Steve Gendreau et al. (2002) West Coast Worm Meeting "Toward the identification of novel regulators of b-catenin signaling in C. elegans"

Emery Dora, Mike Costa, Damian Curtis, Lorena Mora-Blanco, Changyou Chen, Steve Gendreau

[West Coast Worm Meeting, 2002]

The unbridled transcriptional activation of Wnt-responsive genes is a frequent and early event in a number of human tumor types (Polakis, 2000). In an effort to identify novel regulators of Wnt-mediated signaling, we have initiated modifier screens in C. elegans around two key Wnt components, b-catenin (bar-1, Eisenman et al, 1998) and its negative regulator, axin (pry-1, Korswagen et al., 2002).

Kenneth K Lee et al. (2001) International C. elegans Meeting "The expression, lamin-dependent localization, and loss-of-function phenotype for emerin in C. elegans"

Katherine L Wilson, Yosef Gruenbaum, Kenneth K Lee, Jun Liu

[International C. elegans Meeting, 2001]

Emerin is an integral protein of the inner nuclear membrane. In humans, loss of emerin causes the X-linked form of Emery-Dreifuss muscular dystrophy. The emerin gene ( emr-1 ) is conserved in C. elegans . We show that Ce-emerin protein is expressed ubiquitously in C. elegans , and that Ce-emerin localization at the nuclear envelope requires the C. elegans lamin protein, Ce-lamin. We determined the loss-of-function phenotype for emerin in C. elegans , by double-stranded RNA inhibition (RNAi) of the emr-1 gene. In embryos lacking Ce-emerin, we detected normal nuclear envelope localization for Ce-lamin, UNC-84, nucleoporins and Ce-MAN1, which is related to emerin through the conserved LEM-domain. The RNAi-induced loss of Ce-emerin had no apparent effects on development, since Ce-emerin-deficient embryos developed normally into fertile adult nematodes. Furthermore, the RNAi-induced loss of emerin persisted into adulthood, with no detectable phenotype. Based on its lamin-dependent localization at the nuclear envelope, biochemical properties and non-essential phenotype, Ce-emerin closely resembles human emerin. These findings establish C. elegans as a suitable genetic organism in which to dissect the molecular mechanisms of emerin function in vivo .

Miriam Segura et al. (2001) International C. elegans Meeting "In vivo studies of the nuclear function of Barrier-to-Autointegration Factor (BAF)"

Miriam Segura, Katherine L Wilson

[International C. elegans Meeting, 2001]

BAF is an essential, novel DNA-bridging protein of unknown function. We are using C. elegans to determine BAF's function in vivo , and to understand its interactions with a family of inner nuclear membrane proteins termed LEM-domain proteins. The LEM-domain proteins that are conserved in C. elegans include MAN1 and emerin, loss of which causes Emery-Dreifuss muscular dystrophy in humans. To test the hypothesis that BAF interacts with LEM-domain proteins, we stained C. elegans embryos by direct immunofluorescence using BAF-specific antibodies. Endogenous ceBAF is distributed along the nuclear rim, consistent with its association with inner membrane proteins. However, there appeared to be additional BAF staining in the nuclear interior. By double-staining for BAF plus either MAN1, emerin, or lamins in C. elegans embryos, we found that BAF colocalizes at the nuclear envelope with MAN1 and emerin, and that BAF colocalizes with lamins both at the perimeter and interior of the nucleus. These results support our hypothesis, and suggest that BAF is intimately associated with the nuclear lamina. The RNAi phenotype for BAF is early embryonic lethality associated with chromatin defects such as DNA bridging, super-condensation and perhaps nuclear fragmentation. These phenotypes suggest an essential role for BAF in chromatin decondensation and perhaps chromosome segregation.

Jin Liu et al. (2005) International Worm Meeting "Functional proteomic screens of nuclear envelope components"

Daniel A Starr, Peter Hamilton, Jin Liu

[International Worm Meeting, 2005]

Despite the importance of the nuclear envelope in the cell and its implication in a wide variety of diseases, very little is known about the function of individual components that make up this organelle. A recent proteomic study in mammalian cells (Schirmer et al, 2003) identified 67 new integral nuclear membrane components on top of the approximately a dozen components that were previously known. Our BLAST searches identified 51 potential new nuclear envelope components conserved in C. elegans. To gain some new insights into the nuclear envelope, we are using C. elegans as a model system and employing a reverse genetic approach using RNAi against 51 newly identified, evolutionarily conserved components of the nuclear envelope. We are screening progeny of dsRNA-injected animals for post-embryonic defects in nuclear migration and anchorage similar to null phenotypes in unc-83, unc-84, and anc-1. We are also screening for synthetic lethality with emr-1(gk119). Mutations in emerin have been shown to lead to Emery-Dreifuss muscular dystrophy in human, but are silent in worms (Gruenbaum et al, 2002). We confirmed that lem-2(RNAi); emr-1(gk119) animals are synthetic lethal, as previously shown (Liu et al, 2003). Finally, we are cloning the 51 new potential nuclear envelope proteins into GFP fusion vectors under control of the ubiquitous npp-1 promoter (kind gift of A. Schetter and K. Kemphues) to confirm their predicted localization to the nuclear envelope.

Gasser, Susan et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "ferentiation specific positioning of tissue-specific genes during C. elegans development is perturbed by a disease-causing lamin mutation"

Gruenbaum, Yosef, Pike, Brietta, Meister, Peter, Gasser, Susan, Mattout, Anna, Towbin, Benjamin

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

We explored whether gene localization facilitates proper expression during cell type differentiation by studying the subnuclear distribution of cell-type specific promoters during C. elegans development. Low-copy integrated transgenes that contain arrays of LacO sites, and developmentally regulated promoters ( myo -3::mCherry, pha -4::mCherry-H2B, unc -119) were used to analyse cell-type specific nuclear organization. In early embryos, transgenes bearing inactive tissue-specific promoters were randomly distributed in the nucleus. Over the course of differentiation, we observe spatial segregation of the transgenes, based on their transcriptional status. Inactive silent transgenes shift to the nuclear periphery while active promoters are sequestered internally. This is true for four different tissues. As opposed to low-copy transgenes obtained by bombardment, integrated repetitive transgene arrays derived form gonadal plasmid injection accumulate heterochromatic marks (H3K9me3 and H3K27me3). These silent domains attach to the nuclear periphery even in embryos. However, activation of a developmentally regulated promoter overcomes the peripheral anchorage of the large array. We further show that lamin mutations alter the spatial organization of heterochromatic arrays. We introduced a mutation into worm lmn-1 which in humans causes Emery Dreyfuss muscular dystrophy . In a dominant fashion, this mutant lamin interferes specifically with the subnuclear repositioning of a muscle-promoter array. It further interferes with the induction of array-borne myo -3 promoters and confers phenotypes that are both morphologically and phenotypically similar to the human disease. We conclude that nuclear organization is essential for cell type specific transcription patterns and that subnuclear localization of cell-type specific genes is driven by cell-type specific promoters.

Mattout, A. et al. (2013) International Worm Meeting "Tissue integrity and laminopathic phenotypes correlate with subnuclear heterochromatin positioning."

Mattout, A., Gasser, SM.

[International Worm Meeting, 2013]

We have previously shown that wild-type lamin helps organize the subnuclear position of heterochromatin, and that a point mutation in lamin, Y59C, which in humans leads to Emery-Dreifuss muscular dystrophy, impairs the proper muscle-specific redistribution of a heterochromatic array away from the nuclear periphery. We also found that this muscle-specific misorganization of heterochromatin correlated with transcriptional defects and with defective locomotion and muscle integrity (Mattout et al, Curr Biol, 2011). It has remained unclear, however, whether the chromatin misorganization was a cause of the observed physiological defects, or a secondary effect due to an impaired gene expression. In order to clarify this, we took advantage of the cec-4 deletion mutant recently characterized in our laboratory, which specifically releases H3K9me-containing heterochromatin from the nuclear periphery. This release occurs in embryos and does not necessarily provoke transcriptional reactivation. We combined this mutation with expression of the lamin Y59C mutant, in a strain bearing a muscle-specific reporter array, which is heterochromatic due to its large size. We find that combining the cec-4 deletion with the Y59C lamin mutation restores the proper positioning of the muscle-specific heterochromatic array in developing muscle cells, and interestingly, also rescues the impaired locomotion phenotype of the Y59C lamin mutant. This result argues that the spatial reorganization of heterochromatin in the nucleus during differentiation can impact tissue integrity. Indeed, the failed tissue-specific release of heterochromatin in muscle, which stems from a dominant mutation in lamin, may indeed be responsible for the range of phenotypes correlated with lamin-related genetic diseases.

Palmer, Lira et al. (2015) International Worm Meeting "Using C. elegans as a model to study Laminopathies."

Bone, Courtney R., Starr, Daniel, Palmer, Lira

[International Worm Meeting, 2015]

Lamin, the major component of the nucleoskeleton, supports the nuclear envelope and provides strength and structure to the nucleus. Lamin is also required for many nuclear migration events. Three to four lamin proteins are found in vertebrates. Mutations in human LaminA are associated with broad class of disorders termed laminopathies including Hutchinson-Gilford progeria syndrome and Emery-Dreifuss muscular dystrophy. The molecular underpinnings that lead to the varied pathologies associated with laminopathies are poorly understood. C. elegans provides a realtively simplistic model to study lamin proteins in vivo because it contains only a single lamin, LMN-1. Previous studies using C. elegans as a model for studying mutant forms of LMN-1 had two major limitations. First, mutant forms of LMN-1 were overexpressed from extrachromosomal arrays. Second, the mutant forms were always expressed in the wildtype LMN-1 background. We are taking advantage of CRISPR/Cas9 genome editing to precisely introduce point mutations in heterozygous lmn-1 backgrounds at endogenous loci. Our ultimate goal is to simultaneously express wild type and mutant forms of LMN-1 with different fluorescent tags for live imaging. We first attempted to tag LMN-1 at its C-terminus with GFP. The field has previously relied on a LMN-1::GFP protein for live imaging of the nuclear lamina. We were able to generate the GFP tag at the endogenous lmn-1 locus by CRISPR/Cas9. The GFP localized to the nuclear envelope as expected, but we could not homozygous LMN-1::GFP, suggesting it is not fully functional. We are currently using CRISPR/Cas9 to introduce GFP into a number of different positions within LMN-1 with the hope of generating a functional tagged protein. Once such a reagent is generated, mutations in conserved residues will be introduced and their functions tested in vivo. .

Mattout, Anna et al. (2017) International Worm Meeting "Mechanism and function of genome organization in muscle development and integrity."

Kalck, Veronique, Harr, Jennifer C., Gonzalez-Sandoval, Adriana, Mattout, Anna, Gasser , Susan M.

[International Worm Meeting, 2017]

The loss of nuclear structural integrity leads to tissue-specific pathologies in a set of human diseases called laminopathies. These diseases, such as Emery Dreifuss Muscular Dystrophy (EDMD), are late-onset, tissue-specific, and degenerative. A C. elegans mutant that recreates the EDMD phenotypes arises from the introduction of a specific gain-of-function mutation into lamin (Y59C), which affects the proper release of a muscle-specific heterochromatic reporter from the nuclear periphery. We found that this muscle-specific misorganization of heterochromatin correlates with transcriptional defects and with perturbed locomotion and muscle integrity in C. elegans. To identify if chromatin misorganization is a cause or an effect of these physiological defects or if the alteration of gene expression is the primary cause, we took advantage of a cec-4 deletion mutant. The perinuclear C. elegans chromodomain protein-4 (CEC-4) anchors heterochromatin by binding H3K9 methylation. The mutant releases H3K9me containing heterochromatin from the nuclear periphery in embryos and does not alter transcription. In a strain that contains the cec-4 deletion, expresses the lamin Y59C mutant and harbors a muscle-specific heterochromatic reporter, there is a recovery of the proper positioning of the muscle-specific heterochromatic array in muscle. Additionally, we found that the cec-4 mutation rescues the impaired locomotion phenotype of the lamin mutant. This argues that the lamin-induced sequestration or retention of tissue specific genes in a heterochromatic compartment drives defects that can be overcome by reversing perinuclear sequestration. To identify the mechanism behind this phenomenon we are using in vivo biochemical tagging in the LMN-1 Y59C mutant and wild-type counterpart to identify perinuclear components that mediate sequestration of muscle-specific gene promoters. By identifying the proteins involved in this process, we can determine the mechanism in which misorganization of chromatin can lead to a loss of tissue integrity.

Davidovich-Cohen, Maya (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Analyzing the role of specific residues in barrier-to-autointegration factor (BAF) in C.elegans"

Davidovich-Cohen, Maya

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Mutations in the nuclear envelope proteins lamins A/C and emerin cause Emery Dreifuss muscular dystrophy (EDMD). The mammalian barrier to autointegration factor (BAF) is present in protein complexes with lamin A and emerin. However, the role of BAF in EDMD is unknown. Previous studies of human BAF identified many residues in which mutations abolished, reduced or enhanced binding to specific partners. Most of these residues conserved in BAF-1 C. elegans, which is 52% identical to human BAF. In C. elegans early embryos BAF-1 is required for chromatin organization, capture segregated chromosomes within the nascent nuclear envelope and to assemble lamin and emerin proteins in reforming nuclei. In turn, BAF-1 requires lamin and emerin proteins to localize at the nuclear periphery. Animals homozygous for a deletion of the entire baf-1 ORF, in which the maternal su pply of BAF-1 is sufficient to complete embryogenesis and larval stages, revealed that BAF-1 is required for nuclear organization and several different tissue-specific pathways including maturation and survival of the germline, the second and third phases of distal tip cell migration, vulva formation and prevention of premature fusion of cells, suggesting specialized roles for BAF-1 in gene expression. BAF-1 also inhibits premature fusion of seam cells by binding to the promoters of EFF-1 and AFF-1 fusogens. Interestingly, BAF-1 is also required to maintain the integrity of specific muscles in the mid-body and tail regions of the adult animal. The requirement of BAF-1 interactions with emerin, lamin and DNA for these functions is currently being investigated by analysis of BAF-1 that has mutation at specific residues in vitro. We are also testing these mutants for physiological rescue of baf-1 null phenotypes. These results probably have direct implications on the mechanism of human muscular dystrophies caused by mutations in either A-type lamins or the protein emerin

He, M. et al. (2017) International Worm Meeting "Implication of LIN-53 in laminopathies."

Seelk, S., He, M., Tursun, B.

[International Worm Meeting, 2017]

Lamin proteins assemble a complex protein meshwork known as the nuclear lamina of the inner nuclear membrane. Besides their structural role, lamins are essential in multiple cellular functions, such as DNA replication, chromatin organization, gene regulation, and cell differentiation. Mutations in the human LMNA gene encoding for Lamin A/C can result in a diverse set of genetic disorders known as laminopathies. Examples comprise Emery-Dreifuss muscular dystrophy (EDMD) characterized by muscle wasting or Hutchinson-Gilford progeria syndrome (HGPS) causing a severe form of premature ageing. To genetically dissect the pathogenic mechanisms of human laminopathies, we introduced the corresponding mutations of patients from our clinic into the lamin gene lmn-1 of C. elegans. To recapitulate the muscle disorders observed in the patients, the mutated lmn-1 forms were specifically expressed in the body wall muscle cells. The previously described EDMD-causing Y59C (Mattout et al., 2012) was employed as a positive control. Motility assays based on swimming revealed that lamin mutations impair the motility of transgenic worms. Immunostaining for muscle proteins detected structural defects in the internal Myosin and Actin filaments as well as in the attachment structures based on Myotactin and Perlecan proteins. Currently, we are also examining worms with expression of lamin mutations in other tissues such as the intestine. The NuRD (Nucleosome Remodeling and Deacetylase) complex has been implicated in HGPS (Pegoraro et al., 2009). Particularly, the histone chaperone RBBP4 (LIN-53 in C. elegans), a core subunit of NuRD, was identified to be an interactor of Lamin A. Moreover, loss of RBBP4 correlates with global ageing-related chromatin defects in both HGPS and normally aged cells. Notably, depletion of the RBBP4 homolog LIN-53 by RNAi or mutation in worms leads to muscle dysfunction and lifespan shortening. Currently, we are testing whether the expression level and cellular localization of LIN-53, as well as histone modifications, are affected in the patient-derived lamin mutants in order to assess the crosstalk between Lamin and LIN-53 in the development of laminopathies.