Werner, Michael et al. (2019) International Worm Meeting "The epigenetics of nematode mouth form plasticity."

Loschko, Tobias, Werner, Michael, Sommer, Ralf

[International Worm Meeting, 2019]

Early life experiences can affect the development of physiology, behavior, and morphological structures. Post-translational modifications and small RNA-mediated gene silencing are capable of carrying environmental information, yet a holistic understanding of environmental influence remains elusive. We have developed the mouth-form plasticity of the nematode Pristionchus pacificus as a model for investigating the epigenetic mechanisms, temporal boundaries, and ecological consequences of environmental influence. Depending on the conditions experienced as juveniles, an irreversible decision is made in P. pacificus adults to develop either a narrow-mouth bacterial feeding morph, or a wide-mouthed predatory morph with an extra denticle. Here, we combine epigenomic profiling, mass spectrometry, and developmental transcriptomes to identify regulatory elements, post-translational modifications, and genes specific for each mouth form pathway. Transcription of the "switch" gene eud-1 is highly responsive to growth conditions, and CRISPR knockout experiments demonstrate its induction or repression is required for the environment-response phenotype. After the switch decision, we also identify regulatory cascades for hundreds of genes specific to each mouth form, including epigenetic enzymes and genes involved in foraging in the predatory pathway. We also identified the transition between plasticity and canalization using reciprocal transplant experiments, which coincides precisely with the onset of H3K27me3 repression at a multi-gene locus containing eud-1. Finally, we functionally interrogated epigenetic modifications with small molecule inhibitors. The chemotherapeutic drug Trichostatin A (TSA), a histone deacetylase inhibitor, partially coerces development to the predatory morph in conditions that otherwise favor the bacterivorous morph. However, adding the inhibitor after the switch decision fixes the predatory phenotype. These results suggest that histone acetylation acts in the initial environmental response, but is also capable of providing long-term epigenetic memory of juvenile experience. Collectively, our results describe the hills, valleys, and molecular mechanisms of the epigenetic landscape of an organismal phenotype.

Hsu TY et al. (2021) Cells "MUT-7 Provides Molecular Insight into the Werner Syndrome Exonuclease."

Hsu LN, Juang BT, Hsu TY, Chen SY

[Cells, 2021]

Werner syndrome (WS) is a rare recessive genetic disease characterized by premature aging. Individuals with this disorder develop normally during childhood, but their physiological conditions exacerbate the aging process in late adolescence. WS is caused by mutation of the human WS gene (<i>WRN</i>), which encodes two main domains, a 3'-5' exonuclease and a 3'-5' helicase. <i>Caenorhabditis elegans</i> expresses human WRN orthologs as two different proteins: MUT-7, which has a 3'-5' exonuclease domain, and <i>C</i><i>. elegans</i> WRN-1 (CeWRN-1), which has only helicase domains. These unique proteins dynamically regulate olfactory memory in <i>C. elegans</i>, providing insight into the molecular roles of WRN domains in humans. In this review, we specifically focus on characterizing the function of MUT-7 in small interfering RNA (siRNA) synthesis in the cytoplasm and the roles of siRNA in directing nuclear CeWRN-1 loading onto a heterochromatin complex to induce negative feedback regulation. Further studies on the different contributions of the 3'-5' exonuclease and helicase domains in the molecular mechanism will provide clues to the accelerated aging processes in WS.

Michael Werner et al. (2006) Development & Evolution Meeting "Regulation Of Cytokinesis By The Mitotic Spindle In The C.elegans Embryo"

Michael Werner, Michael Glotzer, Ed Munro

[Development & Evolution Meeting, 2006]

The anaphase spindle and asters coordinate actin polymerization and myosin activity so that the cleavage furrow generates two genetically identical daughter cells. Our earlier studies established that two redundant pathways regulate cleavage furrow formation (Dechant and Glotzer 2001). To gain insight into the molecular mechanisms underlying these two pathways, we have followed the dynamics of non-muscle myosin (NMY-2) fused to GFP in wild-type and mutant embryos. Prior to first metaphase, NMY-2 is asymmetrically enriched at the anterior cortex (Munro et al, 2004). We observe that during metaphase, NMY-2 dissociates from the cortex and quickly returns, accumulating in a broad cortical region in the anterior half of the embryo and concentrating at the site of furrow ingression. Recruitment of NMY-2 to the cortex during anaphase requires active RhoA. To determine how the position of the mitotic spindle affects myosin recruitment and furrow formation we destabilized microtubules pharmacologically or by RNAi to generate a small mitotic spindle that is positioned in the posterior and is perpendicular to the A-P axis. During anaphase, NMY-2 accumulates predominantly in the anterior half of the cortex. This accumulation is accompanied by a flow of cortical myosin away from the spindle, towards the anterior where an anterior furrow forms. A second furrow bisects the small spindle at the posterior cortex. These furrows are genetically separable. ZEN-4, a factor required for central spindle assembly, is required for the posterior, but not the anterior furrow. In contrast, the anterior furrow is more sensitive to depletion of regulators of the actomyosin network. These data suggest that mechanistically distinct pathways act simultaneously to position the cleavage furrow. One pathway involves inhibition of cortical myosin recruitment near, and flow away from, regions rich in microtubules. Formation of the anterior furrow requires that the cortical myosin meshwork is organized and non-uniformly distributed, the anterior flow contributes to this non uniform distribution. These data provide direct evidence for astral relaxation. In contrast, the second mechanism involves a local enrichment/activation of NMY-2 close to and dependent on the central spindle, irrespective of the overall pattern of cortical contractility.

Bruskiewich R et al. (1997) International C. elegans Meeting "FUNCTIONAL ANALYSIS OF A WERNER SYNDROME GENE HOMOLOG IN C.ELEGANS"

Wood S, Rose AM, Bruskiewich R

[International C. elegans Meeting, 1997]

Werner syndrome is a human autosomal recessive disorder with a phenotype of apparent premature senescence. The gene locus (WRN) corresponding to the genetic lesion causing this disorder was mapped to the short arm of human chromosome 8 and subsequently positionally cloned last year. Sequence analysis of the gene product suggests that WRN may be a member of the E.coli RecQ DExH DNA helicase family involved in recombination processes in mitotic and meiotic cells; however, this functional role of WRN remains to be confirmed. To this end, we are studying F18C5.2, one of the proposed nematode gene homologs of WRN, in an effort to elucidate the functional nature of this DNA helicase family. Our primary experimental approach at present is to attempt rescue of candidate mutant phenotypes in the vicinity of F18C5.2, by crossing mutant strains against transgenic worms containing cosmid DNA from the region. We are also seeking to confirm whether F18C5.2 lies within an operon and thus, locate the promoter for this gene, as a preliminary step towards the construction of an expression vector system to study gene expression patterns.

Werner, Kristen et al. (2013) International Worm Meeting "Small Molecule Communication: C. elegans and bacterial chemical signals."

Perez, Lark, Werner, Kristen, Semmelhack, Martin, Bassler, Bonnie

[International Worm Meeting, 2013]

Bacterial group behaviors are governed by a process called quorum sensing, in which bacteria produce, secrete, and detect extracellular signal molecules called autoinducers (AIs). Vibrios produce multiple AIs, some enable intra-species communication and others that promote inter-species communication. Vibrio cholerae produces an intra-species AI called CAI-1 that is a 13 carbon long fatty acyl molecule and the interspecies signal called AI-2 that is a boron-containing furanone. The information contained in the AIs is funneled into a shared phosphorelay signaling cascade that controls virulence, biofilm formation, and other traits. The bacteriovorous nematode, Caenorhabditis elegans, also uses small molecules to interpret its environment. A class of C. elegans-derived molecules called ascarosides influence nematode behaviors including attraction, repulsion, and mating. The presence of bacteria stimulates chemotaxis, egg-laying, and feeding in C. elegans, however, the bacteria-produced molecules that the nematode detects to control these phenotypes are largely unknown. We demonstrate that in addition to playing a vital role in quorum-sensing-regulated behaviors in V. cholerae, CAI-1 also influences behavior in C. elegans. C. elegans is more strongly attracted to V. cholerae than to its food source E. coli HB101 and C. elegans prefers V. cholerae that produces CAI-1 over a V. cholerae mutant for CAI-1 production. Consistent with this finding, robust chemoattraction occurs to synthetic CAI-1. CAI-1 is detected by the sensory neuron AWCON. Laser ablation of this cell, but not other amphid sensory neurons, abolished chemoattraction to CAI-1. To define which moieties of CAI-1 are crucial for recognition by C. elegans, we synthesized CAI-1 analogs and tested whether they promote chemoattraction. The fatty-acid chain length as and the precise position of the CAI-1 ketone group are the key features required for mediating CAI-1-directed nematode behavior. Together, these analyses define a bacteria-produced signal and the nematode detection apparatus that permit interkingdom communication.

Lautrup S et al. (2019) Biogerontology "Studying Werner syndrome to elucidate mechanisms and therapeutics of human aging and age-related diseases."

Lautrup S, Stevnsner T, Fang EF, Caponio D, Piccoli C, Chan WY, Cheung HH

[Biogerontology, 2019]

Aging is a natural and unavoidable part of life. However, aging is also the primary driver of the dominant human diseases, such as cardiovascular disease, cancer, and neurodegenerative diseases, including Alzheimer's disease. Unraveling the sophisticated molecular mechanisms of the human aging process may provide novel strategies to extend 'healthy aging' and the cure of human aging-related diseases. Werner syndrome (WS), is a heritable human premature aging disease caused by mutations in the gene encoding the Werner (WRN) DNA helicase. As a classical premature aging disease, etiological exploration of WS can shed light on the mechanisms of normal human aging and facilitate the development of interventional strategies to improve healthspan. Here, we summarize the latest progress of the molecular understandings of WRN protein, highlight the advantages of using different WS model systems, including Caenorhabditis elegans, Drosophila melanogaster and induced pluripotent stem cell (iPSC) systems. Further studies on WS will propel drug development for WS patients, and possibly also for normal age-related diseases.

Sawada D et al. (2023) Aging (Albany NY) "Senescence-associated inflammation and inhibition of adipogenesis in subcutaneous fat in Werner syndrome."

Yamaguchi A, Ide K, Kubota Y, Fujii K, Takasaki A, Nakamura R, Hayashi A, Takayama N, Kitamoto T, Ogata H, Takada-Watanabe A, Ohno T, Iwama A, Kinoshita D, Koshizaka M, Yokote K, Funayama S, Teramoto N, Kaneko H, Eto K, Aono K, Mitsukawa N, Ide S, Endo Y, Sawada D, Shiohama T, Ouchi Y, Maeda Y, Maezawa Y, Takatani T, Kato H, Igarashi K, Minamizuka T, Hamada H, Shoji M

[Aging (Albany NY), 2023]

Werner syndrome (WS) is a hereditary premature aging disorder characterized by visceral fat accumulation and subcutaneous lipoatrophy, resulting in severe insulin resistance. However, its underlying mechanism remains unclear. In this study, we show that senescence-associated inflammation and suppressed adipogenesis play a role in subcutaneous adipose tissue reduction and dysfunction in WS. Clinical data from four Japanese patients with WS revealed significant associations between the decrease of areas of subcutaneous fat and increased insulin resistance measured by the glucose clamp. Adipose-derived stem cells from the stromal vascular fraction derived from WS subcutaneous adipose tissues (WSVF) showed early replicative senescence and a significant increase in the expression of senescence-associated secretory phenotype (SASP) markers. Additionally, adipogenesis and insulin signaling were suppressed in WSVF, and the expression of adipogenesis suppressor genes and SASP-related genes was increased. Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), alleviated premature cellular senescence, rescued the decrease in insulin signaling, and extended the lifespan of WS model of <i>C. elegans</i>. To the best of our knowledge, this study is the first to reveal the critical role of cellular senescence in subcutaneous lipoatrophy and severe insulin resistance in WS, highlighting the therapeutic potential of rapamycin for this disease.

Werner ME et al. (2024) Curr Biol "Mechanical and biochemical feedback combine to generate complex contractile oscillations in cytokinesis."

Ray DD, Jug F, Staddon MF, Breen C, Banerjee S, Werner ME, Maddox AS

[Curr Biol, 2024]

The actomyosin cortex is an active material that generates force to drive shape changes via cytoskeletal remodeling. Cytokinesis is the essential cell division event during which a cortical actomyosin ring closes to separate two daughter cells. Our active gel theory predicted that actomyosin systems controlled by a biochemical oscillator and experiencing mechanical strain would exhibit complex spatiotemporal behavior. To test whether active materials in&#xa0;vivo exhibit spatiotemporally complex kinetics, we imaged the C.&#xa0;elegans embryo with unprecedented temporal resolution and discovered that sections of the cytokinetic cortex undergo periodic phases of acceleration and deceleration. Contractile oscillations exhibited a range of periodicities, including those much longer periods than the timescale of RhoA pulses, which was shorter in cytokinesis than in any other biological context. Modifying mechanical feedback in&#xa0;vivo or in silico revealed that the period of contractile oscillation is prolonged as a function of the intensity of mechanical feedback. Fast local ring ingression occurs where speed oscillations have long periods, likely due to increased local stresses and, therefore, mechanical feedback. Fast ingression also occurs where material turnover is high, in&#xa0;vivo and in silico. We propose that downstream of initiation by pulsed RhoA activity, mechanical feedback, including but not limited to material advection, extends the timescale of contractility beyond that of biochemical input and, therefore, makes it robust to fluctuations in activation. Circumferential propagation of contractility likely allows for sustained contractility despite cytoskeletal remodeling necessary to recover from compaction. Thus, like biochemical feedback, mechanical feedback affords active materials responsiveness and robustness.

Hsu TY et al. (2021) Elife "C. elegans orthologs MUT-7/CeWRN-1 of Werner syndrome protein regulate neuronal plasticity."

L'Etoile ND, Zhang B, Juang BT, Hsu TY

[Elife, 2021]

<i>Caenorhabditis elegans</i> expresses human Werner syndrome protein (WRN) orthologs as two distinct proteins: MUT-7, with a 3'-5' exonuclease domain, and CeWRN-1, with helicase domains. How these domains cooperate remains unclear. Here, we demonstrate the different contributions of MUT-7 and CeWRN-1 to 22G small interfering RNA (siRNA) synthesis and the plasticity of neuronal signaling. MUT-7 acts specifically in the cytoplasm to promote siRNA biogenesis and in the nucleus to associate with CeWRN-1. The import of siRNA by the nuclear Argonaute NRDE-3 promotes the loading of the heterochromatin-binding protein HP1 homolog HPL-2 onto specific loci. This heterochromatin complex represses the gene expression of the guanylyl cyclase ODR-1 to direct olfactory plasticity in <i>C. elegans</i>. Our findings suggest that the exonuclease and helicase domains of human WRN may act in concert to promote RNA-dependent loading into a heterochromatin complex, and the failure of this entire process reduces plasticity in postmitotic neurons.

Werner C et al. (1992) Mol Biochem Parasitol "Characterization of a myosin heavy chain gene from Brugia malayi."

Rajan TV, Werner C

[Mol Biochem Parasitol, 1992]

We have previously shown that an antigen recognized by antibodies in sera of several microfilaremic individuals from a Wuchereria bancrofti endemic area bears strong homology to an invertebrate muscle protein. We have cloned and sequenced the entire gene containing this antigen encoding fragment and present data that confirms that the antigen is myosin heavy chain (MHC). This gene, which we have named Bmmyo-1 extends over 11 kb and has the potential to encode a protein of 1957 amino acids. The coding sequence is interrupted by 14 introns, most of which are larger than those in the myosin gene of the free-living nematode, Caenorhabditis elegans. The protein encoded by this gene bears greatest homology (75.1% identity) to the C. elegans myosin isoform MHC-B, encoded by the unc-54 gene. MHC-B is the major body wall myosin in C. elegans.