Kato Y et al. (2002) J Biol Chem "Determinants of ligand specificity in groups I and IV WW domains as studied by surface plasmon resonance and model building."

Kato Y, Ito M, Tanokura M, Kawai K, Nagata K

[J Biol Chem, 2002]

WW domains are universal protein modules for binding Pro-rich ligands. They are classified into four groups according to their binding specificity. Arg-14 and Arg-17, on the WW domain of Pin1, are thought to be important for the binding of Group IV ligands that have (Ser(P)/Thr(P))-Pro sequences. We have applied surface plasmon resonance to determine the ligand specificity of several WW domains containing Arg-14. Among these WW domains, Rsp5.2 and mNedd4.3 bound only to the Group I ligand containing Pro-Pro-Xaa-Tyr with K(D) values of 11 and 55 microm, respectively. The WW domains of hPin1, Caenorhabditis elegans Pin1 homologue (Y110), PinA, and SspI bound to Group IV ligands with K(D) values ranging from 22 to 700 microm. PinA and SspI do not have Arg-17, unlike Pin1 and Y110. The modeled structures of the WW domains of PinA and SspI revealed that the structure and the network of hydrogen bonds of Loop I, which are also formed in Pin1 and Y110, are conserved. We propose that this configuration of Loop I (referred to as the "p patch") is necessary for binding Group IV ligands and that it can be used to predict the specificity and functions of other WW domains.

Wright GM et al. (2019) Nucleic Acids Res "The nucleosome position-encoding WW/SS sequence pattern is depleted in mammalian genes relative to other eukaryotes."

Cui F, Wright GM

[Nucleic Acids Res, 2019]

Nucleosomal DNA sequences generally follow a well-known pattern with 10-bp periodic WW (where W is A or T) dinucleotides that oscillate in phase with each other and out of phase with SS (where S is G or C) dinucleotides. However, nucleosomes with other DNA patterns have not been systematically analyzed. Here, we focus on an opposite pattern, namely anti-WW/SS pattern, in which WW dinucleotides preferentially occur at DNA sites that bend into major grooves and SS (where S is G or C) dinucleotides are often found at sites that bend into minor grooves. Nucleosomes with the anti-WW/SS pattern are widespread and exhibit a species- and context-specific distribution in eukaryotic genomes. Unlike non-mammals (yeast, nematode and fly), there is a positive correlation between the enrichment of anti-WW/SS nucleosomes and RNA Pol II transcriptional levels in mammals (mouse and human). Interestingly, such enrichment is not due to underlying DNA sequence. In addition, chromatin remodeling complexes have an impact on the abundance but not on the distribution of anti-WW/SS nucleosomes in yeast. Our data reveal distinct roles of cis- and trans-acting factors in the rotational positioning of nucleosomes between non-mammals and mammals. Implications of the anti-WW/SS sequence pattern for RNA Pol II transcription are discussed.

Serizay J et al. (2020) Genome Res "Distinctive regulatory architectures of germline-active and somatic genes in C. elegans."

Dong Y, Serizay J, Chesney M, Cerrato C, Ahringer J, Janes J

[Genome Res, 2020]

RNA profiling has provided increasingly detailed knowledge of gene expression patterns, yet the different regulatory architectures that drive them are not well understood. To address this, we profiled and compared transcriptional and regulatory element activities across five tissues of <i>C. elegans</i>, covering ~90% of cells. We find that the majority of promoters and enhancers have tissue-specific accessibility, and we discover regulatory grammars associated with ubiquitous, germline and somatic tissue-specific gene expression patterns. In addition, we find that germline-active and soma-specific promoters have distinct features. Germline-active promoters have well positioned +1 and -1 nucleosomes associated with a periodic 10-bp WW signal (W = A/T). Somatic tissue-specific promoters lack positioned nucleosomes and this signal, have wide nucleosome depleted regions, and are more enriched for core promoter elements, which differ between tissues. We observe the 10-bp periodic WW signal at ubiquitous promoters in other animals suggesting it is an ancient conserved signal. Our results demonstrate fundamental differences in regulatory architectures of germline and somatic tissue-specific genes, uncover regulatory rules for generating diverse gene expression patterns, and provide a tissue-specific resource for future studies.

Courbard JR et al. (2002) J Biol Chem "Interaction between two ubiquitin-protein isopeptide ligases of different classes, CBLC and AIP4/ITCH."

Birnbaum D, Fiore F, Borg JP, Ollendorff V, Courbard JR, Adelaide J

[J Biol Chem, 2002]

In metazoans, CBL proteins are RING finger type ubiquitin-protein isopeptide (E3) ligases involved in the down-regulation of epidermal growth factor tyrosine kinase receptors (EGFR). Among the three CBL proteins described in humans, CBLC (CBL3) remains poorly studied. By screening in parallel a human and a Caenorhabditis elegans library using the two-hybrid procedure in yeast, we found a novel interaction between Hsa-CBLC and Hsa-AIP4 or its C. elegans counterpart Cel-WWP1. Hsa-AIP4 and Cel-WWP1 are also ubiquitin E3 ligases. They contain a HECT (homologous to E6-AP C terminus) catalytic domain and four WW domains known to bind proline-rich regions. We confirmed the interaction between Hsa-CBLC and Hsa-AIP4 by a combination of glutathione S-transferase pull-down, co-immunoprecipitation, and colocalization experiments. We show that these two E3 ligases are involved in EGFR signaling because both become phosphorylated on tyrosine following epidermal growth factor stimulation. In addition, we observed that CBLC increases the ubiquitination of EGFR, and that coexpressing the WW domains of AIP4 exerts a dominant negative effect on EGFR ubiquitination. Finally, coexpressing CBLC and AIP4 induces a down-regulation of EGFR signaling. In conclusion, our data demonstrate that two E3 ligases of different classes can interact and cooperate to down-regulate EGFR signaling.

Nakajima R et al. (2021) Mol Brain "Similarities of developmental gene expression changes in the brain between human and experimental animals: rhesus monkey, mouse, Zebrafish, and Drosophila."

Hagihara H, Nakajima R, Miyakawa T

[Mol Brain, 2021]

Aim: Experimental animals, such as non-human primates (NHPs), mice, Zebrafish, and Drosophila, are frequently employed as models to gain insights into human physiology and pathology. In developmental neuroscience and related research fields, information about the similarities of developmental gene expression patterns between animal models and humans is vital to choose what animal models to employ. Here, we aimed to statistically compare the similarities of developmental changes of gene expression patterns in the brains of humans with those of animal models frequently used in the neuroscience field.Methods: The developmental gene expression datasets that we analyzed consist of the fold-changes and P values of gene expression in the brains of animals of various ages compared with those of the youngest postnatal animals available in the dataset. By employing the running Fisher algorithm in a bioinformatics platform, BaseSpace, we assessed similarities between the developmental changes of gene expression patterns in the human (Homo sapiens) hippocampus with those in the dentate gyrus (DG) of the rhesus monkey (Macaca mulatta), the DG of the mouse (Mus musculus), the whole brain of Zebrafish (Danio rerio), and the whole brain of Drosophila (D. melanogaster).Results: Among all possible comparisons of different ages and animals in developmental changes in gene expression patterns within the datasets, those between rhesus monkeys and mice were highly similar to those of humans with significant overlap P-value as assessed by the running Fisher algorithm. There was the highest degree of gene expression similarity between 40-59-year-old humans and 6-12-year-old rhesus monkeys (overlap P-value = 2.1 10- 72). The gene expression similarity between 20-39-year-old humans and 29-day-old mice was also significant (overlap P = 1.1 10- 44). Moreover, there was a similarity in developmental changes of gene expression patterns between 1-2-year-old Zebrafish and 40-59-year-old humans (Overlap P-value = 1.4 10- 6). The overlap P-value of developmental gene expression patterns between Drosophila and humans failed to reach significance (30 days Drosophila and 6-11-year-old humans; overlap P-value = 0.0614).Conclusions: These results indicate that the developmental gene expression changes in the brains of the rhesus monkey, mouse, and Zebrafish recapitulate, to a certain degree, those in humans. Our findings support the idea that these animal models are a valid tool for investigating the development of the brain in neurophysiological and neuropsychiatric studies.

Hurd DD (2008) CBE Life Sci Educ "A microcosm of the biomedical research experience for upper-level undergraduates."

Hurd DD

[CBE Life Sci Educ, 2008]

The skill set required of biomedical researchers continues to grow and evolve as biology matures as a natural science. Science necessitates creative yet critical thinking, persuasive communication skills, purposeful use of time, and adeptness at the laboratory bench. Teaching these skills can be effectively accomplished in an inquiry-based, active-learning environment at a primarily undergraduate institution. Cell Biology Techniques, an upper-level cell biology laboratory course at St. John Fisher College, features two independent projects that take advantage of the biology of the nematode Caenorhabditis elegans, a premier yet simple model organism. First, students perform a miniature epigenetic screen for novel phenotypes using RNA interference. The results of this screen combined with literature research direct students toward a singe gene that they attempt to subclone in the second project. The biology of the chosen gene/protein also becomes an individualized focal point with respect to the content of the laboratory. Progress toward course goals is evaluated using written, oral, and group-produced assignments, including a concept map. Pre- and postassessment indicates a significant increase in the understanding of broad concepts in cell biological research.

Ollmann M et al. (2000) Cell "Drosophila p53 is a structural and functional homolog of the tumor suppressor p53."

Whittaker K, Buchman A, Prives C, Young LM, Di Como CJ, Fisher WW, Belvin M, Friedman L, Robertson S, Kopczynski C, Duyk G, Karim F, Demsky M, Ollmann M

[Cell, 2000]

The importance of p53 in carcinogenesis stems from its central role in inducing cell cycle arrest or apoptosis in response to cellular stresses. We have identified a Drosophila homolog of p53 ("Dmp53"). Like mammalian p53, Dmp53 binds specifically to human p53 binding sites, and overexpression of Dmp53 induces apoptosis. Importantly, inhibition of Dmp53 function renders cells resistant to X ray-induced apoptosis, suggesting that Dmp53 is required for the apoptotic response to DNA damage. Unlike mammalian p53, Dmp53 appears unable to induce a G1 cell cycle block when overexpressed, and inhibition of Dmp53 activity does not affect X ray-induced cell cycle arrest. These data reveal an ancestral proapoptotic function for p53 and identify Drosophila as an ideal model system for elucidating the p53 apoptotic pathway(s) induced by DNA damage.

Huang K et al. (2000) Gene "A HECT domain ubiquitin ligase closely related to the mammalian protein WWP1 is essential for Caenorhabditis elegans embryogenesis."

Mosser EA, Bresnick EH, Petcherski AG, Johnson KD, Kimble JE, Vandergon T, Huang K, Jenkins NA, Copeland NG

[Gene, 2000]

The highly conserved ubiquitin/proteasome pathway controls the degradation of many critical regulatory proteins. Proteins are posttranslationally conjugated to ubiquitin through a concerted set of reactions involving activating (E1), conjugating (E2), and ligase (E3) enzymes. Ubiquitination targets proteins for proteolysis via the proteasome and may regulate protein function independent of proteolysis. We describe the cloning and functional analysis of new members of the HECT domain family of E3 ubiquitin ligases. Murine Wwp1 encoded a broadly expressed protein containing a C2 domain, four WW domains, and a catalytic HECT domain. A Caenorhabditis elegans gene was cloned encoding a HECT domain protein (CeWWP1), which was highly homologous to murine and human WWP1. Disruption of CeWwp1 via RNA interference yielded an embryonic lethal phenotype, despite the presence of at least six additional C. elegans genes encoding HECT domain proteins. The embryonic lethality was characterized by grossly abnormal morphogenesis during late embryogenesis, despite normal proliferation early in embryogenesis. CeWWP1 must therefore have unique and nonredundant functions critical for embryogenesis.

Preston S et al. (2017) FASEB J "Deguelin exerts potent nematocidal activity via the mitochondrial respiratory chain."

Sternberg PW, Ansell BRE, Andrews KT, Nowell C, Chang BCH, Hofmann A, Crawford S, Korhonen PK, Baell J, Gijs MAM, Fisher GM, Young ND, Preston S, Mouchiroud L, Gasser RB, Jabbar A, Auwerx J, Davis RA, McGee SL, Cornaglia M

[FASEB J, 2017]

As a result of limited classes of anthelmintics and an over-reliance on chemical control, there is a great need to discover new compounds to combat drug resistance in parasitic nematodes. Here, we show that deguelin, a plant-derived rotenoid, selectively and potently inhibits the motility and development of nematodes, which supports its potential as a lead candidate for drug development. Furthermore, we demonstrate that deguelin treatment significantly increases gene transcription that is associated with energy metabolism, particularly oxidative phosphorylation and mito-ribosomal protein production before inhibiting motility. Mitochondrial tracking confirmed enhanced oxidative phosphorylation. In accordance, real-time measurements of oxidative phosphorylation in response to deguelin treatment demonstrated an immediate decrease in oxygen consumption in both parasitic (Haemonchus contortus) and free-living (Caenorhabditis elegans) nematodes. Consequently, we hypothesize that deguelin is exerting its toxic effect on nematodes as a modulator of oxidative phosphorylation. This study highlights the dynamic biologic response of multicellular organisms to deguelin perturbation.-Preston, S., Korhonen, P. K., Mouchiroud, L., Cornaglia, M., McGee, S. L., Young, N. D., Davis, R. A., Crawford, S., Nowell, C., Ansell, B. R. E., Fisher, G. M., Andrews, K. T., Chang, B. C. H., Gijs, M. A. M., Sternberg, P. W., Auwerx, J., Baell, J., Hofmann, A., Jabbar, A., Gasser, R. B. Deguelin exerts potent nematocidal activity via the mitochondrial respiratory chain.

Tabish M et al. (1999) Biochem J "Organization and alternative splicing of the Caenorhabditis elegans cAMP-dependent protein kinase catalytic-subunit gene (kin-1)."

Tabish M, Clegg RA, Rees HH, Fisher MJ

[Biochem J, 1999]

The cAMP-dependent protein kinase (protein kinase A, PK-A) is multifunctional in nature, with key roles in the control of diverse aspects of eukaryotic cellular activity. In the case of the free-living nematode, Caenorhabditis elegans, a gene encoding the PK-A catalytic subunit has been identified and two isoforms of this subunit, arising from a C-terminal alternative-splicing event, have been characterized [Gross, Bagchi, Lu and Rubin (1990) J. Biol. Chem. 265, 6896-6907]. Here we report the occurrence of N-terminal alternative-splicing events that, in addition to generating a multiplicity of non-myristoylatable isoforms, also generate the myristoylated variant(s) of the catalytic subunit that we have recently characterized [Aspbury, Fisher, Rees and Clegg (1997) Biochem. Biophys. Res. Commun. 238, 523-527]. The gene spans more than 36 kb and is divided into a total of 13 exons. Each of the mature transcripts contains only 7 exons. In addition to the already characterized exon 1, the 5'-untranslated region and first intron actually contain 5 other exons, any one of which may be alternatively spliced on to exon 2 at the 5' end of the pre-mRNA. This N-terminal alternative splicing occurs in combination with either of the already characterized C-terminal alternative exons. Thus, C. elegans expresses at least 12 different isoforms of the catalytic subunit of PK-A. The significance of this unprecedented structural diversity in the family of PK-A catalytic subunits is discussed.