Alexander M. van der Linden

KIN‐29 SIK regulates chemoreceptor gene expression via an MEF2 transcription factor and a class II HDAC

The expression of individual chemoreceptor (CR) genes in Caenorhabditis elegans is regulated by multiple environmental and developmental cues, possibly enabling C. elegans to modulate its sensory responses. We had previously shown that KIN‐29, a member of the salt‐inducible kinase family, acts in a subset of chemosensory neurons to regulate the expression of CR genes, body size and entry into the alternate dauer developmental stage. Here, we show that KIN‐29 regulates these processes by phosphorylating the HDA‐4 class II histone deacetylase (HDAC) and inhibiting the gene repression functions of HDA‐4 and an MEF‐2 MADS domain transcription factor. MEF‐2 binds directly to the CR gene regulatory sequences, and is required only to repress but not activate CR gene expression. A calcineurin phosphatase antagonizes the KIN‐29/MEF‐2‐regulated pathway to modulate levels of CR gene expression. Our results identify KIN‐29 as a new regulator of MEF2/HDAC functions in the nervous system, reveal cell‐specific mechanisms of action of this pathway in vivo and demonstrate remarkable complexity in the regulation of CR gene expression in C. elegans.

Left-right olfactory asymmetry results from antagonistic functions of voltage-activated calcium channels and the Raw repeat protein OLRN-1 in C. elegans

BackgroundThe left and right AWC olfactory neurons in Caenorhabditis elegans differ in their functions and in their expression of chemosensory receptor genes; in each animal, one AWC randomly takes on one identity, designated AWCOFF, and the contralateral AWC becomes AWCON. Signaling between AWC neurons induces left-right asymmetry through a gap junction network and a claudin-related protein, which inhibit a calcium-regulated MAP kinase pathway in the neuron that becomes AWCON.ResultsWe show here that the asymmetry gene olrn-1 acts downstream of the gap junction and claudin genes to inhibit the calcium-MAP kinase pathway in AWCON. OLRN-1, a protein with potential membrane-association domains, is related to the Drosophila Raw protein, a negative regulator of JNK mitogen-activated protein (MAP) kinase signaling. olrn-1 opposes the action of two voltage-activated calcium channel homologs, unc-2 (CaV2) and egl-19 (CaV1), which act together to stimulate the calcium/calmodulin-dependent kinase CaMKII and the MAP kinase pathway. Calcium channel activity is essential in AWCOFF, and the two AWC neurons coordinate left-right asymmetry using signals from the calcium channels and signals from olrn-1.Conclusionolrn-1 and voltage-activated calcium channels are mediators and targets of AWC signaling that act at the transition between a multicellular signaling network and cell-autonomous execution of the decision. We suggest that the asymmetry decision in AWC results from the intercellular coupling of voltage-regulated channels, whose cross-regulation generates distinct calcium signals in the left and right AWC neurons. The interpretation of these signals by the kinase cascade initiates the sustained difference between the two cells.

Genome-Wide Analysis of Light- and Temperature-Entrained Circadian Transcripts in Caenorhabditis elegans

Transcriptional profiling experiments identify light- and temperature-entrained circadian transcripts in C. elegans.

Proteins interacting with Caenorhabditis elegans Gα subunits

To identify novel components in heterotrimeric G-protein signalling, we performed an extensive screen for proteins interacting with Caenorhabditis elegans Gα subunits. The genome of C. elegans contains homologues of each of the four mammalian classes of Gα subunits (Gs, Gi/o, Gq and G12), and 17 other Gα subunits. We tested 19 of the GGα subunits and four constitutively activated Gα subunits in a largescale yeast two-hybrid experiment. This resulted in the identification of 24 clones, representing 11 different proteins that interact with four different Gα subunits. This set includes C. elegans orthologues of known interactors of Gα subunits, such as AGS3 (LGN/PINS), CalNuc and Rap1Gap, but also novel proteins, including two members of the nuclear receptor super family and a homologue of human haspin (germ cell-specific kinase). All interactions were found to be unique for a specific Gα subunit but variable for the activation status of the Gα subunit. We used expression pattern and RNA interference analysis of the G-protein interactors in an attempt to substantiate the biological relevance of the observed interactions. Furthermore, by means of a membrane recruitment assay, we found evidence that GPA-7 and the nuclear receptor NHR-22 can interact in the animal.

The EGL-4 PKG Acts With KIN-29 Salt-Inducible Kinase and Protein Kinase A to Regulate Chemoreceptor Gene Expression and Sensory Behaviors in Caenorhabditis elegans

The regulation of chemoreceptor (CR) gene expression by environmental signals and internal cues may contribute to the modulation of multiple physiological processes and behavior in Caenorhabditis elegans. We previously showed that KIN-29, a homolog of salt-inducible kinase, acts in sensory neurons to regulate the expression of a subset of CR genes, as well as sensory behaviors. Here we show that the cGMP-dependent protein kinase EGL-4 acts partly in parallel with KIN-29 to regulate CR gene expression. Sensory inputs inhibit both EGL-4 and KIN-29 functions, and KIN-29 function is inhibited in turn by cAMP-dependent protein kinase (PKA) activation. EGL-4 and KIN-29 regulate CR gene expression by antagonizing the gene repression functions of the class II HDAC HDA-4 and the MEF-2 transcription factor, and KIN-29, EGL-4, and PKA target distinct residues in HDA-4 to regulate its function and subcellular localization. While KIN-29 acts primarily via MEF-2/HDA-4 to regulate additional sensory signal-regulated physiological processes and behaviors, EGL-4 acts via both MEF-2-dependent and -independent pathways. Our results suggest that integration of complex sensory inputs via multiple signaling pathways allows animals to precisely regulate sensory gene expression, thereby appropriately modulating physiology and behavior.

Hyperactivation of the G12-mediated signaling pathway in Caenorhabditis elegans induces a developmental growth arrest via protein kinase C.

The G(12) type of heterotrimeric G-proteins play an important role in development and behave as potent oncogenes in cultured cells. However, little is known about the molecular nature of the components that act in the G(12)-signaling pathway in an organism. We characterized a C. elegans Galpha subunit gene, gpa-12, which is a homolog of mammalian G(12)/G(13)alpha, and found that animals defective in gpa-12 are viable. Expression of activated GPA-12 (G(12)QL) results in a developmental growth arrest caused by a feeding behavior defect that is due to a dramatic reduction in pharyngeal pumping. To elucidate the molecular nature of the signaling pathways in which G(12) participates, we screened for suppressors of the G(12)QL phenotype. We isolated 50 suppressors that contain mutations in tpa-1, which encodes two protein kinase C isoforms, TPA-1A and TPA-1B, most similar to PKCtheta/delta. TPA-1 mediates the action of the tumor promoter PMA. Expression of G(12)QL and treatment of wild-type animals with PMA induce an identical growth arrest caused by inhibition of larval feeding, which is dependent on TPA-1A and TPA-1B function. These results suggest that TPA-1 is a downstream target of both G(12) signaling and PMA in modulating feeding and growth in C. elegans. Taken together, our findings provide a potential molecular mechanism for the transforming capability of G(12) proteins.

Chemical Genetics Reveals an RGS/G-Protein Role in the Action of a Compound

We report here on a chemical genetic screen designed to address the mechanism of action of a small molecule. Small molecules that were active in models of urinary incontinence were tested on the nematode Caenorhabditis elegans, and the resulting phenotypes were used as readouts in a genetic screen to identify possible molecular targets. The mutations giving resistance to compound were found to affect members of the RGS protein/G-protein complex. Studies in mammalian systems confirmed that the small molecules inhibit muscarinic G-protein coupled receptor (GPCR) signaling involving G-αq (G-protein alpha subunit). Our studies suggest that the small molecules act at the level of the RGS/G-αq signaling complex, and define new mutations in both RGS and G-αq, including a unique hypo-adapation allele of G-αq. These findings suggest that therapeutics targeted to downstream components of GPCR signaling may be effective for treatment of diseases involving inappropriate receptor activation.

Cis-regulatory mechanisms of gene expression in an olfactory neuron type in Caenorhabditis elegans

The generation of cellular diversity is dependent on the precise spatiotemporal regulation of gene expression by both cis‐ and trans‐acting mechanisms. The developmental principles regulating expression of specific gene subsets in individual cell types are not fully understood. Here we define the cis‐regulatory mechanisms driving expression of cell‐selective and broadly expressed genes in vivo in the AWB olfactory neuron subtype in C. elegans. We identify an element that is necessary to drive expression of neuron‐selective chemoreceptor genes in the AWB neurons, and show that this element functions in a context‐dependent manner. We find that the expression of broadly expressed sensory neuronal genes in the AWB neurons is regulated by diverse cis‐ and trans‐regulatory mechanisms that act partly in parallel to the pathways governing expression of AWB‐selective genes. We further demonstrate that cis‐acting mechanisms driving gene expression in the AWB neurons appear to have diverged in related nematode species. Our results provide insights into the cis‐regulatory logic driving cell‐specific gene expression, and suggest that variations in this logic contribute to the generation of functional diversity. Developmental Dynamics 238:3080–3092, 2009.

Shotgun Cloning of Transposon Insertions in the Genome of Caenorhabditis elegans

We present a strategy to identify and map large numbers of transposon insertions in the genome of Caenorhabditis elegans. Our approach makes use of the mutator strain mut-7, which has germline-transposition activity of the Tc1/mariner family of transposons, a display protocol to detect new transposon insertions, and the availability of the genomic sequence of C. elegans. From a pilot insertional mutagenesis screen, we have obtained 351 new Tc1 transposons inserted in or near 219 predicted C. elegans genes. The strategy presented provides an approach to isolate insertions of natural transposable elements in many C. elegans genes and to create a large-scale collection of C. elegans mutants.