected to immunoprecipitation with an anti-FLAG antibody and analyzed by Western blotting with anti-FLAG and anti-HA antibodies. The asterisk indicates non-specific bands that appeared with anti-HA (clone 12CA5). (B) LRRK2 associates with Dll1 via NEURL4 and HERC2. A co-immunoprecipitation assay was performed as in (A). Western blotting was performed with anti-Dll1, anti-LRRK2, anti-NEURL4 and anti-HERC2 antibodies. Longer exposures of X-ray films revealed endogenous proteins that co-immunoprecipitate with FLAG-LRRK2 (Left).</p