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[
International Worm Meeting,
2013]
Proper chromosome organization is important for cell function. A crucial player in packaging DNA into chromosomes is condensin. In C. elegans, two condensin protein complexes promote proper chromosome segregation, and a third regulates X chromosome gene expression. At the heart of each five-subunit condensin complex is a heterodimer of proteins from the SMC (structural maintenance of chromosomes) family of chromosomal ATPases. Eukaryotes have different SMC heterodimer pairs at the core of several complexes involved in chromosome dynamics. Here, we identify a new and unusual SMC protein, SMCL-1 (SMC Like-1), that interacts specifically with condensin SMC subunits and may negatively regulate condensin function. SMCL-1 is atypical in several ways. First, while most SMC proteins bind one other SMC protein, SMCL-1 may interact simultaneously with both members of an SMC heterodimer. Second, although other condensin SMC proteins bind stiochiometrically to all five complex subunits, SMCL-1 preferentially interacts with the SMC subunits. Third, although SMCL-1 associates with condensin SMC proteins, it does not localize to mitotic or X chromosomes like other condensin subunits. Fourth, while mutations of condensin subunits cause lethality, SMCL-1 null mutants are viable. Interestingly, an SMCL-1 null mutant in combination with a condensin hypomorphic mutant partially rescues the lethality of the condensin mutant. This genetic suppression implies that SMCL-1 may negatively regulate condensin function in chromosome segregation and/or X gene regulation. Also, while SMCL-1 shares homology with SMC proteins, it lacks certain domains and has a non-canonical ATPase motif. Together, our results lead us to hypothesize that SMCL-1 negatively regulates the function of one or more condensin complexes by sequestering and preventing activity of their core SMC subunits.
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[
Development & Evolution Meeting,
2008]
The maintenance of genome stability is a fundamental challenge to every organism. In particular, genomic defects in germ cells if transmitted to a fertilized zygote will be present in every cell of the progeny, which could lead to severe developmental defects or even death of the progeny. Here, we report the protein expression pattern and functional requirement for the C. elegans Structural Maintenance of Chromosomes (SMC) 5 and 6 proteins during development and in gametogenesis. We have uncovered a critical role for the SMC-5 and SMC-6 proteins in promoting genome stability in the germ cell lineage of C. elegans. The SMC protein family functions generally to package and maintain the higher-order organization of chromosome. The SMC-5 and SMC-6 proteins were originally identified in yeast for DNA repair function that appeared to be conserved in human cells. However, the impact of SMC-5 and 6 proteins in development is unknown. In order to examine the functions for SMC-5 and SMC-6 in C. elegans, we have generated specific antibodies to the C. elegans SMC-5 and SMC-6 homologs. Using these antibodies, we can demonstrate the association between the C. elegans SMC-5 and SMC-6 proteins in worm extracts and the accumulation of the SMC-5 and 6 proteins in the germ cell lineage. We found that the SMC-5/6 complex accumulated in the primordial germ cells (Z2 and Z3) in embryos. The specific enrichment in the germ cell lineage persisted through development and was also found in the adult germ line, where the SMC-5 and 6 proteins were observed on meiotic chromosomes during gametogenesis and in mature oocytes. Other SMC complexes did not exhibit such a germ line specific accumulation. Consistent with the expression pattern, an
smc-5 deletion mutant strain and wild-type worms after RNAi knockdown of
smc-5 and
smc-6 showed significant reduction in fecundity and other germ line defects. Based on the protein expression pattern and loss-of-function defects for the SMC-5/6 complex in C. elegans and the conserved role for the orthologous complexes in human and yeast for DNA repair, we propose that the C. elegans SMC-5/6 complex functions to prevent mutations or genomic instability in the germ cell lineage. To test this hypothesis, we are examining two experimentally testable predictions drawn from this hypothesis. Preliminary results are consistent with the requirement for the SMC-5/6 complex in reducing the transmission of mutations from gametes to the progeny.
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Mlynarczyk-Evans, S., Zastrow, M., Miley, N., Villeneuve, A., Zhang, Weibin, Chen, G.
[
International Worm Meeting,
2009]
During meiosis, chromosomes assemble specialized structures that promote: 1) establishment and maintenance of homolog pairing, 2) crossing over between homologs and 3) segregation of homologs at the meiosis I division. We are taking several approaches to investigate the dynamic nature of these structures and how their assembly is regulated and coordinated with homolog pairing. During meiotic prophase, axial elements are assembled along the lengths of chromosomes and axes of aligned homologs are connected by the synaptonemal complex (SC). Axial elements are built on a foundation of the cohesin complex, made up of SMC-1, SMC-3, REC-8 and SCC-3. Whereas previous analysis (Chan et al. 2003) had suggested that SMC subunits can load independently of non-SMC subunits, we find that SMC-1 does not localize to chromosomes in
scc-3(
ku263) mutants, implying that SMC loading does require SCC-3. Instead SMC-1 is present in aggregates that also contain SC components SYP-1 and HIM-3. These aggregates are lost in
syp-1 scc-3 double mutants but chromosomal localization of SMC-1 is not restored, implying that SCC-3 plays a positive role in chromosomal loading of cohesin and does not function solely to inhibit premature associations of SC components and cohesin subunits. Our results indicate that SCC-3 is involved in the proper chromosomal assembly of cohesin and SC. Prior to SC assembly, homolog pairing is accompanied by nuclear reorganization of chromosomes into a clustered configuration. Such nuclear reorganization is absent in pairing-defective
hal-2 mutants. In addition to defective pairing,
hal-2 mutants load SYP-1 incorrectly on unpaired homologs. Pairing at the pairing centers is substantially restored in
hal-2;
syp-2 double mutants, implying that incorrect loading of SYP proteins is partially responsible for inhibiting homolog pairing.
hal-2;
syp-2 mutants lack the extended region of clustered chromosome configuration seen in syp mutants, however, implying that HAL-2 has additional roles in promoting normal chromosome organization beyond inhibiting association of SYP proteins with unpaired homologs. Thus, we have identified HAL-2 as a novel component of the meiotic machinery involved in coordination of early meiotic events. While EM images give an impression of the SC as a static scaffold-like structure, recent findings suggest that the SC is more dynamic than previously thought. We will conduct FRAP analysis to investigate SC protein dynamics, using strains expressing GFP- and mCherry-tagged SC components that correctly localize to chromosomes.
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[
International C. elegans Meeting,
2001]
The highly conserved s tructural m aintenance of c hromosome (SMC) proteins serve essential roles in chromosome segregation and condensation in mitosis and meiosis. The typical eukaryotic SMC proteins function within a multimeric complex, containing a pair of SMC heterodimer and other non-SMC subunits. In C. elegans , the SMC proteins have been adapted for a third essential process, dosage compensation. This process equalizes X-linked gene products between XX hermaphrodites and XO males. A dosage compensation specific SMC protein, DPY-27, along with mitotic and meiotic proteins form a complex that reduces gene expression from the hermaphrodite X chromosomes. The s ex-determination and d osage c ompensation (SDC) proteins in hermaphrodites target this d osage c ompensation specific SMC c omplex (DCC) to the X chromosomes. We are interested in identifying and characterizing the components of the SMC complexes in C. elegans to elucidate their functions in dosage compensation and other chromosome dynamics. The SMC protein, MIX-1, serves dual roles in dosage compensation and in mitotic chromosome condensation. Immunoprecipitation of MIX-1 from embryonic extract co-purified a 200-kDa protein, encoded by the ORF Y110A7A.1. Injections of dsRNA against this gene resulted in embryonic lethality. Analysis of the RNAi-treated embryos revealed severe chromosomal abnormalities. Subsequent biochemical experiments demonstrated that Y110A7A.1 associates exclusively with the mitotic SMC complex and not with the DCC. Reiterative BLAST searches found that Y110A7A.1 is distantly related to the XCAP-D2 subunit of the Xenopus mitotic 13 S condensin. Hence we refer to this protein as CeCAP-D2. Interestingly, CeCAP-D2 is also homologous to the dosage compensation protein, DPY-28, which is found in the DCC and not in the mitotic SMC complex. The CeCAP-D2 and DPY-28 proteins may be paralogs, which raises an intriguing possibility that these two proteins may have similar functions within their respective SMC complex. Work is in progress to identify additional proteins that co-precipitate with MIX-1 and CeCAP-D2. Aside from identifying protein co-factors by immunoprecipitation, we are also interested in defining the X sequences bound by the SDC proteins and the DCC. We have developed a chromatin precipitation (ChIP) protocol to show interaction between the SDC proteins and the
her-1 gene, an autosomal gene regulated by SDC proteins. We have examined the DNA precipitated by SDC-2 and SDC-3 antibodies, and found by PCR and southern blot analyses that genomic
her-1 DNA is specifically co-precipitated. We are applying this approach to identify the X sequences bound by the SDC proteins.
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[
International Worm Meeting,
2003]
Dramatic changes in chromosome structure ensure accurate chromosome segregation during cell division. We are studying how the conserved protein complex condensin promotes chromosome organization and segregation in C. elegans. Condensin includes proteins of the SMC (structural maintenance of chromosomes) family, chromosomal ATPases required for a wide variety of chromosome dynamics. C. elegans contains two condensin-like complexes, one with a conserved function in chromosome segregation, and another without mitotic function that regulates X-linked gene expression during dosage compensation. Both complexes share the protein MIX-1, which performs its dual roles in combination with different protein partners. The mitotic C. elegans condensin contains a pair of conserved SMC proteins (MIX-1 and SMC-4), positively supercoils DNA in vitro, and promotes mitotic chromosome organization and segregation in vivo. Time-lapse analysis of MIX-1 or SMC-4 depleted embryos carrying histone-GFP revealed defects in chromosome condensation at prometaphase, yet a surprising degree of compaction by metaphase. Depletion of SMC-4 or MIX-1 caused failed mitotic and meiotic chromosome segregation. Interestingly, SMC-4 and MIX-1 localized to condensed chromosomes in a pattern that resembled that of centromere proteins. Depletion of SMC-4 or MIX-1 did not prevent the mitotic association of centromere proteins such as HCP-3/CENP-A, but did disrupt their restricted bi-polar localization towards the spindle poles. Thus, condensin may help build and orient the centromere as it organizes mitotic chromosome structure. We have performed mass spectrometry to identify proteins immunoprecipitated with SMC-4 or centromere component antibodies. Identified proteins were assayed for function by RNAi in a strain carrying histone-GFP and tubulin-GFP. Proteins in the SMC-4 IP include homologs of condensin subunits as well as previously uncharacterized factors that produce striking chromosome segregation phenotypes by RNAi. This proteomic approach appears to be productive for further exploring condensin function and for identifying novel chromosome segregation factors.
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[
West Coast Worm Meeting,
2002]
As a cell divides, a complete copy of the genome must be faithfully segregated to each new daughter cell. Dramatic changes in chromosome structure are necessary to ensure accurate chromosome separation. We are studying how a conserved protein complex called condensin promotes chromosome condensation and segregation. Condensin contains proteins of the SMC (structural maintenance of chromosomes) family, conserved chromosomal ATPases required for various aspects of chromosome dynamics. C. elegans contains two condensin-like complexes, one with a conserved function in chromosome segregation, and another without mitotic function that regulates X-linked gene expression during dosage compensation. Both complexes share the protein MIX-1, which performs its dual roles in combination with different proteins partners. The mitotic C. elegans condensin, like condensin in other organisms, contains a pair of conserved SMC proteins (MIX-1 and SMC-4), positively supercoils DNA in vitro, and promotes mitotic chromosome structure and segregation in vivo. Time-lapse analysis of MIX-1 or SMC-4 depleted embryos carrying histone-GFP revealed defects in chromosome condensation at prometaphase, yet a surprising degree of compaction by metaphase. SMC-4 or MIX-1 RNAi caused failed sister chromatid separation in both mitosis and meiosis II, although defects in homolog separation at meiosis I were not observed. We are currently investigating the meiotic localization and function of this complex in more detail in both sperm and oocytes. Interestingly, SMC-4 and MIX-1 localized to the outer faces of condensed mitotic chromosomes in a pattern that resembled that of centromere proteins, and required the AIR-2/Aurora-B kinase for this localization. RNAi of SMC-4 or MIX-1 did not prevent the mitotic association of centromere proteins such as HCP-3/CENP-A, but did disrupt their restricted localization towards the spindle poles. Thus, condensin may help build and position the centromere as it organizes mitotic chromosome structure. We are performing mass spectrometry to identify proteins immunoprecipitated with SMC-4 or centromere component antibodies, to determine whether condensin physically interacts with centromere proteins and to identify other members of the SMC-4/MIX-1 complex.
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[
International C. elegans Meeting,
1999]
Members of the highly conserved SMC (structural maintenance of chromosomes) protein family are involved in several aspects of chromosome dynamics. From yeast to man, SMC proteins are essential for processes such as mitotic chromosome condensation, sister chromosome cohesion, and recombinational DNA repair. We are investigating the C. elegans homologs of this chromosomal protein family, and have found that they play critical roles in dosage compensation, mitosis, and meiosis. The SMC proteins DPY-27 and MIX-1 are part of the dosage compensation protein complex that binds to hermaphrodite X chromosomes and downregulates their transcription. In addition, MIX-1 associates with all mitotic chromosomes in both sexes and is essential for their proper segregation. A third SMC family member, HIM-1, appears to function in meiosis, perhaps for sister chromatid cohesion (see abstract from A. Chan, D. Pasqualone, T. Wu, and B.J. Meyer.) Characterization of the only other C. elegans SMC homolog, SLP-2 (SMC-like protein 2) is presented here. SLP-2 RNA interference produces dead embryos with abnormal DNA bodies, most likely resulting from defective mitotic chromosome segregation. Transgenic worms carrying extra wild type copies of the SLP-2 gene exhibit lethality, with a high incidence of males among the survivors. This Him phenotype implies that misexpression of SLP-2 also disrupts chromosome segregation during meiosis. Preliminary immunolocalization studies indicate that the SLP-2 protein is not specifically localized to the X, but rather is associated with all chromosomes in both the germline and in embryos. To investigate potential associations between SLP-2 and other SMC proteins, co-immunoprecipitation studies were performed. In embryonic extracts, SLP-2 co-immunoprecipitates with MIX-1, but not with DPY-27. Based on these results, we speculate that while DPY-27 is a partner for MIX-1 within the dosage compensation protein complex, SLP-2 is the partner for MIX-1 in a complex that executes mitotic chromosome segregation. We hope that continued study of the composition and function of SMC protein complexes will provide a greater understanding of their influence on chromosome mechanics.
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[
International C. elegans Meeting,
1997]
Members of the highly conserved SMC protein family participate in various aspects of chromosome dynamics in organisms ranging from bacteria to humans. SMC proteins function primarily in the structural maintenance and segregation of condensed mitotic chromosomes. In addition, a role for some SMC family members in DNA recombination has been demonstrated. In C. elegans, four SMC homologs have been identified thus far. Two of these homologs, DPY-27 and MIX-1 (see abstract, Lieb et al.), are members of the dosage compensation protein complex that specifically localizes to the X chromosomes in hermaphrodites to reduce their transcript levels. This repression may be achieved by a DPY-27/MIX-1-mediated alteration of higher order X chromosome structure. In addition, MIX-1 localizes to all mitotic chromosomes and is required for their segregation. The characterization of the two remaining SMC homologs, SLP-1 and SLP-2 (SMC-like proteins), is reported here. Specific antibodies to both of these proteins show a similar localization pattern as revealed by indirect immunofluorescence in situ. SLP-1 and SLP-2 colocalize with chromosomal DNA in interphase nuclei, but they do not localize to condensed metaphase chromosomes. Interestingly, SLP-1 shows a non-uniform and occasionally punctate distribution within interphase nuclei. This punctate localization pattern persists and appears to surround condensed chromosomes during metaphase. These observations suggest that SLP-1 may function within particular chromosome domains. In addition, RNA interference studies of SLP-2 reveal catastrophic effects on chromosome segregation in early embryonic divisions, suggesting that SLP-2 is required for this process. Continued study of these two proteins at the cytological, biochemical and genetic levels will further elucidate their function in chromosome dynamics and will broaden our overall understanding of the SMC protein family.
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[
West Coast Worm Meeting,
2000]
From bacteria to man, the highly conserved SMC (structural maintenance of chromosomes) protein family is required for chromosome segregation and cell division. In C. elegans SMC proteins also direct X chromosome dosage compensation. We are studying the composition and function of SMC protein complexes and how individual SMC proteins participate in more than one chromosomal process. For example, how does the SMC protein MIX-1, essential for both mitosis and dosage compensation, achieve its dual function within a single cell? MIX-1 requires the SMC protein DPY-27 for its role in dosage compensation and X localization, but DPY-27 plays no role in mitosis. Thus, it seemed likely that MIX-1 would have a different SMC partner for mitosis. Searching the C. elegans genome revealed another SMC homolog, SLP-2 (SMC-like protein-2.) The RNAi phenotype and protein localization of SLP-2 suggested its involvement in mitosis. Like MIX-1, SLP-2 RNAi produces dead embryos with defects such as chromatin bridges and abnormally large nuclei. Time-lapse microscopy shows a failure in chromosome segregation, and fluorescent in situ hybridization reveals nuclei with abnormally high DNA content. Thus, loss of SLP-2 prevents chromosome segregation, but not DNA replication and cell cycle progression. SLP-2 co-localizes with MIX-1 on mitotic chromosomes in embryos and in the germline. SLP-2 and MIX-1 surround chromosomes as they condense, then appear on the poleward face of chromosomes aligned at metaphase, where they remain until they disappear at telophase. The idea that MIX-1 partners with SLP-2 for mitosis, but with DPY-27 for dosage compensation, is further supported by immunoprecipitation (IP) results. Co-IP from embryonic extracts is observed between SLP-2 and MIX-1, but not between SLP-2 and DPY-27. Moreover, IPs show that SLP-2 and MIX-1 are part of a large protein complex. The identities of these subunits are being explored by mass spectrometry. One subunit (see R. Chan, et al.) shares homology with both a conserved component of the mitotic complex in other organisms, and a component of the C. elegans dosage compensation complex. It will be interesting to learn if the mitotic and dosage compensation complexes share additional components, and to what extent the biochemical activities of these complexes are related.
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[
International C. elegans Meeting,
2001]
The ordered compaction of chromosomes into two condensed and paired sister chromatids is a structural problem of all mitotic cells. If chromosomes fail to establish cohesion or to condense, they can become entangled, and break or missegregate at anaphase. The highly conserved SMC (structural maintenance of chromosomes) protein family directs these critical aspects of chromosome segregation. SMC proteins generally associate with an SMC partner and non-SMC proteins to form large complexes. An SMC complex called condensin was described in other organisms for its role in condensing mitotic chromosomes. In C. elegans , a homologous complex directs X dosage compensation. We are studying the composition and function of SMC complexes, and have found individual SMC proteins that participate in more than one chromosomal process. For example, MIX-1 is a condensin SMC homolog essential for both mitosis and dosage compensation. How does MIX-1 achieve its dual function within a single cell? MIX-1 requires the SMC protein DPY-27 for its role in dosage compensation and its X localization, but DPY-27 plays no role in mitosis. Thus, it seemed likely that MIX-1 would have a different SMC partner for mitosis. Searching the genome revealed another DPY-27-like SMC protein, CeSMC-4. The RNAi phenotype and protein localization of CeSMC-4 indicate its involvement in mitosis. Like MIX-1, CeSMC-4 RNAi produces dead embryos with defects such as anaphase chromatin bridges and extremely large nuclei. In support of the idea that CeSMC-4 and MIX-1 are SMC partners for mitosis, MIX-1 protein is undetectable in CeSMC-4 RNAi embryos. Using histone::GFP and time-lapse microscopy, we see that chromosomes are poorly organized as they condense at prophase and metaphase, remain entangled at anaphase, and eventually become pulled into one cell which continues more cycles of apparent replication without division. Meiotic chromosome segregation defects are also observed. In contrast to the expectation from studies in Xenopus, loss of C. elegans condensin causes chromosomes to be poorly organized but still substantially compacted, and the more dramatic phenotype is instead the chromatin bridges at anaphase. CeSMC-4 and MIX-1 associate with chromosomes only during stages of the cell cycle when chromosomes are condensed. The mitotic kinase AIR-2, responsible for the mitosis-specific phosphorylation of histone H3, is required for CeSMC-4 and MIX-1 localization. CeSMC-4 and MIX-1 proteins do not coat the entire metaphase plate, but rather appear on the poleward face in a punctate pattern coincident with the centromere protein HCP-3, and internal to the kinetochore protein HIM-10. However, localization of these proteins is not interdependent. Immunoprecipitation (IP) experiments substantiate that MIX-1 partners with CeSMC-4 for mitosis, but DPY-27 for dosage compensation. Co-IP from embryonic extracts is observed between CeSMC-4 and MIX-1, but not between CeSMC-4 and DPY-27. Moreover, IPs show that CeSMC-4 and MIX-1 are part of a large protein complex whose additional components are being investigated by mass spectrometry. One subunit called CeCAP-D2 (see R. Chan et al.) shares homology with both a conserved non-SMC component of the mitotic complex in other organisms, and a component of the C. elegans dosage compensation complex. In collaboration with the Cozzarelli lab, the mitotic complex was shown to drive ATP-dependent positive DNA supercoiling in vitro, a conserved activity thought to reflect its role in chromosome condensation. It will be interesting to learn if the mitotic and dosage compensation complexes share additional components, and to what extent the biochemical activities of these two condensin-like complexes are related.