REV-ERBα Participates in Circadian SREBP Signaling and Bile Acid Homeostasis

The nuclear receptor REV-ERBα shapes the daily activity profile of Sterol Response Element Binding Protein (SREBP) and thereby participates in the circadian control of cholesterol and bile acid synthesis in the liver

Le rôle du Récepteur Nucléaire Orphelin REV-ERBα dans le métabolisme circadien des lipides et des acides biliaires

Chez les mammifères presque tous les processus physiologiques sont sujets à des oscillations journalières. Cette chronologie est gérée par un système circadien, composé d'une horloge principale résidant dans le noyau suprachiasmatique (NSC) de l'hypothalamus et d'oscillateurs périphériques existant dans quasiment toutes les cellules. Comme les horloges circadiennes peuvent mesurer le temps d'une façon seulement approximative (circadien vient des mots latins "circa diem"), elles doivent être remises à l'heure chaque jour afin de rester en résonance avec le temps géophysique. Cette synchronisation se fait d'une façon hiérarchique. La phase de l'horloge centrale est synchronisée par des signaux photiques perçus par la rétine et transmis via le tractus rétino-hypothalamique aux neurones du NSC. Ce dernier synchronise les oscillateurs périphériques par plusieurs voies de signalisation chimiques et physiques. Pour plusieurs organes, les cycles de prises de nourriture sont en faitle Zeitgeber dominant pour la mise à l'heure des horloges périphériques. Ceci est en parfait accord avec le rôle important de celles-ci dans la coordination temporelle du métabolisme. Par exemple, les horloges des hépatocytes déterminent les fenêtres de temps pendant lesquelles les glucides, les acides gras, le cholestérol et les acide biliaires sont synthétisés et/ou métabolisés. D'un point de vue moléculaire, l'horloge circadienne fonctionne selon un modèle de boucle de rétrocontrôle négatif de la transcription et de la traduction impliquant notamment les protéines Period (PER1, PER2) et Cryptochrome (CRY1, CRY2). Ces protéines inhibent rythmiquement leur propre transcription en interferant avec l'hétérodimère activateur CLOCK:BMAL1. L'expression circadienne du gène codant pour le récepteur nucléaire REV-ERB⍺ est régulé par un mécanisme similaire, en ce sens qu'elle est respectivement activée et réprimée par les hétérodimères CLOCK:BMAL1 et PER:CRY. L'accumulation périodique de REV-ERB⍺ provoque la répression cyclique du gène essentiel de l'horloge, Bmal1 couplant ainsi les pôles positifs et négatifs de l'oscillateur circadien. De même, REV-ERB⍺ régule l'expression circadienne de gènes sous le contrôle de l'horloge mais qui ne sont pas directement impliqués dans le bon fonctionnement de son mécanisme. Les expériences menées durant mon travail de thèse m'ont permi de mettre en évidence que le facteur de transcription circadien REV-ERB⍺, couple l'horloge circadienne au métabolisme hépatique, notamment à la synthèse des lipides et des acides biliaires

Genome-Wide Analysis of SREBP1 Activity around the Clock Reveals Its Combined Dependency on Nutrient and Circadian Signals

In mammals, the circadian clock allows them to anticipate and adapt physiology around the 24 hours. Conversely, metabolism and food consumption regulate the internal clock, pointing the existence of an intricate relationship between nutrient state and circadian homeostasis that is far from being understood. The Sterol Regulatory Element Binding Protein 1 (SREBP1) is a key regulator of lipid homeostasis. Hepatic SREBP1 function is influenced by the nutrient-response cycle, but also by the circadian machinery. To systematically understand how the interplay of circadian clock and nutrient-driven rhythm regulates SREBP1 activity, we evaluated the genome-wide binding of SREBP1 to its targets throughout the day in C57BL/6 mice. The recruitment of SREBP1 to the DNA showed a highly circadian behaviour, with a maximum during the fed status. However, the temporal expression of SREBP1 targets was not always synchronized with its binding pattern. In particular, different expression phases were observed for SREBP1 target genes depending on their function, suggesting the involvement of other transcription factors in their regulation. Binding sites for Hepatocyte Nuclear Factor 4 (HNF4) were specifically enriched in the close proximity of SREBP1 peaks of genes, whose expression was shifted by about 8 hours with respect to SREBP1 binding. Thus, the cross-talk between hepatic HNF4 and SREBP1 may underlie the expression timing of this subgroup of SREBP1 targets. Interestingly, the proper temporal expression profile of these genes was dramatically changed in Bmal1−/− mice upon time-restricted feeding, for which a rhythmic, but slightly delayed, binding of SREBP1 was maintained. Collectively, our results show that besides the nutrient-driven regulation of SREBP1 nuclear translocation, a second layer of modulation of SREBP1 transcriptional activity, strongly dependent from the circadian clock, exists. This system allows us to fine tune the expression timing of SREBP1 target genes, thus helping to temporally separate the different physiological processes in which these genes are involved

Circadian clock characteristics are altered in human thyroid malignant nodules

Context: The circadian clock represents the body's molecular time-keeping system. Recent findings revealed strong changes of clock gene expression in various types of human cancers. Objective: Due to emerging evidence on the connection between the circadian oscillator, cell cycle, and oncogenic transformation, we aimed to characterize the circadian clockwork in human benign and malignant thyroid nodules. Design: Clock transcript levels were assessed by quantitative RT-PCR in thyroid tissues. To provide molecular characteristics of human thyroid clockwork, primary thyrocytes established from normal or nodular thyroid tissue biopsies were subjected to in vitro synchronization with subsequent clock gene expression analysis by circadian bioluminescence reporter assay and by quantitative RT-PCR. Results: The expression levels of the Bmal1 were up-regulated in tissue samples of follicular thyroid carcinoma (FTC), and in papillary thyroid carcinoma (PTC), as compared with normal thyroid and benign nodules, whereas Cry2 was down-regulated in FTC and PTC. Human thyrocytes derived from normal thyroid tissue exhibited high-amplitude circadian oscillations of Bmal1-luciferase reporter expression and endogenous clock transcripts. Thyrocytes established from FTC and PTC exhibited clock transcript oscillations similar to those of normal thyroid tissue and benign nodules (except for Per2 altered in PTC), whereas cells derived from poorly differentiated thyroid carcinoma exhibited altered circadian oscillations. Conclusions: This is the first study demonstrating a molecular makeup of the human thyroid circadian clock. Characterization of the thyroid clock machinery alterations upon thyroid nodule malignant transformation contributes to understanding the connections between circadian clocks and oncogenic transformation. Moreover, it might help in improving the thyroid nodule preoperative diagnostics

Functional annotation clustering of putative SREBP1 targets with a different temporal expression profile.

<p>Enriched GO categories were identified in three distinct sets of rhythmic SREBP1 target genes (P<0.05), based of their phase of expression. To define the three intervals of time we calculated the shortest time range containing the phases of at least 50% of the genes belonging to clusters A1, A2 or A3. For each set, the total number of genes is indicated and the number of genes with annotation is indicated in parentheses. For each functional cluster, only the most significant associated GO term is shown in the table, with the corresponding Modified Fisher Exact P-value.</p

Dynamics of SREBP1 binding.

<p>(A) C57BL/6 mice were fed only during the night (ZT12-ZT24) for one week before collecting liver every 4 hours for one day. Chromatin from 5 mice was pooled at each time point and ChIP with an antibody against SREBP1 was performed. Peaks were positioned where the signal for SREBP1 was at least a four-fold in comparison to the input signal in at least one time point. The heat-map represents SREBP1 binding to all its targets along the time. Hierarchical clustering was done using Pearson correlation scores and identified four major clusters (A, B, C and D). The color scale is indicated below. In the column on the right, black lines indicate that Pol II was detected in the same site as SREBP1 in at least one time point, as assessed in our previous ChIP-seq data set <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004155#pgen.1004155-LeMartelot2" target="_blank">[24]</a>. (B) Two SREBP1 binding sites were identified in the proximity of <i>Srebp1</i> gene, at a distance of −30 and +299 nucleotides from the <i>Srebp1c</i> and <i>Srebp1a</i> TSS, respectively. Graphs represent the fitting to a cosine function of experimental data obtained on these peaks (black dots), in order to calculate the phase of the binding (dashed line), its interval of confidence (dotted lines) and the associated P-value. (C) Histogram of binding phase frequency in clusters A, B and C, for peaks with a P-value of the amplitude <0.1. None of the peaks belonging to cluster D met this requirement. (D) mRNA expression of <i>Srebp1c</i> was evaluated by qPCR in livers from C57BL/6 mice at the indicated ZT time (n = 5). Data are normalized using 36B4 as housekeeping gene. (E) Hepatic nuclear extracts from C57BL/6 mice were subjected to western blot analysis to detect the nuclear SREBP1. U2AF was used as loading control. Each sample is a pool of 5 livers. (F) Quantification of the Western Blot was performed by densitometry, using ImageJ software.</p

Pol II recruitment on SREBP1 target genes is not always synchronized with SREBP1 binding.

<p>(A) The heat-maps represent the recruitment of Pol II to the promoter (left) and to the gene body (middle) of SREBP1 putative target genes along the day, as assessed in our previous ChIP-seq data set <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004155#pgen.1004155-LeMartelot2" target="_blank">[24]</a>. In parallel, we evaluated hepatic gene expression by microarray analysis in the same samples (right). Hierarchical clustering was done applying a Pearson correlation scores to the data describing Pol II recruitment on the promoters of SREBP1 target genes (left). Three major clusters of genes displaying a different temporal binding profile of Pol II were identified (A1, A2, and A3). The genes are ordered in the three heat-maps according to this clustering. (B) Gene expression data from microarray analysis were fit to a cosine function to estimate the phase of expression (peak time of the fit) of SREBP1 target genes. The graph shows the smoothing of phase distributions of the genes belonging to the three clusters (green line for A1, blue line for A2, magenta line for A3). Only genes with a P-value<0.05 are plotted. Dotted lines define three time intervals containing the most recurrent phases associated to the genes belonging to the clusters A1, A2 and A3. (C) HNF4 binding was tested on randomly selected SRE identified in our SREBP1 ChIP-seq. <i>Gnat1</i> and <i>Anks4b</i> SREs belong to cluster A3 and contain a HNF4 putative binding sites. In contrast, <i>Ldlr</i> and <i>Insig1</i> SREs do not contain in their sequence a HNF4 motif and belong to cluster A2. NEG and POS were used as negative and positive control loci and correspond to two regions of <i>Cyp7a1</i> promoter, localized at −1500 and −150 from the TSS, respectively <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004155#pgen.1004155-Kir1" target="_blank">[69]</a>. The graph shows the mean ± SEM of three independent experiments. * indicates P-value<0.01 vs. NEG. Statistical analysis was performed by one-way ANOVA followed by Bonferroni post-test. Primer sequences are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004155#pgen.1004155.s012" target="_blank">Table S8</a>.</p

Functional annotation clustering of putative SREBP1 targets using DAVID tools.

<p>Enriched GO categories were identified in four distinct sets of SREBP1 target genes exhibiting a different combination of binding sites for SP1, NFY and/or HNF4. In total, 219 out of 223 SREBP1 putative target genes have a functional annotation (the number of annotated genes for each set is indicated in parentheses). The analysis using DAVID groups the GO categories in functional related clusters. For each enriched cellular process, only the most significant associated GO term is shown in the table, with the corresponding Modified Fisher Exact P-value. The complete list of all GO terms enriched in each functional cluster for all the groups is available in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004155#pgen.1004155.s008" target="_blank">Table S4</a>.</p