Michel, Agnes et al. (2009) International Worm Meeting "Mechanism of X-chromosome dosage compensation: probing X-chromosome higher-order structure in C. elegans ."

Pickle, Cathy, Meyer, Barbara, Michel, Agnes

[International Worm Meeting, 2009]

Males and females differ in their number of X chromosomes, yet both require a similar level of X-chromosome products. Dosage compensation is an essential, universal, chromosome-wide regulatory process that equalizes the somatic expression of X-linked genes between males and females/hermaphrodites. A dosage compensation complex (DCC) is directed to both X chromosomes of C. elegans hermaphrodites to reduce gene expression by half. DCC binds to discrete, dispersed cis-acting elements on X. Of the many sites bound by DCC, few can serve as DCC recruitment sites (recruitment element on X, rex). We have shown that the recruiting ability is at least partially conferred by a consensus motif (MEX) that is shared among rex sites. Most sites (dependent on X, dox) fail to recruit the DCC autonomously, therefore must be bound through a different mechanism. The similarity of DCC to condensin, a conserved protein complex essential for chromosome compaction, resolution, and segregation, suggests that DCC loading along X is associated with changes in chromosome structure. Long distances separate rex from dox sites, implying that long-range interactions are important for DCC distribution. We have recovered animals that carry homozygous deletions of rex sites. These strains will be used to test the hypothesis that DCC binding to rex sites is necessary for DCC binding to dox sites via a mechanism of long-range interaction. Chromatin immunoprecipitation experiments using DCC-specific antibodies will allow us to determine whether dox sites become unoccupied in the rex deletion strains. In parallel, Chromosome Conformation Capture will be used to determine whether the rex and dox sites contained in two well-characterized 190kb regions interact at long distances. Finally, we will establish the network of interactions of all the DCC binding sites present in a large representative portion of the X chromosome, using the method of Chromosome Conformation Capture Carbon Copy. Swiss National Science Foundation PBGEA-119304 and PA00P3_124165 / 1 NIH R01-GM30702.

Wheeler, Bayly et al. (2015) International Worm Meeting "Regulation of nuclear organization and long-range gene expression by condensin."

Meyer, Barbara J., Frokjaer-Jensen, Christian, Anderson, Erika, Jorgensen, Erik, Wheeler, Bayly, Bian, Qian

[International Worm Meeting, 2015]

The relationship between chromosome structure, nuclear positioning, and long-range gene regulation is poorly understood. To explore this relationship, we dissected X-chromosome-wide gene regulation enacted by a dosage compensation complex (DCC), which represses X transcription in hermaphrodites to balance gene expression between sexes. We inserted transgenes throughout the genome and queried their expression to determine whether different transcriptional environments exist on X and autosomes. Transgenes integrated on X were dosage compensated regardless of position, meaning their expression was equal in wild-type males and hermaphrodites but elevated in dosage-compensation-defective hermaphrodites. This result indicates the X chromosome is broadly permissive for repression, and endogenous genes that escape have special features enabling them to overcome this repression. In contrast, we found no chromosome-wide mechanism to balance X expression with that of autosomes, given that transgenes on X were expressed at half the level of transgenes on autosomes. Repression of X transgenes was independent of their proximity to DCC recruitment sites (rex), highlighting the long-range mechanism of regulation employed by the DCC. We already showed that changes in higher order X-chromosome structure accompany repression of X-linked genes, so we next explored whether spatial positioning of X influences dosage compensation. We first addressed a model of others that rex sites target X to the nuclear periphery in males to increase gene expression, and DCC binding to rex sites in hermaphrodites helps relocate X to the interior, thereby repressing X. Using FISH, we found for both sexes that neither endogenous rex sites on X nor ectopically inserted rex sites on autosomes were preferentially located at the nuclear periphery. Furthermore, though rex insertions on autosomes recruit the DCC, the expression of adjacent genes was not elevated in DCC-depleted animals. These observations disfavor the proposed model. Instead, we found that pairs of distant rex sites interact in a DCC-dependent manner, and interacting rex sites are preferentially located at the nuclear periphery compared to non-interacting sites. Interacting rex pairs associate with nuclear pores, not the lamina. We propose the nuclear pore might act as a scaffold to promote rex site interactions, which in turn influence gene expression by remodeling higher order chromosome structure.

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.

Albritton, Sarah et al. (2017) International Worm Meeting "Long-distance cooperation and hierarchy between recruitment sites specify the X chromosome for dosage compensation."

Albritton, Sarah, Ercan, Sevinc, Winterkorn, Lara, Street, Lena, Kranz, Anna-Lena

[International Worm Meeting, 2017]

Dosage compensation mechanisms specifically target gene regulatory complexes to the X chromosomes for transcriptional regulation. However, it remains unclear how X is specified, as the DNA sequence motifs shown to be important for binding of dosage compensation machineries are themselves not X-specific. Here, we analyzed binding of the C. elegans dosage compensation complex (DCC) through a series of experiments that include deletion and ectopic insertion of recruitment site sequences. Our data suggest that DCC recruitment is initiated by a small number of primary recruitment sites characterized by clusters of the 12-bp recruitment motif and overlap with high occupancy transcription factor target (HOT) regions, two features that fully explain X-specificity. SDC-2, the protein essential for hermaphrodite-specific recruitment of the DCC during early embryogenesis, is required to maintain open chromatin specifically at the primary recruitment sites whose DNA sequence encodes for high intrinsic nucleosome occupancy. Along the X, the primary recruitment sites are interspersed by secondary, weaker DCC recruitment sites. While insertion of a secondary recruitment element on an autosome failed to fully recruit the DCC, the same element was capable of recruitment at an ectopic locus on the X, suggesting that the function of the weaker recruitment sites are X-dependent. On the autosome, insertion of multiple recruitment elements in tandem or at a distance (>30 kb) increased DCC recruitment, demonstrating that recruitment sites cooperate over long distances. On the X, deletion of single recruitment sites resulted in reduced DCC binding across several megabases flanked by topologically associating domain (TAD) boundaries, suggesting that DCC recruitment and spreading occurs within defined X chromosomal domains. Our work illustrates a fundamental strategy for specifically targeting large chromosomal domains for co-regulation, which involves hierarchy and long-distance cooperativity between functional genomic elements.

Fassio, C.A. et al. (2011) International Worm Meeting "Chromosome-wide gene regulation and higher-order chromosome structure in C. elegans dosage compensation."

Michel, A., Crane, E., Uzawa, S., Fassio, C.A., Meyer, B.J.

[International Worm Meeting, 2011]

Dosage compensation, the essential process that equalizes X-linked gene expression between sexes, provides an excellent system to examine the complex mechanisms that control gene expression across large chromosomal domains. In C. elegans, the condensin-like dosage compensation complex (DCC) reduces transcriptional activity of both hermaphrodite X chromosomes by half to equal expression from the single male X1. The DCC binds two classes of sites on X: rex sites (recruitment element on X), which recruit the DCC autonomously2.3 and dox sites (dependent on X), which robustly bind the DCC only when attached to X3. Long-range interactions between rex and dox sites appear important for full DCC occupancy at dox sites3. The homology of DCC components to condensin suggests that the DCC facilitates long-range regulation of X-linked gene expression by restructuring the X chromosome. Interactions between rex sites may facilitate loading of the DCC and/or targeting of the complex to dox sites. Consistent with this hypothesis, pairs of rex sites interact more frequently in XX and mutant XO embryos in which the DCC is loaded onto X than in XO embryos, in which the dosage compensation does not occur. Additionally, I have shown that the DCC binds an ectopic rex site integrated on the X chromosome. This ectopic binding site is in close proximity to an endogenous rex site that is located over 2 Mb away more often than a control integration sequence. These results further suggest that DCC occupancy imposes structural changes on the X chromosome. Using 5C, I am asking how altered DCC occupancy at specific loci affects the frequencies of long-range interactions among rex sites and between rex and dox sites. These data will show whether increased DCC occupancy corresponds to increased interaction frequencies between binding sites. I am also performing RNA-seq to to ask how localized changes in DCC occupancy affect transcriptional activity of X-linked genes and determine how transcription is influenced by DCC binding. These experiments address the transcriptional consequences of DCC occupancy at defined sites and determine the role of DCC-mediated changes in chromosome structure in this process. 1 Meyer BJ. Curr Opin in Genet & Dev (2010); 2 Jans J. et al. Genes & Dev. (2009); 3 Pferdehirt R. el al. Genes & Dev. (2011).

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.