Chow JC et al. (2010) Cold Spring Harb Perspect Biol "Nuclear organization and dosage compensation."

Heard E, Chow JC

[Cold Spring Harb Perspect Biol, 2010]

Dosage compensation is a strategy to deal with the imbalance of sex chromosomal gene products relative to autosomes and also between the sexes. The mechanisms that ensure dosage compensation for X-chromosome activity have been extensively studied in mammals, worms, and flies. Although each entails very different mechanisms to equalize the dose of X-linked genes between the sexes, they all involve the co-ordinate regulation of hundreds of genes specifically on the sex chromosomes and not the autosomes. In addition to chromatin modifications and changes in higher order chromatin structure, nuclear organization is emerging as an important component of these chromosome-wide processes and in the specific targeting of dosage compensation complexes to the sex chromosomes. Preferential localization within the nucleus and 3D organization are thought to contribute to the differential treatment of two identical homologs within the same nucleus, as well as to the chromosome-wide spread and stable maintenance of heterochromatin.

Chow, Elly S et al. (2011) International Worm Meeting "Cis-regulating network in the ALA neuron."

Van Buskirk, Cheryl, Chow, Elly S, Sternberg, Paul

[International Worm Meeting, 2011]

The ALA neuron is so far the only neuron known to be involved in regulating the EGF-induced sleep-like state in C. elegans. We have previously discovered a network of three homeobox-containing transcriptional regulatory proteins that regulate expression of let-23/EGFR and other ALA-expressed genes. By comparing multiple nematode genomic sequences, we have identified highly conserved regions in the vicinity of coding sequence of two transcription factors (ceh-14 and ceh-17) and three of the functional genes (plc-3, ida-1, ver-3). These elements contain a MEME motif and a cluster of putative homeobox transcription factor binding sites. We analyzed 150 bp genomic elements for their ability to confer cell-type specific gene expression on the pes-10 basal promoter using a reporter gene assay. In each case, the element directed expression in the ALA neuron specifically, except in the case of the plc-3 element, expression is also found in two vulva cells. These elements thus contain ALA-specific regulatory sequences. We believe these elements may serve as probes for finding ALA -expressing genes in the genome. To verify our hypothesis, we continue to identify ALA-specific elements in all the known genes expressed in the ALA. To identify additional ALA-expressed genes, we also seek to profile enriched transcripts in the ALA neuron. We hope to identify crucial cis-regulatory elements and the logic of gene expression regulation in the ALA neuron.

Chow, Elly S. et al. (2013) International Worm Meeting "Deciphering a genetic regulatory network of the ALA neuron."

Van Buskirk, Cheryl, Schwarz, Erich M., Sternberg, Paul W., Chow, Elly S.

[International Worm Meeting, 2013]

The single, unpaired C. elegans ALA neuron responds to EGF-receptor activation and induces sleep-like behavior, including cessation of feeding and locomotion. We previously discovered a regulatory network comprising one LIM homeodomain transcription factor, ceh-14, and two paired-like homeodomain transcription factors, ceh-10 and ceh-17, that co-regulate EGF signaling pathway components, let-23/EGFR and phospholipase C-g, as well as other ALA-expressed genes. We sought to identify crucial cis-regulatory elements and the logic of gene regulation in the ALA neuron. We identified an 11-bp motif (the ALA-motif) from the regulatory regions of eight known ALA-expressing genes. The ALA-motif is necessary and sufficient to drive green fluorescent protein expression in the ALA neuron when attached to a basal promoter in a reporter construct. This motif harbors putative transcription binding sites for LIM homeodomain Lhx3 (an ortholog of ceh-14) and a paired-like homeodomain (like ceh-17 and ceh-10). Genetic analysis showed that the ALA-motif::GFP expression requires ceh-14 and ceh-17. A genome-wide search identified a set of 597 genes that have the ALA-motif in conserved neighboring non-coding DNA sequences, which may include regulatory regions. Of four neuropeptide genes in this set, we found a least one ALA-motif in the intron or in the 5' non-coding region that are sufficient to drive reporter expression in the ALA. Concurrently, we characterized the ALA transcriptomes from wild-type animals and ceh-14 mutants. We detected 8110 genes in the ALA and a set of these genes overlaps with motif-searched genes, which are also ceh-14-dependent. These results demonstrate that cell-specific cis-regulatory motif can identify co-expressed genes and single cell RNA-seq analysis in wild-type and mutant animals can sort out genes that are sharing the same regulatory inputs. Our combined approach may be used to identify the cis-regulatory network of a cell.

Chow YL et al. (2018) Genes Cells "Transgenerational lipid-reducing activity of benzylisoquinoline alkaloids in Caenorhabditis elegans."

Chow YL, Sato F

[Genes Cells, 2018]

Epigenetic mechanisms allow for transgenerational memory of an ancestor's environment and can affect the gene expression, physiology and phenotype of that ancestor's descendants, independent of DNA sequence alteration. Among many model organisms, Caenorhabditis elegans has been instrumental in studies of transgenerational inheritance, most of which have focused on the effects of external stressors of the parent worm on the lifespan and stress resistance of future generations. In this work, we used Nile red staining of accumulated lipids in C. elegans to investigate the transgenerational effect of two benzylisoquinoline alkaloids, namely, berberine and sanguinarine. Our results showed that a reduction in Nile red fluorescence can be propagated to subsequent worm generations. Using mutant worms, we found that the transgenerational effect requires the ASH-2 component of the histone H3K4me3 complex and the HRDE-1 worm Argonaute protein. Ash-2 is also required for transgenerational inheritance of the xenobiotic response in the worm. Our study offers new insights into transmissible drug effects across multiple generations and suggests the importance of such analyses in the drug development process. This article is protected by copyright. All rights reserved.

Chow K-L et al. (1991) Worm Breeder's Gazette "Seeking Trans- and Cis- regulators of Transposon Tc1"

Emmons SW, Chow K-L

[Worm Breeder's Gazette, 1991]

Recent studies of transposon Tc1 have focused on the Tc1 open reading frame product, TcA, and its function in transposition. Since different levels of transposition activity have been detected in different worm strains and tissues, the cellular environment appears to influence the transposon function. Unpublished data from our laboratory have shown that Tc1 transcripts are about 1.3 Kb in length and start 5' to the ORF at multiple sites between 137 and 380 bp ( Radice & Emmons, per. comm.). We propose that transposition activity may correlate with Tc1 transcription activity. In this study, we examined the 5' internal sequence and flanking region of Tc1 tor interaction with cellular factors in order to see whether transcriptional signals and activity can be identified in the transposon or its flanking sequence. We subcloned a 258 bp fragment containing 138 bp of the 5' end of Tc1 element plus 120 bp of flanking DNA from a genomic clone pCE2001 ( see figure), and examined the association of this fragment with cellular factors by gel retardation assays and DNase I . A worm whole cell extract was made from the C. elegans Bergerac strain, which is active tor both Tc1 somatic excision as well as germline transposition and excision. Using from 1 to 20 g of extract protein tor each assay, we were able to detect substantial binding activity to the Tc1 DNA probe in the extract. However, competition with specific and non- specific cold DNA showed that the binding activity was not specific to the Tc1 probe. DNase I footprinting gave a more promising result. Using probes of the same 258 bp fragment labelled at either end and a whole cell extract, we were able to identify a number of protected regions and DNase I hypersensitive sites (see figure). The protected regions were mapped to sequences outside Tc1 in the flanking DNA. Two DNase I hypersensitive sites were detected in the presence or absence of protein binding (+/-), one near the end of the Tc1 element and one inside the element. An additional hypersensitive site within the element (+) was detected only in the presence of protein extract. This additional hypersensitive site strongly argues for specific DNA binding activity in the extract which can cause DNA conformational changes to the Tc1 sequence. Currently, we are trying to further define the binding regions. We are also aware of the DNA binding activity of transposase TcA and have analyzed the specificity of its binding to the Tc1 sequence. We adopted an immunoprecipitation approach using mixed labelled DNA fragments containing plasmid sequences or Tc1 sequences. DNA was incubated with either worm whole cell extract, E. coli extract or extract from E. coli expressing TcA to allow interaction with DNA binding protein. Incubation was followed by addition of purified antibody against TcA (from Ron Plasterk), and then protein A sepharose which interacts with the Ab. A series of washes with buffer eliminated any nonspecific binding of DNA to the protein recognized by the Ab (presumably TcA). Running the remaining bound fragments on an agarose gel revealed the specificity of the binding activity. The result showed that the binding activity recognized by the Ab was present in the worm extract and in the extract of E. coli expressing TcA but absent in the extract of E. coli not expressing TcA. This result indicated that we were examining the binding activity of TcA in E. coli and suggested that the same was true for the worm extract. This would be the first demonstration of the presence of TcA in worms. The activity however was nonspecific. All the DNA fragments, with or without Tc1 sequences, were precipitated. It therefore supports the finding of Ron Plasterk that TcA is a non-specific DNA binding protein. However, this does not eliminate the possibility that TcA, in concert with additional factors, is involved in the regulation of Tc1 transcription. To further investigate the potential transcriptional activity associated with the 5' region of Tc1, we have made a reporter construct linking the -galactosidase gene with a 750 bp Tc1 5' flanking and internal sequence from pCE2001, and will test for transcriptional activity by transformation into the worms. The result will give us some idea whether transcriptional signals are present in this DNA fragment. If so, deletion analysis will allow localization of these signals to the Tc1 or to flanking genomic sequences, and shed light on the possible control of transposon activity. [See Figure 1]

KL Chow (1999) International C. elegans Meeting "Dosage of ram-5 gene is critical for sensory ray morphogenesis in C. elegans"

KL Chow

[International C. elegans Meeting, 1999]

During normal tissue morphogenesis, cell adhesion and repellent molecules have to be expressed in the right level at the right place and right time. Deviation from the normal expression level may not be tolerated for tissue function nor animal vitality. In our study of ram-5 mutation, it was noticed that ram-5 mutant animals rescued by transformation constantly threw progeny with missing ray phenotype. A strong correlation of missing ray phenotype and the rescue activity was established. We speculate that there was a causative relationship between the ram-5 gene dosage and missing ray phenotype and thus employed both genetic and molecular approaches to test this notion. In the genetic analysis, two stable transgenic lines, KC61 and KC62 were used. These stable lines carried truncated and wild type ram-5 genes in a him-5 background. They showed 18% and 100% ray loss respectively. When heterozygous mutant transgenic worms were generated from them to lower the transgene dosage, the percentage of ray loss dropped to 0% and 53% respectively. A positive correlation between ray loss and transgene copy number was established in these lines by Southern analysis. This result argues that high level of ram-5 product might interfere the regular ray assembly process. When truncated ram-5 gene product was expressed in him-5 worms, both swollen ray tip and lumpy ray phenotype were observed. However, when the WT ram-5 gene was injected into the him-5 worm, it did not give these phenotypes. The results suggest that truncated RAM-5 protein can compete with the WT RAM-5 protein for its interacting components to form inactive complexes. By lowering of functional complex level, swollen ray tip or lumpy ray phenotype was resulted. Further over-expression of the truncated protein would lead to severe ray loss. We propose that ram gene products function as a multi-protein complex in a specific stoichiometry. Alteration of the ratio of these proteins will adversely affect the formation of functional complexes. As a result, ray cell assembly cannot take place and rays fail to attach to the cuticle to establish proper morphology.

Chow FW et al. (2019) Nucleic Acids Res "Secretion of an Argonaute protein by a parasitic nematode and the evolution of its siRNA guides."

Koutsovoulos G, Abreu-Goodger C, Ovando-Vazquez C, Neophytou K, Claycomb JM, Laetsch DR, Chow FW, Bermudez-Barrientos JR, Blaxter M, Buck AH, Kumar S, Robertson E

[Nucleic Acids Res, 2019]

Extracellular RNA has been proposed to mediate communication between cells and organisms however relatively little is understood regarding how specific sequences are selected for export. Here, we describe a specific Argonaute protein (exWAGO) that is secreted in extracellular vesicles (EVs) released by the gastrointestinal nematode Heligmosomoides bakeri, at multiple copies per EV. Phylogenetic and gene expression analyses demonstrate exWAGO orthologues are highly conserved and abundantly expressed in related parasites but highly diverged in free-living genus Caenorhabditis. We show that the most abundant small RNAs released from the nematode parasite are not microRNAs as previously thought, but rather secondary small interfering RNAs (siRNAs) that are produced by RNA-dependent RNA Polymerases. The siRNAs that are released in EVs have distinct evolutionary properties compared to those resident in free-living or parasitic nematodes. Immunoprecipitation of exWAGO demonstrates that it specifically associates with siRNAs from transposons and newly evolved repetitive elements that are packaged in EVs and released into the host environment. Together this work demonstrates molecular and evolutionary selectivity in the small RNA sequences that are released in EVs into the host environment and identifies a novel Argonaute protein as the mediator of this.

Chow, Elly S. et al. (2013) International Worm Meeting "Single-cell transcriptomic analysis identifies quiescence-inducing neuropeptides."

Sternberg, Paul W., Schwarz, Erich M., Chow, Elly S.

[International Worm Meeting, 2013]

Neuropeptides are found throughout animal nervous systems and are important in regulating many behaviors and physiological functions. We used the nematode Caenorhabditis elegans to explore the interaction of neuropeptides and a conserved sleep regulator, Epidermal Growth Factor Receptor (EGFR) signaling, in a prolonged and reversible sleep-like state in young adults, when animals are normally active. RNA-seq reveals that 12 neuropeptide-encoding genes in C. elegans are enriched in the ALA neuron. Using conditional activation of neuropeptides and genetic screening, we identified a behavioral quiescence regulatory system consisting of two FMRFamide (Phe-Met-Arg-Phe-NH2)-like neuropeptide encoding genes, flp-13 and flp-24, and a neuropeptide-related receptor, npr-22, orthologous to human neuropeptide Y receptor (24.7% identity) and human opiate receptor-like 1 (19% identity). Overexpression of flp-13 and flp-24 reduces locomotion, feeding, and sensory responsiveness. In particular, hs:FLP-13 animals are inert to tactile and chemosensory stimuli while hs:FLP-24 animals exhibit delayed responses to them; and joint induction of hs:FLP-13 and hs:FLP-24 is stronger than either alone, suggesting functional redundancy. Loss of npr-22 function blocks the effects of neuropeptide-induced quiescence behavior. Our results indicate that nematodes and vertebrates share conserved neural signals that regulate sleep.

Chow YL et al. (2013) Biosci Biotechnol Biochem "Screening of isoquinoline alkaloids for potent lipid metabolism modulation with Caenorhabditis elegans."

Sato F, Chow YL

[Biosci Biotechnol Biochem, 2013]

Metabolic syndrome and related disorders are increasingly prevalent in contemporary society, and thus pose the need for potent agents to control lipid accumulation in the body. This study indicates that Caenorhabditis elegans was effective in screening for potent lipid metabolism modulators with berberine as a model compound. Among the various isoquinoline alkaloids tested, sanguinarine, a benzophenanthridine alkaloid, was found to be the most potent. Sanguinarine, like berberine, reduced lipid accumulation through AMP-activated protein kinase activation. Analysis of AMPK (aak-1 and aak-2) RNAi worms revealed that effects were aak-2-dependent. Characterization of worms with knockdown nhr-49, a hormone nuclear receptor gene that functions as a key regulator of fat consumption, showed that both alkaloids were effective even in these markedly lipid-accumulating nhr-49 RNAi worms, suggesting that they predominantly affect lipid synthesis, rather than fatty acid -oxidation. The versatility of C. elegans for the purpose of lipid-modulating chemical screening and characterization of the underlying mechanisms is discussed.

KL Chow (1999) International C. elegans Meeting "Cloning and characterization of C. elegans ram-5: strucutral organization and expression pattern of the gene"

KL Chow

[International C. elegans Meeting, 1999]

In C. elegans , nine pairs of bilateral sensory rays are formed during the late L4 larval stage. This morphogenesis process involves diverse cellular activities such as the formation of junctions with correct neighbors, cell migration, changes in cell-shape and cell-volume. A number of mutations were isolated to affect this morphogenetic process. The major group of these loci, the ram 's, showed a lumpy ray phenotype when they are mutated. In mutant ram animals, tight cylindrical morphology unique to individual ray is not maintained. Instead, rays are swollen with highly variable morphology. We attempted to characterize the morphogenesis process by cloning these ram genes. ram-5 mutation representing one of them was rescued by cosmid transformation. Three overlapping cosmids, H01A2, H02I07 and T24C2 could restore the wild type phenotype in ram-5 mutants. The result suggested that ram-5 gene was located in the overlapping region. Deletion analyses defined a minimal rescuing fragment of 9.5 Kb. More detailed deletion study suggested that the ram-5 gene was encoded in the minus strand, and the promoter was defined in a 2.5 Kb region on this genomic fragment. The identity of this locus will be further confirmed by genomic mapping of mutant alleles and RNA interference. It is interesting to note that a number of these deletion constructs caused a ray loss phenomenon when they carried high copy of the transgene (see the accompanying abstract by Yu and Chow). While no known cDNA was isolated in the library screened and the EST database, we used mutational strategy, inverse PCR, 5' and 3' RACE to define the transcript(s). Sequence analysis predicts that the encoded protein is a transmembrane glycoprotein with a short domain sharing similarity with cuticulin, a nematode cuticular protein. Promoter reporter constructs in transgenic animals revealed that ram-5 was expressed in ray structural cells in the male tail and a number of neuronal cells in the amphids, and pharyngeal region. These expression patterns suggest that this gene may have additional neuronal functions in worm.