A combinatorial regulatory signature controls terminal differentiation of the dopaminergic nervous system in C. elegans

15 páginas, 7 figuras, 3 tablas.Terminal differentiation programs in the nervous system are encoded by cis-regulatory elements that control the expression of terminal features of individual neuron types. We decoded the regulatory information that controls the expression of five enzymes and transporters that define the terminal identity of all eight dopaminergic neurons in the nervous system of the Caenorhabditis elegans hermaphrodite. We show that the tightly coordinated, robust expression of these dopaminergic enzymes and transporters ("dopamine pathway") is ensured through a combinatorial cis-regulatory signature that is shared by all dopamine pathway genes. This signature is composed of an Ets domain-binding site, recognized by the previously described AST-1 Ets domain factor, and two distinct types of homeodomain-binding sites that act in a partially redundant manner. Through genetic screens, we identified the sole C. elegans Distalless/Dlx ortholog, ceh-43, as a factor that acts through one of the homeodomain sites to control both induction and maintenance of terminal dopaminergic fate. The second type of homeodomain site is a Pbx-type site, which is recognized in a partially redundant and neuron subtype-specific manner by two Pbx factors, ceh-20 and ceh-40, revealing novel roles of Pbx factors in the context of terminal neuron differentiation. Taken together, we revealed a specific regulatory signature and cognate, terminal selector-type transcription factors that define the entire dopaminergic nervous system of an animal. Dopaminergic neurons in the mouse olfactory bulb express a similar combinatorial transcription factor collective of Ets/Dlx/Pbx factors, suggesting deep phylogenetic conservation of dopaminergic regulatory programs.This work was funded by EMBO post-doctoral fellowships and Marie Curie Funds (to M.D. and N.F.), the New York Stem Cell Foundation Fellowships and the Spanish Government (SAF2011-26273) (to N.F), the NIH (R01NS039996-05; R01NS050266-03 to O.H.; R01GM30997 to M.C.; R01GM054510 to R.S.M.; and F32GM099160 to N.A.), and the Stavanger University Hospital (to M.D.). N.F is a NARSAD Young Investigator. O.H. is an Investigator of the Howard Hughes Medical Institute.Peer reviewe

G Protein-Coupled Receptor Kinase-2 (GRK-2) Controls Exploration Through Neuropeptide Signaling in Caenorhabditis Elegans

Animals alter their behavior in manners that depend on environmental conditions as well as their developmental and metabolic states. For example, C. elegans is quiescent during larval molts or during conditions of satiety. By contrast, worms enter an exploration state when removed from food. Sensory perception influences movement quiescence (defined as a lack of body movement), as well as the expression of additional locomotor states in C. elegans that are associated with increased or reduced locomotion activity, such as roaming (exploration behavior) and dwelling (local search). Here we find that movement quiescence is enhanced, and exploration behavior is reduced in G protein-coupled receptor kinase grk-2 mutant animals. grk-2 was previously shown to act in chemosensation, locomotion, and egg-laying behaviors. Using neuron-specific rescuing experiments, we show that GRK-2 acts in multiple ciliated chemosensory neurons to control exploration behavior. grk-2 acts in opposite ways from the cGMP-dependent protein kinase gene egl-4 to control movement quiescence and exploration behavior. Analysis of mutants with defects in ciliated sensory neurons indicates that grk-2 and the cilium-structure mutants act in the same pathway to control exploration behavior. We find that GRK-2 controls exploration behavior in an opposite manner from the neuropeptide receptor NPR-1 and the neuropeptides FLP-1 and FLP-18. Finally, we show that secretion of the FLP-1 neuropeptide is negatively regulated by GRK-2 and that overexpression of FLP-1 reduces exploration behavior. These results define neurons and molecular pathways that modulate movement quiescence and exploration behavior

Recruitment and function of transcriptional protein complexes

Regulation of gene expression opperates mainly at the level of transcription and results as a consequence of the co-ordinated activity of two categories of regulatory elements, promoters and activators. In this study the in vivo role of both these elements in the recruitment and function of transcriptional complexes was examined. Firstly, the contribution of the promoter architecture in such phenomena was studied. It was revealed that the quality of the TATA element plays a role in nucleosome modifications and selection of coactivators. More importantly this study revealed the existence of a co-transcriptional mechanism that determines the levels of Gcn5-dependent histone acetylation within promoter nucleosomes. Secondly, studies on the contribution of the activator for the recruitment and function of transcriptional complexes revealed a role of the activation domains in determining the transcriptional requirement for specific SAGA components. In addition, a novel function of the Spt3 component of SAGA and of the Mot1 ATPase in nucleosome remodeling was demonstrated. In the last part the study of the contribution of cis-elements revealed the ability of activators to associate in vivo with non promoter euhromatic sites. This association was shown to result in the functional recruitement of SAGA and Swi2/Snf2 complexes. This assemblage of coactivator complexes was proven not to be productive because of the absence of core promoter elements other than the TATA box that are required for stable recruitement of the Mediator.Η ρύθμιση της γονιδιακής έκφρασης επιτελείται κυρίως στο επίπεδο της μεταγραφής των γονιδίων και είναι αποτέλεσμα της συνδυαστικής σχέσης των ρυθμιστικών στοιχείων cis και trans, δηλαδή των υποκινητών και ενεργοποιητών αντίστοιχα. Στη συγκεκριμένη εργασία διερευνήθηκε ο ρόλος των στοιχείων αυτών στη στρατολόγηση και δράση μεταγραφικών συμπλόκων in vivo. Αρχικά αντιμετωπίστηκε το ερώτημα της συνεισφοράς της αρχιτεκτονικής του υποκινητή σε τέτοια φαινόμενα. Προέκυψαν συμπεράσματα σε σχέση με την πιθανότητα συμμετοχής της ποιότητας του στοιχείου ΤΑΤΑ σε θέματα νουκλεοσωμικής αναδιαμόρφωσης, τροποποίησης των ιστονών αλλά και επιλογής για μεταγραφικούς συνενεργοποιητές. Από τέτοιες παρατηρήσεις αναδείχθηκε η ύπαρξη ενός συν-μεταγραφικού μηχανισμού που καθορίζει τα επίπεδα της Gcn5-εξαρτόμενης ακετυλίωσης των υποκινητών. Σε ένα δεύτερο επίπεδο αντιμετωπίστηκε το ακριβώς αντίστροφο ερώτημα της συνεισφοράς του ενεργοποιητή σε θέματα στρατολόγησης και δράσης μεταγραφικών συμπλόκων. Προέκυψαν συμπεράσματα σε σχέση με το ρόλο της θέσης ενεργοποίησης των ενεργοποιητών στον καθορισμό της απαίτησης και του τρόπου δράσης συστατικών του συμπλόκου SAGA ενώ αποκαλύφθηκε ένας μηχανισμός δράσης της υπομονάδας Spt3 του συμπλόκου αυτού, καθώς και της ΑΤΡάσης Μot1, στη νουκλεοσωμική αναδιαμόρφωση. Τέλος, η μελέτη του ρόλου των cis στοιχείων των υποκινητών αποκάλυψε τη δυνατότητα πρόσδεσης ενεργοποιητών σε ευχρωματινικές θέσεις μη υποκινητών μέσα στο γένωμα. Η πρόσδεση αυτή έχει σαν αποτέλεσμα τη στρατολόγηση λειτουργικών συμπλόκων SAGA και Swi2/Snf2 ενώ η συγκρότηση των μεταγραφικών αυτών συμπλόκων δείχθηκε ότι δεν είναι παραγωγική λόγω της απουσίας από τις θέσεις αυτές cis στοιχείων των υποκινητών, ανεξάρτητων του στοιχείου ΤΑΤΑ, που χρειάζονται για τη στρατολόγηση του συμπλόκου του Μediator

Gcn4 occupancy of open reading frame regions results in the recruitment of chromatin-modifying complexes but not the mediator complex

Eukaryotic transcriptional activators usually recognize short DNA motifs, which are not only located within promoter regions, but also scattered throughout the genome. Assuming that the function of activators at non-promoter regions is wasteful and perhaps harmful, one can ask whether such binding is somehow prevented or if transcription is blocked at a downstream step. Here, we show that the yeast transcriptional activator Gcn4 is associated in vivo with several non-promoter euchromatic sites. This association results in the recruitment of the SAGA (Spt3/Ada/Gcn5/acetyltransferase) complex and the consequent activity of the Gcn5 histone acetyltransferase. The functional recruitment of the Swi/Snf nucleosome-remodelling complex was also evident at sites located in positioned nucleosomes. We show that this assemblage of coactivator complexes is not productive because of the absence of core promoter elements, other than the TATA box, that are required for stable mediator recruitment

Caenorhabditis elegans HIF-1 Is Broadly Required for Survival in Hydrogen Sulfide

Hydrogen sulfide is common in the environment, and is also endogenously produced by animal cells. Although hydrogen sulfide is often toxic, exposure to low levels of hydrogen sulfide improves outcomes in a variety of mammalian models of ischemia-reperfusion injury. In Caenorhabditis elegans, the initial transcriptional response to hydrogen sulfide depends on the hif-1 transcription factor, and hif-1 mutant animals die when exposed to hydrogen sulfide. In this study, we use rescue experiments to identify tissues in which hif-1 is required to survive exposure to hydrogen sulfide. We find that expression of hif-1 from the unc-14 promoter is sufficient to survive hydrogen sulfide. Although unc-14 is generally considered to be a pan-neuronal promoter, we show that it is active in many nonneuronal cells as well. Using other promoters, we show that pan-neuronal expression of hif-1 is not sufficient to survive exposure to hydrogen sulfide. Our data suggest that hif-1 is required in many different tissues to direct the essential response to hydrogen sulfide

Post-TATA Binding Protein Recruitment Clearance of Gcn5-Dependent Histone Acetylation within Promoter Nucleosomes

Transcriptional activation of eukaryotic genes often requires the function of histone acetyltransferases (HATs), which is expected to result in the hyperacetylation of histones within promoter nucleosomes. In this study we show that, in Saccharomyces cerevisiae, the steady-state levels of Gcn5-dependent histone acetylation within a number of transcriptionally active promoters are inversely related to the rate of transcription. High acetylation levels were measured only when transcription was attenuated either by TATA element mutations or in a strain carrying a temperature-sensitive protein component of RNA polymerase II. In addition, we show that in one case the low levels of histone acetylation depend on the function of the Rpd3 histone deacetylase. These results point to the existence of an unexpected interplay of two opposing histone-modifying activities which operate on promoter nucleosomes following the initiation of RNA synthesis. Such interplay could ensure rapid turnover of chromatin acetylation states in continuously reprogrammed transcriptional systems

A Role for Gcn5-Mediated Global Histone Acetylation in Transcriptional Regulation

Transcriptional activators often require histone acetyltransferases (HATs) for full activity. The common explanation is that activators directly recruit HATs to gene promoters to locally hyperacetylate histones and thereby facilitate transcription complex formation. However, in addition to being targeted to specific loci, HATs such as Gcn5 also modify histones genome-wide. Here we provide evidence for a role of this global HAT activity in regulated transcription. We show that activation by direct recruitment of the transcriptional machinery neither recruits Gcn5 nor induces changes in histone acetylation yet can strongly depend on Gcn5 at promoters showing a high basal state of Gcn5-mediated histone acetylation. We also show that Gcn5 dependency varies among core promoters and is influenced by the strength of interaction used to recruit the machinery and by the affinity of the latter for the core promoter. These data support a role for global Gcn5 HAT activity in modulating transcription independently of its known coactivator function

Spt3 and Mot1 cooperate in nucleosome remodeling independently of TBP recruitment

We have investigated the requirements for nucleosome remodeling upon transcriptional induction of the GAL1 promoter. We found that remodeling was dependent on two SAGA complex components, Gcn5 and Spt3. The involvement of the latter was surprising as its function has been suggested to be directly involved in TATA-binding protein (TBP) recruitment. We demonstrated that this novel function was in fact independent of TBP recruitment and this was further validated using a Gal4-driven synthetic promoter. Most importantly, we showed that the involvement of Spt3 in chromatin remodeling was independent of transcription, as it was also observed for a nonpromoter nucleosome located next to an activator-binding site. In an effort to explore how the Spt3 function was elicited, we found that Mot1, an ATPase of the Snf2 family that genetically interacts with Spt3, was also required for nucleosome remodeling independently of TBP recruitment. Interestingly enough, Spt3 and Mot1 were recruited on the GAL1 promoter as well as on the nonpromoter site in an interdependent manner. These findings show that the two proteins cooperate in nucleosomal transactions

Dopamine negatively modulates the NCA ion channels in <i>C</i>. <i>elegans</i>

<div><p>The NALCN/NCA ion channel is a cation channel related to voltage-gated sodium and calcium channels. NALCN has been reported to be a sodium leak channel with a conserved role in establishing neuronal resting membrane potential, but its precise cellular role and regulation are unclear. The <i>Caenorhabditis elegans</i> orthologs of NALCN, NCA-1 and NCA-2, act in premotor interneurons to regulate motor circuit activity that sustains locomotion. Recently we found that NCA-1 and NCA-2 are activated by a signal transduction pathway acting downstream of the heterotrimeric G protein G_q and the small GTPase Rho. Through a forward genetic screen, here we identify the GPCR kinase GRK-2 as a new player affecting signaling through the G_q-Rho-NCA pathway. Using structure-function analysis, we find that the GPCR phosphorylation and membrane association domains of GRK-2 are required for its function. Genetic epistasis experiments suggest that GRK-2 acts on the D2-like dopamine receptor DOP-3 to inhibit G_o signaling and positively modulate NCA-1 and NCA-2 activity. Through cell-specific rescuing experiments, we find that GRK-2 and DOP-3 act in premotor interneurons to modulate NCA channel function. Finally, we demonstrate that dopamine, through DOP-3, negatively regulates NCA activity. Thus, this study identifies a pathway by which dopamine modulates the activity of the NCA channels.</p></div