Zhiyong Shao et al. (2007) International Worm Meeting "VHL-1-independent pathways that regulate the HIF-1 hypoxia-inducible factor."

Zhiyong Shao, Chuan Shen, Jo Anne Powell-Coffman

[International Worm Meeting, 2007]

Animals sense and must respond to changing levels of environmental oxygen. In mammals and in C. elegans, the hypoxia-inducible factor (HIF) transcription complex controls most of the gene expression changes that allow adaptation to hypoxia (low oxygen). In C. elegans, the heterodimeric HIF complex is composed of two proteins: HIF-1 (orthologous to mammalian HIF alpha subunit) and AHA-1 (orthologous to mammalian ARNT / HIF beta) (Jiang H et al., 2001 PNAS 98: 7916; Powell-Coffman JA et al., 1998 PNAS 95: 2844). The EGL-9 / VHL-1 pathway that targets HIF-1 for oxygen-dependent degradation has been well characterized (Epstein et al. 2001, Cell 107: 43). Recently, we have presented evidence that EGL-9 represses expression of HIF-1 target genes via VHL-1-independent pathway(s) (Shen et al., Genetics 174: 1205). A major focus of our current work is to understand the functions of EGL-9 and interacting proteins in this pathway. We are testing the hypothesis that the two EGL-9 pathways can be uncoupled by specific mutations in egl-9 or its related genes. Towards this goal, we have designed genetic screens to identify mutations that cause over-expression of HIF-1 target genes in a vhl-1-defective background. We have termed genes mutable to this phenotype rhy: regulators of hypoxia-inducible factor. We have isolated multiple mutations in the rhy-1 gene, caused by chemical mutagen or MOS1 transposon insertion. RHY-1 encodes a multi-pass transmembrane protein that represses HIF-1 transcriptional activity. We have also isolated a MOS1 insertion in egl-9 and mutations in additional rhy genes that map elsewhere in the genome. At the meeting, we will describe our progress towards understanding EGL-9 and RHY-1 function and the characterization of additional rhy genes.

Zhiyong Shao et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Dual Functions of EGL-9: Defining the Molecular Mechanisms by which EGL-9 inhibits HIF-1 Transcriptional Activity"

Zhiyong Shao, Jo Anne Powell-Coffman

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

In mammals and in C. elegans, the hypoxia inducible transcription factor (HIF-1) mediates most of the gene expression changes induced by hypoxia (Jiang H et al., 2001 PNAS 98: 7916; Shen et al., 2005 JBC 280: 20580). EGL-9 has a central role in HIF regulatory networks. EGL-9 hydroxylates the proline in the LXXLAP motif of HIF-1, using oxygen as a co-substrate. This promotes HIF-1 degradation through a ubiquitination/proteasome pathway that requires the VHL-1 E3 ligase. When oxygen levels are low, EGL-9 hydroxylase activity is inhibited, and HIF-1 is stabilized (Epistein et al. 2001 Cell 107: 43). In prior studies, we have shown that EGL-9 functions through at least one additional pathway to inhibit HIF-1. In animals carrying a deletion mutation in vhl-1, loss-of-function mutations in egl-9 have no effect on HIF-1 protein levels but dramatically up-regulate expression of HIF-1 target genes (Shen et al., 2006 Genetics 174: 1205). In the past year, we have conducted a series of molecular genetic experiments to distinguish between alternative hypotheses for EGL-9 function. Our data support a model in which EGL-9 has two distinct functions: (1) hydroxylation and destabilization of HIF-1 via the well-characterized VHL-1 pathway; and (2) repression of HIF-1 transcriptional activity by a novel pathway that does not require HIF-1 hydroxylation. We gratefully acknowledge funding from the NIH (GM078424).

Yanjun, Shi et al. (2021) International Worm Meeting "A gut neuroendocrine signal regulates synaptic assembly in the brain"

Yanjun, Shi, Zhiyong , Shao, Lu, Qin

[International Worm Meeting, 2021]

The gut-brain axis plays an essential role in regulating neural development in response to internal environmental stimuli, such as microbes or nutrients. Defects in gut-brain communication can lead to various neurological disorders. However, it remains unknown whether gut plays an intrinsic role in regulating neuronal development. Through a genetic screen in C. elegans, we uncovered that an intrinsic Wnt-endocrine pathway in gut regulates synaptic development and neuronal activity in brain. Specifically, the Wnt signaling upregulates the expression of the neuropeptide NLP-40 in the gut and facilitates presynaptic assembly through the neuronal expressed GPCR AEX-2 receptor during development. The NLP-40 acts most likely through modulating neuronal activity and promoting synaptic protein trafficking. Our study reveals a novel role of gut in synaptic development in the brain and provides additional molecular basis for gut-brain interactions. Key words: Gut-brain axis; Synaptic development; Wnts; Endocrine; Neuropeptide/NLP-40; GPCR/AEX-2; Neuronal activity

Clarke LE et al. (2013) Cell "Glia keep synapse distribution under wraps."

Barres BA, Clarke LE

[Cell, 2013]

The wiring of the nervous system requires that axons navigate to the correct targets and maintain their correct positions during developmental growth. In this issue, Shao etal. (2013) now reveal a crucial new role for glia in preserving correct synaptic connectivity during developmental growth.

Zhiwei Zhai et al. (2007) International Worm Meeting "RNAi screens provide insights to cellular processes and pathways that regulate the C. elegans hypoxia-inducible factor HIF-1."

Yi Zhang, Zhiyong Shao, Zhiwei Zhai, Jo Anne Powell-Coffman

[International Worm Meeting, 2007]

The hypoxia-inducible transcription factor HIF-1 is a critical regulator of cellular and systemic responses to low oxygen levels. Our objective is to identify and decipher the regulatory networks that modulate HIF-1 function in C. elegans. Towards this goal, we developed a visual assay for HIF-1 function, in which a hif-1-dependent promoter drives GFP expression in transgenic animals (Shen et al., Genetics 174: 1205). We then screened a genome wide RNAi library (from Geneservice) to identify genes that regulate expression of the reporter. We identified 187 genes for which RNAi inactivation increased expression of the reporter, including many genes with predicted roles in metabolism, mitochondrial function, stress response, or protein turnover. To further evaluate these RNAi phenotypes, we are assaying the expression of HIF-1 protein. These data will provide insight to the metabolic pathways that modulate EGL-9 enzymatic activity and oxygen-dependent degradation of HIF-1. Interestingly, some of the mitochondrial genes that we identified as potential regulators of HIF-1 had previously been shown to modulate lifespan. We find that loss-of-function mutations in hif-1 do not decrease longevity, and lifespan extension by RNAi inactivation of select mitochondrial genes do not require hif-1 function. These data are in general agreement with current models, in which hypoxic stress increases both HIF-1 levels and daf-16 expression, and the two transcription factors have independent as well as shared targets that enable response to stress.

Yi Zhang et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "HIF-1 and Hypoxia Response Networks"

Yi Zhang, Zhiwei Zhai, Zhiyong Shao, Dingxia Feng, Jo Anne Powell-Coffman

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

In aerobic organisms, cells must be poised to sense and respond to small changes in oxygen availability. Most of the critical transcriptional responses to hypoxia (low oxygen) in animals are controlled by evolutionarily conserved transcription complexes called hypoxia inducible factors (HIF). HIF DNA-binding complexes are heterodimeric. While the HIFbeta proteins have other partners, the HIFalpha subunits appear to be dedicated to oxygen-dependent regulation of gene expression. In many types of tumors, over-expression of HIF correlates with aggressive cancer and poor patient outcome, so it is critically important to understand the cellular networks that modulate and attenuate HIF-1 activity. C. elegans has proven to be an excellent model for deciphering the cellular circuits that regulate HIF stability and function. The C. elegans orthologs of human HIFalpha, HIFbeta, VHL, and the PHD prolyl hydroxylases are encoded by the hif-1, aha-1, vhl-1, and egl-9 genes, respectively. The EGL-9 enzyme hydroxylates a specific proline residue on HIF-1, in a reaction that requires molecular oxygen. Hydroxylated HIF-1 binds VHL-1 and is targeted for proteasomal degradation. Genetic analyses have demonstrated that EGL-9 also inhibits expression of HIF-1 target genes via VHL-1-independent pathways. One of the current goals of our research group is to understand and characterize these VHL-1-independent functions of EGL-9. We have initiated several complementary strategies to identify genes that regulate or interact with HIF-1. At the meeting, we will describe some of these studies, including forward genetic screens, RNAi screens, purification of HIF-1 complexes, and yeast two-hybrid analyses. These data have provided insights to HIF-1 regulation and function. We gratefully acknowledge funding from NIH (GM078424) and from a Bailey Research Career Development Award to JAPC.

Shao H et al. (2014) Bio Protoc "Detection of piggyBac-mediated Transposition by Splinkerette PCR in Transgenic Lines of Strongyloides ratti."

Lok JB, Shao H

[Bio Protoc, 2014]

Splinkerette PCR (spPCR) is a newly developed and efficient method to ascertain and characterize genomic insertion sites of transgenes. The method described in this protocol was successfully applied to confirm piggyBac transposon-mediated integration of transgenes into chromosomes of the parasitic nematode Strongyloides ratti. This work is described in detail in Shao et al. (2012) and presented here in a simplified diagram (Figure 1). Using this method, chromosomal loci of integration were determined based on target site and 5'- and 3' flanking sequences. Therefore, spPCR can be a useful method to confirm integrative transgenesis in functional genomic studies of parasitic nematodes. Potter and Luo (2010) contains a protocol for use of spPCR to detect and map piggyBac transposon-mediated chromosomal integrations in Drosophila, and was the source of our method for Strongyloides. The splinkerette- and piggyBac-specific oligos described in that reference could be used without modification in Strongyloides. For interested readers, a general review of the biology of parasitic nematodes in the genus Strongyloides may be found in Viney and Lok (2007), and a methods-based article on S. stercoralis as an experimental model, with information on transgenesis, may be found in Lok (2007).

Moorthy S et al. (1996) East Coast Worm Meeting "A NOVEL C. ELEGANS ADDUCIN: A WHOLE NEW CAN OF WORMS"

Bennett V, Chen LS, Moorthy S

[East Coast Worm Meeting, 1996]

Vertebrate adducin is an actin-binding protein which can promote assembly of a spectrin-actin complex and can also act as a barbed-end actin capping protein. Biochemical and biophysical characterization in our lab has shown that active adducin is probably a tetramer consisting of two alpha subunits tightly complexed with either beta or gamma subunits. Adducin binds calmodulin and is an in vivo substrate for protein kinases C and A, all of which modulate adducin's spectrin-actin binding activity in different ways in in vitro assays. Nevertheless, vertebrate adducin has never been definitively linked to any known mutant phenotype. However mutations in a Drosophila adducin homologue, hu-li tai shao (hts), result in a female sterile phenotype in which egg chambers have fewer nurse cells, with abnormal actin distribution in the ring canals. Here we report preliminary results of our attempt to develop C. elegans as a genetic model system for adducin. We made a database search for C. elegans adducin and originally identified two partial cDNAs from Yuji Kohara's EST sequences. The first clone, yk26f2, which we call add-1 (C. elegans Adducin 1), maps to cosmid F39C12 on the left arm of LGX, close to lon-2. The 659 residue predicted protein shows significant homology to vertebrate adducin and hts in the head, neck and tail regions which suggests it may behave like the "canonical" adducins. Interestingly though, yk120b7(add-2) which lies on Cosmid F57F5 on the right arm of LGV, shows a novel structure. The C-terminal 261 residues show homology to the adducin "head" which normally lies at the N-terminus of other adducins. Instead Cad-2 has a novel 350 residue N-terminus that shows no homology in a BLAST search to known proteins. Cad-2 may use its adducin "head" to interact with Cad-1 in a heteromeric manner similar to that demonstrated for vertebrate adducins. But it also raises the exciting prospect that the adducin "head" domain may actually be a component of multiple proteins with different functions, in a manner analogous to that demonstrated for the kinesin and myosin head domains. We aim to resolve these possibilities using C. elegans as a model system.