Greiss S et al. (2011) J Am Chem Soc "Expanding the genetic code of an animal."

Chin JW, Greiss S

[J Am Chem Soc, 2011]

Genetic code expansion, for the site-specific incorporation of unnatural amino acids into proteins, is currently limited to cultured cells and unicellular organisms. Here we expand the genetic code of a multicellular animal, the nematode Caenorhabditis elegans.

Greiss S et al. (2008) BMC Genomics "Transcriptional profiling in C. elegans suggests DNA damage dependent apoptosis as an ancient function of the p53 family."

Rothblatt J, Schumacher B, Gartner A, Grandien K, Greiss S

[BMC Genomics, 2008]

ABSTRACT: BACKGROUND: In contrast to the three mammalian p53 family members, p53, which is generally involved in DNA damage responses, and p63 and p73 which are primarily needed for developmental regulation, cep-1 encodes for the single C. elegans p53-like gene. cep-1 acts as a transcription activator in a primordial p53 pathway that involves CEP-1 activation and the CEP-1 dependent transcriptional induction of the worm BH3 only domain encoding genes egl-1 and ced-13 to induce germ cell apoptosis. EGL-1 and CED-13 proteins inactivate Bcl-2 like CED-9 to trigger CED-4 and CED-3 caspase dependent germ cell apoptosis. To address the function of p53 in global transcriptional regulation we investigate genome-wide transcriptional responses upon DNA damage and cep-1 deficiency. RESULTS: Examining C. elegans expression profiles using whole genome Affymetrix GeneChip arrays, we found that 83 genes were induced more than two fold upon ionizing radiation (IR). None of these genes, with exception of an ATP ribosylase homolog, encode for known DNA repair genes. Using two independent cep-1 loss if function alleles we did not find genes regulated by cep-1 in the absence of IR. Among the IR-induced genes only three are dependent on cep-1, namely egl-1, ced-13 and a novel C. elegans specific gene. The majority of IR-induced genes appear to be involved in general stress responses, and qRT-PCR experiments indicate that they are mainly expressed in somatic tissues. Interestingly, we reveal an extensive overlap of gene expression changes occurring in response to DNA damage and in response to bacterial infection. Furthermore, many genes induced by IR are also transcriptionally regulated in longevity mutants suggesting that DNA damage and aging induce an overlapping stress response. CONCLUSIONS: We performed genome-wide gene expression analyses which indicate that only a surprisingly small number of genes are regulated by CEP-1 and that DNA damage induced apoptosis via the transcriptional induction of BH3 domain proteins is likely to be an ancient DNA damage response function of the p53 family. Interestingly, although the apoptotic response to DNA damage is regulated through transcriptional activity of CEP-1, other DNA damage responses do not appear to be regulated on the transcriptional level and do not require the p53 like gene cep-1.

Greiss S et al. (2008) Genes Dev "C. elegans SIR-2.1 translocation is linked to a proapoptotic pathway parallel to cep-1/p53 during DNA damage-induced apoptosis."

Gartner A, Ahmed S, Hall J, Greiss S

[Genes Dev, 2008]

Caenorhabditis elegans SIR-2.1, a member of the sirtuin family related to Saccharomyces cerevisiae Sir2p, has previously been implicated in aging. The mammalian homolog SIRT1 plays important roles in multiple cellular processes including transcriptional repression and stress response. We show that sir-2.1 is essential for the execution of apoptosis in response to DNA damage, and that sir-2.1 genetically acts in parallel to the worm p53-like gene cep-1. This novel cep-1-independent proapoptotic pathway does not require the daf-16 FOXO transcription factor. Cytological analysis of SIR-2.1 suggests a novel mechanism of apoptosis induction. During apoptosis SIR-2.1 changes its subcellular localization from the nucleus to the cytoplasm and transiently colocalizes with the C. elegans Apaf-1 homolog CED-4 at the nuclear periphery. SIR-2.1 translocation is an early event in germ cell apoptosis and is independent of apoptosis execution and cep-1, raising the possibility that SIR-2.1 translocation is linked to the induction of DNA damage-induced apoptosis.

Davis L et al. (2018) Methods Mol Biol "Genetic Encoding of Unnatural Amino Acids in C. elegans."

Davis L, Greiss S

[Methods Mol Biol, 2018]

Site-specific incorporation of unnatural amino acids (UAAs) has greatly expanded the toolkit available to study biological phenomena in single cells. However, to address questions involving complex cellular interactions such as development, ageing, and the functions of the nervous system it is often necessary to use multicellular model organisms. The nematode Caenorhabditis elegans was the first organism to have its genetic code expanded. Due to its small size, ease of cultivation, and excellent UAA incorporation efficiency, C. elegans makes an ideal model organism to apply UAAs as tools to investigate the functioning of multicellular systems.Here, we describe methods to generate transgenic C. elegans capable of UAA incorporation, as well as how to deliver unnatural amino acids and test incorporation. Furthermore, we describe methods to uncage photosensitive unnatural amino acid derivatives.

Xi, Z. et al. (2019) International Worm Meeting "Light-inducible caspase as high-precision cell ablation tool in C. elegans."

Baxter, K., Xi, Z., Greiss, S., Davis, L.

[International Worm Meeting, 2019]

As the fundamental units of the physiological activities, C. elegans cells generally function through sophisticated intercellular collaborations. Ablation is one important strategy of studying this functional organization. By comparing the experimental outputs of intact worms and worms that have lost specific cell(s), researchers are able to figure out the overall role of the target cell(s). Among the many methods of targeted cell killing, laser ablation is the earliest and the most used. Reactive oxygen species generating proteins stepped into the ablation practice in the last decade. However, none of them simultaneously guarantee high spatial resolution and temporal control in the removal procedure. Also, the accompanying necrosis could interfere with subsequent physiology assays. Hence we designed and made a novel ablation tool via light trigger as well as apoptotic pathway, aiming at improving spatiotemporal control and reducing side effects to the surroundings of the target. The strategy is to express in anticipated cells a modified caspase protein that can be activated optically in vivo. Practically, we firstly replaced the critical cysteine in the catalytic site of pre-introduced CASPASE-3 protein by an amber stop codon. With the help of genetic elements of code expansion, the stop codon was then able to encode a light-reactive artificial amino acid (namely, photocaged cysteine or PCC). Only after the exogenous PCC supplied incorporate into the polypeptide, will the caspase mRNA go through complete translation. However, as its catalytic site is still masked by the caging group of PCC, the caspase stays inactive. When we illuminate the target cell with UV, the incorporated PCC molecule transforms into cysteine, allowing the caspase to restore its function and induce cell apoptosis. The UV light is long-wave, so its use will not interfere with other optogenetic tools that work with visible light. Also the low intensity required by the uncaging poses little optical threat to worms. We now have succeeded in incorporating photocaged cysteine into both fluorescent reporter and modified CASPASE-3 expressed in different types of neurons with satisfactory efficiency. Up to this stage, we have not observed any cell death due to accidental caspase activation. Global illumination of UV is then shown to rescue caspase function and kill only the neurons that have incorporated PCC. By using microscope mounted UV laser, we can selectively target and kill a single neuron of its neuronal pair in adult worms with little off-target or damage. The illumination takes less than two minutes and can be performed from larvae stages to adulthood. And most treated cells are gone after 12 hours. The above results altogether show that the light-inducible caspase is promising for large-scale assays on the neuron(s) within complex network.

Radman I et al. (2013) PLoS One "Efficient and rapid C. elegans transgenesis by bombardment and hygromycin B selection."

Radman I, Chin JW, Greiss S

[PLoS One, 2013]

We report a simple, cost-effective, scalable and efficient method for creating transgenic Caenorhabditis elegans that requires minimal hands-on time. The method combines biolistic bombardment with selection for transgenics that bear a hygromycin B resistance gene on agar plates supplemented with hygromycin B, taking advantage of our observation that hygromycin B is sufficient to kill wild-type C. elegans at very low concentrations. Crucially, the method provides substantial improvements in the success of bombardments for isolating transmitting strains, the isolation of multiple independent strains, and the isolation of integrated strains: 100% of bombardments in a large data set yielded transgenics; 10 or more independent strains were isolated from 84% of bombardments, and up to 28 independent strains were isolated from a single bombardment; 82% of bombardments yielded stably transmitting integrated lines with most yielding multiple integrated lines. We anticipate that the selection will be widely adopted for C. elegans transgenesis via bombardment, and that hygromycin B resistance will be adopted as a marker in other approaches for manipulating, introducing or deleting DNA in C. elegans.

Davis, L. et al. (2019) International Worm Meeting "Precise Spatio-temporal Control of Gene Expression by Laser Illumination."

Greiss, S., Passlack, J., Davis, L.

[International Worm Meeting, 2019]

Precise control of gene expression is crucial for studying biological processes. However, the ability to control where genes are expressed is limited by a dearth of highly specific promoters in C. elegans. Furthermore, only very limited tools exist for temporal control of gene activity. This often makes it impossible to study gene function in single cells and/or at specific time points. The utility of genetic tools, such as for example optogenetic channels, also depend heavily on the ability to target them precisely to cells of interest and is often hampered by off-target expression. We have taken advantage of genetic code expansion to develop a method for precisely controlling gene expression both spatially and temporally using low intensity UVA light. The wavelength used does not harm the animals. We show that our method can be used to irreversibly switch on gene expression in a temporally controlled manner in C. elegans neurons by illumination. We observe no induction without illumination or when subjecting the animals to visible light, making our method compatible with most available optogenetic tools. Furthermore, we show that we can limit expression to single cells by targeting them with a microscope mounted UVA laser. The required illumination times are in the range of seconds. We demonstrate our method by using it to induce the expression of ChR2 in single C. elegans neurons, allowing us to, for example, separate individual members of neuronal pairs or clusters without the need for cell specific promoters. We believe that our method, using genetic code expansion to introduce 'on' and 'off' light-inducible switches for any gene of interest, will be of wide interest to C. elegans researchers who wish to exert fine control over the spatial and temporal dynamics of gene expression.

Pourkarimi E et al. (2012) Cell Death Differ "Evidence that CED-9/Bcl2 and CED-4/Apaf-1 localization is not consistent with the current model for C. elegans apoptosis induction."

Greiss S, Pourkarimi E, Gartner A

[Cell Death Differ, 2012]

In C. elegans, the BH3-only domain protein EGL-1, the Apaf-1 homolog CED-4 and the CED-3 caspase are required for apoptosis induction, whereas the Bcl-2 homolog CED-9 prevents apoptosis. Mammalian B-cell lymphoma 2 (Bcl-2) inhibits apoptosis by preventing the release of the Apaf-1 (apoptotic protease-activating factor 1) activator cytochrome c from mitochondria. In contrast, C. elegans CED-9 is thought to inhibit CED-4 by sequestering it at the outer mitochondrial membrane by direct binding. We show that CED-9 associates with the outer mitochondrial membrane within distinct foci that do not overlap with CED-4, which is predominantly perinuclear and does not localize to mitochondria. CED-4 further accumulates in the perinuclear space in response to proapoptotic stimuli such as ionizing radiation. This increased accumulation depends on EGL-1 and is abrogated in ced-9 gain-of-function mutants. CED-4 accumulation is not sufficient to trigger apoptosis execution, even though it may prime cells for apoptosis. Our results suggest that the cell death protection conferred by CED-9 cannot be solely explained by a direct interaction with CED-4.

Vinci CR et al. (2007) J Biol Chem "Recognition of age-damaged (R,S)-adenosyl-L-methionine by two methyltransferases in the yeast Saccharomyces cerevisiae."

Vinci CR, Clarke SG

[J Biol Chem, 2007]

The biological methyl donor, S adenosylmethionine (AdoMet), can exist in two diastereoisomeric states with respect to its sulfonium ion. The "S" configuration, (S,S)AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the "R" form, producing (R,S)AdoMet. As of yet, (R,S)AdoMet has no known physiological function and may inhibit cellular reactions. In this study, two enzymes have been found in Saccharomyces cerevisiae that are capable of recognizing (R,S)AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, previously identified as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine respectively. We find here that Sam4 recognizes both (S,S) and (R,S)AdoMet, but its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet while no activity is seen with the S,S form. R,S-specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)AdoMet in these organisms.

Fanning S et al. (2018) Mol Cell "Lipidomic Analysis of -Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment."

Lou Y, Haque A, Freyzon Y, Farese RV, Terry-Kantor E, Hofbauer HF, Termine D, Welte MA, Barrasa MI, Imberdis T, Noble T, Lindquist S, Clish CB, Jaenisch R, Pincus D, Nuber S, Sandoe J, Kohlwein SD, Kim TE, Ho GPH, Ramalingam N, Walther TC, Baru V, Selkoe D, Srinivasan S, Landgraf D, Soldner F, Dettmer U, Fanning S, Becuwe M, Newby G

[Mol Cell, 2018]

In Parkinson's disease (PD), -synuclein (S) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in S or lipid/fattyacid homeostasis affect each other. Lipidomic profiling of human S-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of S dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased S yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in S-overexpressing rat neurons. In a C.elegans model, SCD knockout prevented S-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on S homeostasis: in human neural cells, excess OA caused S inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for S-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.