Mao X et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Hypoxic Adaptation and Survival Controlled by the Unfolded Protein Response"

Mao X, Crowder C

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

The Unfolded Protein Response (UPR) is a cellular reaction that enables organisms to ward off deleterious stresses. Here we investigated if hypoxia can induce the UPR in C elegans and if this response is beneficial to survival after hypoxia. A short exposure to tunicamycin, a UPR inducing reagent, was able to provide a delayed protective response for worms against subsequent hypoxic exposure. Canonical UPR pathway components IRE-1, XBP-1, and ATF-6 are required for this hypoxic protection. Protection due to hypoxic reconditioning (HP), after a short pre-exposure to hypoxia itself, requires IRE-1 and GCN-2 but not other UPR components. We found that the role of IRE-1 in hypoxia and HP is complex. We tested four ire-1 alleles for their baseline hypoxic sensitivity and ability to adapt to hypoxia after HP: v33 (null) and ok799 (deletion loss of function) homozygous mutants are HP defective but have normal baseline hypoxic sensitivity whereas zc14 (missense) and tm400 (reduction of function) have a normal HP response but are hypoxia resistant at baseline. v33 heterozygotes phenocopy zc14. These results show that hypoxic sensitivity and adaptation are quite sensitive to the level and nature of IRE-1 signaling. In order to investigate if IRE-1 is also important for hypoxic survival in mammals, we knockdowned ERN-1 and ERN-2 (mouse ire-1 homologs) expression with lentiviral shRNAs in primary mouse hippocampal neurons and found significant protection from hypoxic cell death. These data indicate that the UPR has a conserved role in hypoxic death in C. elegans and mammals.

Mao X et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Proteostasis, a potent modulator of hypoxic injury and adaptation"

Mao X, Scott B, Mabonn M, Anderson L, Crowder C

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Hypoxic cellular injury is the leading cause of human mortality in the United States. However, no therapy has been approved to ameliorate hypoxic injury. With the aim of a better understanding of the determinants of hypoxic cell death, we have performed a variety of screens to identify genes regulating the sensitivity of C. elegans to hypoxic death. Our screens have uncovered a large number of genes whose activity significantly promotes C. elegans death following hypoxia. While at least 200 C. elegans genes have a reduction of function phenotype of weak to moderate hypoxia resistance, we have discovered only a handful of mutations/RNAi's that strongly protect C. elegans from hypoxic death. The two strongest hypoxia resistant phenotypes were found in alleles of genes that function in proteostasis. We found that hypoxia induces a response to protein misfolding, suggesting that the response to unfolded protein may actively regulate hypoxic sensitivity. Indeed, mutations in a subset of the ER unfolded protein response genes block adaptive responses to hypoxic injury in C. elegans. Knockdown of homologous genes in mouse shows that modulation of proteostasis can also increase survival of mouse brain neurons after hypoxic injury. Modulation of proteostasis is a promising strategy for protection from hypoxic cellular injury.

Hodgkin, Jonathan et al. (2009) International Worm Meeting "Analysis of breakage and meiotic instability in attached-X-chromosome strains."

Hodgkin, Jonathan, O'Rourke, Delia, Jethwa, Nirmal

[International Worm Meeting, 2009]

The isolation of an attached X chromosome (X^X), consisting of two apparently intact X chromosomes joined at their left telomeres, was previously reported (Hodgkin and Albertson 1995, Genetics 141: 527). In contrast to attached X chromosome constructs in other organisms, and also to X-autosome fusions in C. elegans, this X^X configuration is unstable, frequently breaking down to give an apparently normal X chromosome, or else an X chromosome with a deletion of the left end, or an X chromosome carrying a duplication of the left end. The extent of these deletions and duplications is variable, and they are generated at high frequency (in at least 5% of all X^X oogenic meioses). X^X strains therefore provide a useful source of aberrations in this region of the genome, which contains both a meiotic pairing site and numerator sites for sex determination. It seemed likely that X^X breakage was occurring at meiosis, and might be dependent on the meiotic recombination machinery. To test this hypothesis, X^X was crossed onto a series of meiosis-defective backgrounds (him-1, him-3, him-5, him-6, him-8, him-14, xnd-1, etc.). X^X breakage, as measured by the production of self-progeny males, was significantly or greatly reduced in most of these backgrounds, indicating that the breakage events are partly or wholly dependent on meiotic recombination. Previous FISH studies using YAC probes indicated that the end junction between the two X chromosomes was symmetrical and involved no deletion. We attempted to clone the junction fragment by single primer PCR, in order to determine its exact structure. However, PCR amplification was not successful, perhaps because the junction may include a substantial stretch of telomere repeats or local rearrangements. Further investigation of the structure and properties of the end junction is in progress.

Chun Yi Huang et al. (2005) International Worm Meeting "A new player in the C. elegans programmed cell death pathway"

Yi-Chun Wu, Tsung-Yuan Hsu, Ruey-Hwa Chen, Chun Yi Huang, Pei-ju Lu, Jia-Yun Chen

[International Worm Meeting, 2005]

Human CED-X was identified as a strong interacting protein with the death domain of DAPK (death associated protein kinase) in a yeast two-hybrid screen. To investigate the potential involvement of CED-X in apoptosis, we study the function of its C. elegans homolog ced-x. Inactivation of ced-x by either RNAi or a deletion mutation resulted in decreased numbers of cell corpses during embryogenesis. Ectopic killing caused by transcriptional overexpression of egl-1 or ced-4 but not ced-3 in touch cells was suppressed by the ced-x(0) mutation. Therefore, ced-x genetically acts downstream of or in parallel to egl-1 and ced-4 and upstream of or in parallel to ced-3 in the programmed cell death pathway. In addition, the observation that the ced-x(0) mutation partially suppressed ectopic cell deaths caused by the icd-1(RNAi) mutation indicates that ced-x genetically acts downstream of or in parallel to icd-1. To further explore CED-X function, we generated antibodies against CED-X. The immunostaining signal revealed a filamentous CED-X localization pattern in the cytosol during embryogenesis. We are currently investigating if CED-X is associated with organelles or cytoskeleton. In summary, our results show that ced-x is important for promoting or proper execution of programmed cell death in C. elegans. As human ced-x can functionally substitute C. elegans ced-x in a ced-x mutant, the function of ced-x in apoptosis is likely conserved in evolution.

Lau, Alyssa et al. (2015) International Worm Meeting "Deciphering the mechanism of X-upregulation in C. elegans dosage compensation."

Lau, Alyssa, Csankovszki, Gyorgyi, Zhu, Kevin

[International Worm Meeting, 2015]

In C. elegans dosage compensation is required to balance X-linked gene expression to autosomes. It is hypothesized that an unknown mechanism causes X upregulation in both sexes. This mechanism balances the X to autosomal expression in males, but creates X overexpression in hermaphrodites. Therefore, to restore the balance, hermaphrodites downregulate gene expression two-fold on both X chromosomes. While many studies have focused on X chromosome downregulation, the mechanism of X upregulation is not known.Using 3D FISH microscopy to measure the volume of chromosome territories we found that the X chromosome, but not chromosome 1, territories in males are unexpectedly decondensed. We found that this X chromosome decondensation requires the activity of the histone acetyltransferase, MYS-1 (homologous to human TIP60). Interestingly, MYS-1 acetylates H4K16, the key factor responsible for male-specific X-upregulation in Drosophila melanogaster dosage compensation. This suggests that H4K16ac may be responsible for higher gene expression levels on the male X in both species. Depleting other members of a putative C. elegans TIP60-like complex led to similar X phenotypes as MYS-1 depletion. We hypothesize that a TIP60-like complex decondenses the X chromosome in C. elegans males, and this decondensation contributes to upregulation of gene expression on the chromosome. By analyzing X chromosome volumes in young male embryos we determined that the developmental time window for this male X chromosome decondensation occurs around the 30-to-50-cell stage, surprisingly the same stage as the downregulation process in hermaphrodites. Overall, our data indicates that histone acetylation by the MYST family HAT MYS-1 may play an important role in the highly debated X upregulation mechanism in C. elegans. .

Albritton, Sarah et al. (2011) International Worm Meeting "Regulation of X chromosome transcription in Caenorhabditis species."

Albritton, Sarah, Ercan, Sevinc

[International Worm Meeting, 2011]

Animals with different numbers of X chromosomes in males and females possess mechanisms to compensate for the difference in the X-linked gene dose between the two sexes. In addition to the X chromosome dose difference between the sexes, the presence of a single-copy X chromosome per two-copy diploid autosomes creates an important problem for males, because all X-linked genes are haploinsufficient compared to the autosomal genes. In C. elegans, hermaphrodites (XX) contain two Xes, whereas males (XO) contain a single X, therefore facing X haploinsufficiency. By performing microarray analysis of RNA abundance in XX and XO worms, we observed that the overall transcript levels from the X chromosome in both XX and XO animals is similar to that of overall expression from autosomes. This suggests that transcription from the single X in XO L3 hermaphrodites (TY2205, her-1(e1520) sdc-3(y126) V; xol-1(y9) X) is increased approximately two-fold. The mechanism of this upregulation is unclear. We had shown that the X chromosome promoters have higher GC content compared to the autosomes (Ercan et al 2010), suggesting a DNA-encoded mechanism of transcriptional regulation. We will study X upregulation by comparing transcription of orthologus genes that are on the X versus autosomes in four Caenorhabditis species. Ercan S, Lubling Y, Segal E, Lieb JD. High nucleosome occupancy is encoded at X-linked gene promoters in C. elegans. Genome Res. 2011 Feb;21(2):237-44. PMID:21177966.

Satoru Uzawa et al. (2007) International Worm Meeting "Visualization of the Spreading of the Dosage Compensation Complex on X by Microscopy."

Satoru Uzawa, Barbara Meyer

[International Worm Meeting, 2007]

For proper development of XX and XO animals, the dosage of X-chromosome-linked genes have to be compensated. In free-living soil nematode C. elegans, the dosage compensation of the X-linked genes is accomplished by reducing the gene expression from the X chromosomes in XX hermaphrodites by half. This process is done by the molecular machinery known as the dosage compensation complex (DCC), which resembles condensin complex. DCC loads specifically onto the X chromosome in early embryos (~20 cell stage) and remains on the X chromosome throughout the life of the hermaphrodite. The DCC is recruited onto the X chromosome through specific DNA sequences on the X chromosomes, rex (recruitment element on X). The X chromosome regions that lack a rex site receive DCC through spreading from other rex sites in cis. The nature and molecular mechanism of the spreading of the DCC on the X chromosomes is unknown. We are trying to visualize this spreading process through various microscopic methods using FISH and IF double staining for high resolution images and live cell imaging for time domain analysis.

LaMunyon CW et al. (1995) International C. elegans Meeting "SPERM COMPETITION IN HERMAPHRODITIC CAENORHABDITIS SPECIES"

Ward S, LaMunyon CW

[International C. elegans Meeting, 1995]

As in C. elegans, male sperm take precedence over hermaphrodite sperm after C. briggsae worms mate. However, in C. briggsae the initial outcross progeny are principally hermaphrodites and become male-biased with time. Thus, X-bearing male sperm have a striking fertilization advantage over non-X-bearing male sperm. Results from controlled matings show that the X-bearing sperm neither outnumber nor activate faster than their non-X-bearing counterparts. The advantage (meiotic drive) of the X-bearing sperm is therefore due to a competitive edge over non-X-bearing sperm. Thus, in C. briggsae, male X-bearing sperm outcompete male non-X-bearing sperm which themselves outcompete hermaphrodite sperm. While we do not know the mechanism underlying the competitive effect of the X chromosome, we do have evidence from artificial insemination that the precedence of male sperm over hermaphrodite sperm in C. elegans is independent of both seminal fluid and the mode of sperm activation (male or hermaphrodite). Male sperm precedence in hermaphroditic Caenorhabditis maximizes outcrossing after mating, and the X-bearing sperm advantage in C. briggsae delays the associated cost of making males which are by themselves incapable of reproduction.

Albritton, Sarah E. et al. (2013) International Worm Meeting "Gene movement between X and autosomes and its effect on transcription."

Kranz, Anna-Lena, Ercan, Sevinc, Albritton, Sarah E.

[International Worm Meeting, 2013]

In Caenorhabditis species, dosage compensation acts to reduce transcriptional output of both female/hermaphrodite X chromosomes by one half. This balances X expression between the sexes, but potentially gives females the same problem faced by males: functional monosomy of the X chromosome. It has been proposed that a mechanism evolved to upregulate X expression to balance X and autosomal transcription, thereby overcoming male monosomy. However, owing to biased gene content and tissue-specific regulation of the X, direct comparison of X and autosomal transcription is difficult. In order to more directly compare X and autosomal transcription we looked at expression of 1:1 orthologs that are differentially located on the X or an autosome between two nematode species. Our work focused on four species: C. elegans, C. briggsae, C. remanei, and Pristionchus pacificus. The C. elegans and C. briggsae genomes are well assembled and annotated. The genomes of C. remanei and P. pacificus have been sequenced, but their genes have not yet been assigned to chromosomal locations. Since our analysis depends on comparing differentially located orthologs, we first needed to map genes to either the X or autosomes. We took a read-depth-variation approach. We performed genomic DNA-seq in males and females/hermaphrodites of C. brenneri, C. remanei and P. pacificus. C. briggsae males and hermaphrodites were also sequenced as controls. Genes located on the X chromosome were expected to have a 1:2 ratio of sequencing coverage between males and hermaphrodites (X:XX) and all autosomal genes a 1:1 ratio. Our analysis yielded a list of X and autosomal genes for each of the four nematode species and allowed the identification of differentially located 1:1 orthologs. Comparison of X and autosomal transcription showed no bias towards male upregulation of X-located orthologs.

Custer, Laura et al. (2011) International Worm Meeting "Examining the kinetics of chromatin modifications associated with dosage compensation onset."

Custer, Laura, Csankovszki, Gyorgyi

[International Worm Meeting, 2011]

Dosage compensation (DC) is a specialized gene regulation process to equalize gene expression along the entire X chromosome between XX hermaphrodites and XO males. In hermaphrodites, expression from each X chromosome is down-regulated two-fold to equal the expression of the single male X chromosome. Dosage compensation is achieved by the dosage compensation complex, which localizes to the X chromosomes in hermaphrodites beginning around the time of gastrulation. Our lab has previously shown reduced levels of histone H4 lysine 16 acetylation (H4K16Ac), a mark associated with active transcription, on the adult hermaphrodite X chromosomes in a dosage compensation complex-dependent manner. We have also observed a dramatic increase in the levels of histone H4 lysine 20 monomethylation (H4K20me1) and stalled RNA Polymerase II associated with the X chromosomes in adult hermaphrodites. This study examines chromatin modifications on the X chromosome(s) during development, including time points before and after the onset of DC. We predicted that H4K16Ac levels on the X chromosomes would equal, or even exceed, autosomal levels in pre-gastrulation embryos. However, by quantification of immunofluorescent signals, H4K16Ac is reduced on the X chromosomes of hermaphrodite embryos even before the onset of DC. Male embryos also have reduced H4K16Ac levels on their single X chromosome. H4K16Ac levels remain reduced on the X chromosomes in dosage compensation complex mutants, suggesting that another mechanism is involved in X chromosome silencing in early embryos. Within the adult germline, the X chromosome(s) are silenced by MES-4 and the MES-2/3/6 complex, which resembles the Polycomb repressive complex. Mutations in the mes genes restore H4K16Ac levels on the X chromosomes in early embryos to the levels observed on autosomes. Dosage compensation onset coincides with the transition from plasticity to differentiation, a process regulated by the MES proteins. We are examining the relationship between Polycomb's function in X chromosome silencing, its role in differentiation, and the onset of DC. Together our results describe a time course of H4K16Ac reduction and other chromatin modifications on the X chromosomes during development and suggest that the onset of differentiation coincides with a transition from MES protein-mediated X chromosome repression to DC.