Feng H et al. (2013) Genesis "The Caenorhabditis elegans homeobox gene ceh-19 is required for MC motorneuron function."

Feng H, Hope IA

[Genesis, 2013]

Simplicity has made C. elegans pharyngeal development a particularly well-studied subject. Nevertheless, here we add the previously uncharacterized homeobox gene F20D12.6/ceh-19 to the set of transcription factor genes involved. GFP reporter assays revealed that ceh-19 is expressed in three pairs of neurons, the pharyngeal pace-maker neurons MC, the amphid neurons ADF and the phasmid neurons PHA. ceh-19(tm452) mutants are viable and fertile, but grow slightly slower, produce less progeny over a prolonged period, and live longer than the wild type. These phenotypes are likely due to the moderately reduced pharyngeal pumping speed arising from the impairment of MC activity. MC neurons are still born in the ceh-19 mutants but display various morphological defects. ceh-19 expression in MC is completely lost in progeny from animals subject to RNAi for pha-4, which encodes an organ-specifying forkhead transcription factor. CEH-19 is required for the activation in MCs of the excitatory FMRFamide-like neuropeptide-encoding gene flp-2. A regulatory pathway from pha-4 through ceh-19 to flp-2 is thereby defined. The resilience of MC identity in the absence of CEH-19 may reflect the buffering qualities of transcription factor regulatory networks.

Feng H et al. (2012) Methods Mol Biol "Expression pattern analysis of regulatory transcription factors in Caenorhabditis elegans."

Hope IA, Feng H, Craig HL

[Methods Mol Biol, 2012]

Expression pattern data are fundamental to understanding transcriptional regulatory networks and the biological significance of such networks. For Caenorhabditis elegans, expression pattern analysis of transcription factor genes, with cellular resolution, typically involves generation of transcription factor gene/reporter gene fusions. This is followed by the creation of C. elegans strains transgenic for, and determination of expression patterns driven by, these fusions. Physiologically relevant regulatory relationships between transcription factors are both inferred from their expression patterns, in combination with protein-DNA interaction data, and evidenced from alterations of expression patterns when networks are disturbed.

Feng H et al. (2012) Gene "A regulatory cascade of three transcription factors in a single specific neuron, DVC, in Caenorhabditis elegans."

Feng H, Walhout AJ, Reece-Hoyes JS, Hope IA

[Gene, 2012]

Homeobox proteins are critical regulators of developmental gene transcription and cell specification. Many insights into transcriptional regulation have been gained from studies in the nematode Caenorhabditis elegans. We investigated the expression and regulation of the C. elegans homeobox gene ceh-63, which encodes a single-homeodomain transcription factor of 152 amino acids. ceh-63 is expressed in the interneuron DVC in both sexes, from late embryogenesis through adulthood, and two pairs of uterine cells in reproductive hermaphrodites only. A reporter gene fusion, encoding GFP fused to the full-length CEH-63, also drove weak inconsistent expression in additional unidentified cells in the head and tail. A potential ceh-63 null mutant had no obvious abnormalities, except for a possible increase in subtle defects of the DVC axon projection. No behavioural responses were observed upon either laser ablation of DVC or activation of DVC through light stimulation of channelrhodopsin-2 specifically expressed in this neuron. The function of DVC therefore remains enigmatic. A transcriptional regulatory cascade operating in DVC was defined from the LIM-homeodomain protein CEH-14 through CEH-63 to the helix-turn-helix transcription factor MBR-1. Both CEH-14 and CEH-63 individually bound the mbr-1 promoter in a yeast one-hybrid assay. A model is proposed suggesting that CEH-14 activates ceh-63 and then along with CEH-63 co-ordinately activates mbr-1.

Feng H et al. (2006) J Biol Chem "Conserved domains subserve novel mechanisms and functions in DKF-1, a Caenorhabditis elegans protein kinase D."

Rubin CS, Ren M, Feng H

[J Biol Chem, 2006]

Protein kinase D (PKD) isoforms are effectors in signaling pathways controlled by diacylglycerol (DAG). PKDs contain conserved DAG binding (C1a, C1b), PH and S/T kinase domains. However, properties of conserved domains may vary within the context of distinct PKD polypeptides. Such functional/structural malleability (plasticity) was explored by studying C. elegans D kinase family-1 (DKF-1), a PKD that governs locomotion in vivo. Phorbol ester binding with C1b alone activates classical PKDs by relieving C1-mediated inhibition. In contrast, C1a avidly ligates PMA and anchors DKF-1 at plasma membrane. C1b binds PMA (moderate affinity) and co-operates with C1a in targeting DKF-1 to membranes. Mutations at a "Pro(11)" position in C1 domains are inactivating; kinase activity is minimal at PMA concentrations that stimulate wild type (WT) DKF-1 ~10-fold. DKF-1 mutants exhibited unchanged, maximum kinase activity after cells were incubated with high PMA concentrations. Titration in situ revealed that translocation and activation of WT and mutant DKF-1 are tightly and quantitatively linked at all PMA concentrations. C1 domains are positive regulators of phosphotransferase activity that dock DKF-1 with pools of activating lipid. A PH domain inhibits kinase activity in classical PKDs. DKF-1 PH module neither inhibits catalytic activity, nor binds phosphoinositides. The PH module is an obligatory, positive regulator of DKF-1 activity that is compromised by mutation of K(298) or W(396). Phosphorylation of T(588) switches on DKF-1 kinase activity. Persistent phosphorylation of T(588) (activation loop) promotes ubiquitinylation and proteasome-mediated degradation of DKF-1. Each DKF-1 domain displays novel properties indicative of functional malleability (plasticity).

Feng H et al. (1999) Nat Cell Biol "CUL-2 is required for the G1-to-S-phase transition and mitotic chromosome condensation in Caenorhabditis elegans."

Zhong W, Punkosdy G, Seabolt EK, Zhou L, Feng H, Gu S, Kipreos ET

[Nat Cell Biol, 1999]

The human cullin protein CUL-2 functions in a ubiquitin-ligase complex with the von Hippel-Lindau (VHL) tumour suppressor protein. Here we show that, in Caenorhabditis elegans, cul-2 is expressed in proliferating cells and is required at two distinct points in the cell cycle, the G1-to-S-phase transition and mitosis. cul-2 mutant germ cells undergo a G1-phase arrest that correlates with accumulation of CKI-1, a member of the CIP/KIP family of cyclin-dependent-kinase inhibitors. In cul-2 mutant embryos, mitotic chromosomes are unable to condense, leading to unequal DNA segregation, chromosome bridging and the formation of multiple nuclei.

Feng H et al. (2006) J Biol Chem "Characterization of a novel protein kinase D: Caenorhabditis elegans DKF-1 is activated by translocation-phosphorylation and regulates movement and growth in vivo."

Hall DH, Ren M, Rubin CS, Feng H, Wu SL

[J Biol Chem, 2006]

Protein kinase D (PKD) isoforms are protein kinase C (PKC) effectors in diacylglycerol (DAG)-regulated signaling pathways. Key physiological processes are placed under DAG control by the distinctive substrate specificity and intracellular distribution of PKDs. Comprehension of PKDs'' roles in homeostasis and signal transduction requires further knowledge of regulatory interplay among PKD and PKC isoforms, analysis of PKC-independent PKD activation and characterization of functions controlled by PKDs in vivo. C. elegans and mammals share conserved signaling mechanisms, molecules and pathways Thus, characterization of C. elegans PKDs could yield insights into regulation and function that apply to all eukaryotic PKDs. C. elegans DKF-1 (D Kinase Family-1) contains tandem, DAG binding (C1) modules, a PH domain and S/T protein kinase segment, which are homologous with domains in classical PKDs. DKF-1 and PKDs have similar substrate specificities. PMA switches on DKF-1 catalytic activity in situ by promoting phosphorylation of a single amino acid T(588), in the activation loop. DKF-1 phosphorylation and activation are unaffected when PKC activity is eliminated by inhibitors. Both phosphorylation and kinase activity of DKF-1 are extinguished by substituting A for T(588), Q for K(455) ("kinase dead") or incubating with protein phosphatase 2C. Thus, DKF-1 is a PMA-activated, PKC-independent D kinase. In vivo, dkf-1 gene promoter activity is evident in neurons. Both dkf-1 gene disruption (null phenotype) and RNAi-mediated depletion of DKF-1 protein cause lower body paralysis. Targeted DKF-1 expression corrected this locomotory defect in dkf-1 null animals. Supra-physiological expression of DKF-1 limited C. elegans growth to ~60% of normal length.

Feng H et al. (2007) J Biol Chem "Properties, regulation, and in vivo functions of a novel protein kinase D: Caenorhabditis elegans DKF-2 links diacylglycerol second messenger to the regulation of stress responses and life span."

Feng H, Ren M, Chen L, Rubin CS

[J Biol Chem, 2007]

Protein kinase D (PKD) isoforms are protein kinase C (PKC) effectors in signaling cascades controlled by diacylglycerol (DAG). All PKDs are regulated by DAG/PMA-binding C1 domains and an activation loop (A-loop). To understand how PKD isoforms diversify DAG signaling networks, it is essential to determine redundant and novel properties of their regulatory domains; characterize factors controlling PKD gene expression; and discover their in vivo physiological roles. Studies on a novel PKD, C. elegans DKF-2 (D kinase family-2), addressed these topics. The C1b domain mediates PMA-induced translocation and activation of DKF-2. However, when DAG is elevated, C1a and C1b contribute equally to targeting/activation of DKF-2. DKF-2 C1 domains do not inhibit catalytic activity; they mediate delivery of DKF-2 to a membrane where PKC phosphorylates S(925) and S(929) in the A-loop. This potently stimulates DKF-2 catalytic activity. Phosphorylation of S(925) alone switches on 70% of maximal kinase activity. Persistent phosphorylation of S(929) tags DKF-2 for proteasomal degradation; P-Ser(925) plays a minor role in DKF-2 degradation. GATA enhancer sequences govern DKF-2 expression in intestine in vivo. Adult lifespan increases 40% in animals lacking DKF-2. In thermally stressed WT animals, the DAF-16 transcription factor is segregated from nuclei of adult intestinal cells. In contrast, DAF-16 enters adult intestinal nuclei of DKF-2 deficient, thermally-stressed animals, where it can trigger gene transcription that protects against various insults. The results suggest a mechanism for increased longevity and show that a PKD links DAG signals to regulation of stress responses and lifespan.

H Feng et al. (1999) International C. elegans Meeting "cul-2 and the vhl Tumor Suppressor Gene: A genetic study"

H Feng, R Barstead, G Moulder, ET Kipreos

[International C. elegans Meeting, 1999]

Inactivation of the Von Hippel-Lindau tumor suppressor gene ( VHL ) causes cancers of the kidney, brain and retina in humans. The VHL protein associates with Elongin B and Elongin C in a VBC complex. VHL was initially thought to inhibit transcription by sequestering Elongin B and C from the Elongin (SIII) complex, composed of Elongins A, B and C. However, the concentration of Elongin B and C is reported to be 100 to 1000-fold higher than that of VHL in cells (Conaway et al., 1998, BBA 1377: M49) making it unlikely that VHL can sequester them. Currently, how VHL functions to suppress tumor formation is unknown. Recently, VHL was found to bind to human CUL-2 (Pause et al., 1997, PNAS 94: 2155). cul-2 is a member of the cullin gene family. Cullin homologs Cdc53 and CUL-1 are involved in the degradation of Cyclins and CKIs to regulate cell cycle progression. In cul-2 ( ek1 ) mutants, germ cells are arrested in G1 phase, and this is correlated with a higher level of CKI-1, a member of the CIP/KIP family of cyclin-dependent kinase inhibitors. Our results suggest that cul-2 is required for the G1 to S phase transition, potentially functioning by degrading CKI-1. Interestingly, overexpression of wild type VHL in human cells also induces a G1 phase cell cycle arrest and increases the level of the CKI p27KIP1 (Kim et al., 1998, BBRC 253: 672). Based on these observations, we propose a model in which VHL negatively regulates CUL-2, which negatively regulates CKIs, which negatively regulates the cell cycle. Since a VHL homolog is present in C. elegans we are undertaking a genetic study of cul-2 and vhl . Both the vhl ( ok161 ) allele and the cul-2 ( ek1 ) allele were obtained by PCR screening for deletions in EMS mutagenized animals, by G.M./R.B. and E.T.K., respectively. We are trying to gain insights into the (potential) genetic interaction of vhl and cul-2 by making a strain homozygous for vhl ( ok161 ) and heterozygous for cul-2 ( ek1 ) and analyzing the resulting homozygous progeny. We are in the final stage of making this strain and we will present the double mutant phenotype at the meeting.

Kipreos ET et al. (2000) East Coast Worm Meeting "Investigation of cul-2 E3 complexes and Interaction between cul-2 and vhl"

Kipreos ET, Feng H

[East Coast Worm Meeting, 2000]

The ubiquitin-proteasome proteolytic pathway plays a critical role in the regulation of cell cycle progression. Ubiquitin ligases (E3s) function in this pathway to target substrates for degradation. There are two major types of E3 complexes, the APC and Cullin/Ring finger complexes. The cullin/Ring finger complexes contain a cullin, a RBX1 ring finger protein, a SKP1-like molecule, and a substrate-binding adaptor. The first described cullin member, CUL-1, functions to allow the cell cycle exit. Our recent study (Feng et al, Nature cell Biology, Vol.1(8), 1999) described the function of another member of the cullin family, CUL-2. CUL-2 is a positive cell cycle regulator that is required for the G1 to S phase transition, functioning in part by negatively regulating CKI-1 (a cyclin-dependent kinase inhibitor). Surprisingly, CUL-2 is also required for mitotic chromosome condensation. In humans, CUL-2 was found in an E3 complex with the von Hippel-Lindau (VHL) tumor suppressor, Elongin C (a SKP1 like protein), Elongin B (a ubiquitin like protein), and RBX1. This CUL-2/VCB complex functions to degrade hypoxia inducible transcription factor 1a. In order to gain insights into the composition of the CUL-2 E3 complexes that regulate CKI-1 degradation and chromosome condensation, we took advantage of the RNAi technique to look for genes which phenocopy cul-2 after RNAi. RNAi of Elongin C gave phenotypes similar to cul-2. We think that it is likely that Elongin C functions with CUL-2 in a complex to regulate the cell cycle. However, VHL does not appear to function with CUL-2 in cell cycle regulation since the inactivation of this gene either by RNAi or deletion mutant (obtained by Gary Moulder in Robert Barsteads lab) doesnt phenocopy cul-2. In addition, in humans, a removal of VHL decreases the level of the CKI p27, while in contrast overexpression of VHL increases p27. We propose that there are adaptor proteins other than VHL in CUL-2 complexes that are required for cell cycle regulation. VHL may sequester CUL-2 from other CUL-2 complexes containing different adaptor molecules. The absence of VHL may lead to an increase of these CUL-2 complexes, causing rapid degradation of CKIs, thereby contributing to more cell division and tumor formation.

Thomas JH et al. (1994) Worm Breeder's Gazette "lin-36, a Class B Synthetic Multivulva Gene, Encodes a Novel Protein"

Thomas JH, Horvitz HR

[Worm Breeder's Gazette, 1994]

lin-36, a Class B Synthetic Multivulva Gene, Encodes a Novel Protein Jeffrey H. Thomas and H. Robert Horvitz, HHMI, Dept. Biology, MIT, Cambridge, MA 02139, USA