Meneely, Philip M. et al. (2009) International Worm Meeting "HIM-8 interacts with SUN-1."

Daou, Maral, Thompson, Scott, Meneely, Philip M., Burch, Lauren

[International Worm Meeting, 2009]

During homolog recognition and pairing in meiosis, pairing centers at or near one end of each meiotic chromosome are localized to the nuclear envelope. SUN-1 is an evolutionarily conserved protein located in the inner nuclear membrane that appears to be directly involved in mediating telomere attachment in mice and yeast; in worms, a direct interaction between pairing center proteins and SUN-1 at the nuclear envelope has not yet been proved (1, 2). In sun-1 null mutants, none of the meiotic chromosomes are paired but the pairing centers are localized to the nuclear envelope (2). HIM-8 is a C2H2 zinc finger protein that binds specifically to the pairing center on the X chromosome and is necessary for the pairing of the X chromosome and for synapsis (3). When a zinc finger is mutated in him-8, such as in e1489 or the tm611 deletion, the protein does not bind to the X pairing center, the X chromosomes do not pair, but the chromosomes are still located at the nuclear envelope. Thus, pairing and nuclear envelope attachment may be separable activities for the pairing center proteins. The autosomes are paired normally in him-8 mutants. The three ZIM proteins, which are highly related to HIM-8 in sequence, mediate pairing of the autosomes (4). We find that SUN-1 and HIM-8 physically associate with each other in a yeast two-hybrid assay. Preliminary evidence suggests a similar physical interaction occurs between SUN-1 and at least one of the ZIM proteins. This suggests a model whereby meiotic chromosomes are tethered to the nuclear envelope for pairing via a direct interaction between SUN-1 in the inner membrane and HIM-8 or one of the ZIM proteins on each specific chromosome. 1.Penkner et al. 2007. Develop. Cell 12: 873-885 2.Fridkin, et al., 2009. Cell Mol. Life Sci. 66: (in press) 3.Phillips et al. 2005 Cell 123: 1051-1063 4.Phillips and Dernburg 2006 Develop. Cell 11: 817-829.

Aya Sato et al. (2006) Development & Evolution Meeting "The role of the nuclear envelope in homologous chromosome pairing and synapsis during meiosis"

Abby Dernburg, Carolyn Phillips, Aya Sato, Roshni Kasad

[Development & Evolution Meeting, 2006]

Pairing and recombination between homologous chromosomes are essential for accurate segregation of chromosomes during meiotic prophase. Specific chromosome sites known as Pairing Centers (PCs) are essential for homolog pairing and synapsis in C. elegans. Our laboratory has identified a family of four related proteins, HIM-8, ZIM-1, ZIM-2, and ZIM-3, that bind to the PCs on each chromosome and mediate homologous synapsis. Interestingly, foci of these proteins are associated with the nuclear envelope during early meiotic prophase, suggesting that they might tether the chromosomes to the nuclear envelope. We have found that two nuclear envelope proteins in C.elegans, SUN-1 and ZYG-12, form distinct patches at the nuclear envelope in transition zone nuclei in which homologous chromosomes are pairing, and that these SUN-1/ZYG-12 patches co-localize with HIM-8, ZIM-1, -2, and -3. To dissect the role of the nuclear envelope in homologous chromosome pairing and synapsis, we have carried out functional analysis of SUN-1 and ZYG-12 by genetic, cytological, and biochemical approaches. SUN-1 is a germline enriched nuclear envelope protein. ZYG-12 displays SUN-1-dependent localization at the nuclear envelope during meiosis, similar to its localization requirement in mitosis.4 We also found that sun-1 mutations genetically interact with X chromosome PC deficiencies, and that cosuppression of zyg-12 results in a Him phenotype. Both sun-1 and zyg-12 deletion mutants are sterile and show severe defects in germline mitosis, as well as meiosis. However, HIM-8 foci are still associated with the nuclear envelope in these mutants, suggesting that the role of SUN-1 and ZYG-12 is not simply to tether chromosomes to the envelope. We are currently investigating whether these proteins interact with other cellular components to mediate their roles in meiosis.

Il Minn et al. (2007) International Worm Meeting "Centrosome Attachment to the Nucleus Requires the SUN-1 Mediated Localization of ZYG-12 to the Outer Membrane of the Nuclear Envelope."

Il Minn, Christian J. Malone

[International Worm Meeting, 2007]

The localization of proteins specifically to the outer membrane of the nuclear envelope is thought to be required for attachment of the centrosome to the nucleus. The outer membrane of the nuclear envelope is contiguous with the ER and has been considered to be identical to the ER. Thus, the mechanism of specific outer nuclear membrane localization is unknown. We and others have recently identified a novel class of proteins with KASH domains that specifically localize to the outer membrane of the nuclear envelope via an interaction with SUN proteins. We have shown that two isoforms of the C. elegans protein ZYG-12, which contain KASH and transmembrane domains, depend on a SUN protein, SUN-1 (also called MTF-1), to localize to the nuclear envelope. ZYG-12 is required for attachment of the centrosome to the nucleus in C. elegans. To map the domains of ZYG-12 necessary for SUN-1 mediated localization, we expressed C-terminal fragments of ZYG-12, which contain KASH and transmembrane domains. These C-terminal fragments are sufficient for nuclear envelope localization in a SUN-1 dependent manner. We have investigated the membrane topology of both ZYG-12 and SUN-1. Both epitope accessibility and fluorescent protease protection assays using in vivo nuclei in combination with proteinase K protection assays using microsomes revealed that ZYG-12 and SUN-1 are type II membrane proteins of the outer and inner membrane of the nuclear envelope, respectively. In support of ZYG-12 being a type II membrane protein of the outer nuclear envelope, ZYG-12 physically interacts with cytoplasmic dynein via the N-terminus of ZYG-12 and is required for the localization of dynein to the nucleus. To investigate the molecular mechanism of SUN-1 dependent ZYG-12 localization, we asked if the proteins physically interact. Our data indicate that SUN-1 directly interacts with the KASH domain of ZYG-12. The interaction occurs between the KASH domain and the internal domain distinct from the SUN domain of SUN-1 as mapped by a membrane based two-hybrid assay and a pull-down assay. ZYG-12 at the outer nuclear membrane is necessary for centrosome attachment. Our results suggest that the inner nuclear membrane protein SUN-1 confines localization of ZYG-12 to the outer nuclear membrane via direct binding in the lumen of the nuclear envelope.

Matthew McGee et al. (2007) International Worm Meeting "KASH and SUN proteins bridge the nuclear envelope to transfer force from the cytoskeleton to the nucleoskeleton during nuclear positioning."

Matthew McGee, Daniel Starr

[International Worm Meeting, 2007]

Nuclear positioning, which includes both migration and anchorage of the nucleus, is an important process in C. elegans development. The outer nuclear membrane (ONM) KASH proteins UNC-83 and ANC-1 are required for nuclear migration and anchorage, respectively. The inner nuclear membrane (INM) SUN protein UNC-84 is required for both nuclear migration and anchorage. The UNC-83 and ANC-1 KASH domains are thought to interact with the UNC-84 SUN domain in the perinuclear space. This allows force to be transferred from the cytoskeleton to the nucleoskeleton during nuclear positioning. KASH protein localization is dependent on SUN proteins, but SUN protein localization is not dependent on KASH proteins. Using a membrane-bound yeast two-hybrid system (Igor Stagljar, Dualsystems Biotech), we have shown that the KASH domain directly interacts with two separable domains of UNC-84, the SUN and linker domains. This interaction is required for nuclear migration and anchorage. Using a molecular genetics approach, we found that the UNC-84 SUN domain is required for KASH protein localization and function in vivo. We also demonstrated that point mutations in the KASH domain disrupt the function but not the localization of UNC-83. This suggests a stronger interaction, presumably to allow a large transfer of force during nuclear positioning, is required to move or anchor the nucleus than is required to simply localize the KASH proteins to the ONM (McGee, et al. Mol Biol Cell. 2006:1790-801). This KASH and SUN interaction appears to be conserved across eukaryotes. To identify residues in the linker domain that are required for interaction with the UNC-83 KASH domain, we are performing a mutational analysis of the UNC-84 linker domain in the membrane-bound two-hybrid system. We are also using this two-hybrid system in a screen to test whether there are unidentified binding partners with the SUN domain of UNC-84. In order to test the conservation of function between human and C. elegans KASH and SUN domains, we are testing whether the human domains in the C. elegans KASH and SUN proteins localize and are functional. We have found that a human KASH domain in UNC-83 is able to partially rescue UNC-83 function in migrating hyp-7 nuclei and localize to the nuclear envelope. We have also found that this hyp-7 rescue is dependent on the UNC-84 SUN domain. This suggests that the human KASH domain is able to interact with the UNC-84 SUN domain in vivo. Further experiments are testing the functional rescue of human SUN domains in UNC-84.

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.

Erin C. Tapley et al. (2007) International Worm Meeting "Characterizing the UNC-83/UNC-84 interaction in vitro.."

Erin C. Tapley, Daniel A. Starr

[International Worm Meeting, 2007]

Nuclear migration and positioning is important to a wide variety of eukaryotes. For example, mammalian skeletal muscles must specialize nuclei at the neuromuscular junction, and in the developing human brain nuclei must migrate toward the cortex. Using C. elegans, we have identified two proteins that function in nuclear migration: UNC-83 and UNC-84. UNC-84 is a member of a family of proteins that share a conserved C-terminal SUN domain and that localize to the inner nuclear membrane (INM). UNC-83 is a member of a family of proteins that share a conserved C-terminal KASH domain and that localize to the outer nuclear membrane (ONM). Using genetic methods, we have shown that both the SUN and KASH domains are essential for nuclear migration in vivo and that a stronger SUN-KASH interaction is required for nuclear migration than for nuclear localization. Membrane bound yeast two hybrid (MBYTH) assays show that the SUN domain of UNC-84 directly interacts with the KASH domain of UNC-83 in the perinuclear space and that two independent domains, SUN and linker, of UNC-84 interact with UNC-83s KASH domain (for further details see McGee et. al 2006). We propose that the SUN-KASH interaction creates a structural bridge across the nuclear envelope that allows the transfer of force from the nuclear lamina to the cytoskeleton during migration. To further characterize the KASH-SUN and KASH-SUN-linker interactions biochemically, we have expressed and purified several MBP-6His fusion proteins from E. coli. Previous transmembrane (TM) prediction programs suggest that UNC-84 has one TM domain located at residues 512-532, which is consistent with our model that the N-terminus of UNC-84 is nucleoplasmic and the C-terminus resides in the perinuclear space. The largest fusion protein, which contains H568-V1111 is insoluble. TopPred2, a TM prediction program, suggests that UNC-84 may have as many as five TM domains. Three of these TM domains reside within the previously described linker domain: 685-705, 719-739, and 763-783. Previous data from our MBYTH suggest that the region between 742-780 is important for membrane targeting. Smaller fusion proteins, D739-V1111 and R783-V1111, which lack either one or both of the last two predicted TMs have also been expressed and purified. Both R783-V1111 and the SUN domain fusion protein T931-V1111 are completely soluble. Soluble SUN-linker and SUN fusion proteins will be used to test the interaction with a custom synthesized UNC-83 KASH peptide in vitro. As the linker domain may be vital for force transfer, it is essential to narrow down its functional domain. To do this, we first need to clearly establish where UNC-84s TM domains are located. Protease protection assays using Promegas TNT ® T7 Quick for PCR , S35 methionine and canine microsomes are currently being performed.