MAN1 and emerin have overlapping function(s) essential for segregation and cell division in Caenorhabditis elegans

Jun Liu*, Kenneth K. Lee†, Miriam Segura-Totten†, Ester Neufeld‡, Katherine L. Wilson†§, and Yosef Gruenbaum‡§¶

*Department of Molecular Biology and Genetics, 439 Biotechnology Building, Cornell University, Ithaca, NY 14853; †Department of Cell Biology, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205; and ‡Department of Genetics, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

Communicated by Roger D. Kornberg, Stanford University School of Medicine, Stanford, CA, February 11, 2003 (received for review December 11, 2002) Emerin and MAN1 are LEM domain-containing integral membrane family member, named SANE (16), has not yet been localized of the vertebrate . The function of MAN1 definitively. The LEM domains of LAP2 and emerin mediate is unknown, whereas emerin is known to interact with nuclear their direct binding to a chromatin named barrier-to- lamins, barrier-to-autointegration factor (BAF), nesprin-1␣, and a autointegration factor (BAF) (reviewed in ref. 17). All LEM transcription repressor. Mutations in emerin cause X-linked reces- proteins tested (LAP2␣,LAP2␤, and emerin) also have a sive Emery–Dreifuss muscular dystrophy. Emerin and MAN1 ho- separate domain that confers direct binding to A or B type mologs are both conserved in Caenorhabditis elegans, but loss of lamins (3, 18, 19). Ce-emerin has no detectable phenotype. We therefore used C. Only three LEM proteins are conserved in Caenorhabditis elegans to test the hypothesis that Ce-MAN1 overlaps functionally elegans: Ce-MAN1 and Ce-emerin, which have a transmembrane with Ce-emerin. Supporting this model, Ce-MAN1 interacted di- domain, and lem-3, which does not (2, 13). The small number of rectly with Ce-lamin and Ce-BAF in vitro and required Ce-lamin for LEM proteins in C. elegans and the presence of conserved its nuclear envelope localization. Interestingly, RNA interference- encoding BAF and one B type lamin facilitate the study of their mediated removal of Ϸ90% of Ce-MAN1 was lethal to Ϸ15% of functions and interactions in vivo. Reducing the level of either embryos. However, in the absence of Ce-emerin, Ϸ90% reduction lamin or BAF homologs in C. elegans causes abnormal nuclear of Ce-MAN1 was lethal to all embryos by the 100-cell stage, with structure, catastrophic exit from mitosis (chromosome misseg- a phenotype involving repeated cycles of anaphase chromosome regation and anaphase chromosome bridging), and early embry- bridging and cytokinesis [‘‘cell untimely torn’’ (cut) phenotype]. onic lethality (20, 21). In contrast, elimination of emerin has no Immunostaining showed that the anaphase-bridged chromatin detectable phenotype in C. elegans (22). In humans, emerin is specifically retained a mitosis-specific phosphohistone H3 epitope expressed in nearly all tissues, but the null phenotype is restricted and failed to recruit detectable Ce-lamin or Ce-BAF. These findings to skeletal muscles, cardiac function, and major tendons (23), show that LEM domain proteins are essential for cell division and suggesting that the unaffected tissues may express protein(s) that that Ce-emerin and Ce-MAN1 share at least one and possibly overlap functionally with emerin. multiple overlapping functions, which may be relevant to Emery– MAN1 and emerin are the only integral membrane LEM Dreifuss muscular dystrophy. proteins in C. elegans, with similar biochemical extraction prop- erties and cell cycle dynamics (13). We therefore used C. elegans amins form stable networks of filaments located near the to determine whether MAN1 has any functional overlap with Linner membrane of the nucleus and in the nuclear interior emerin. Our results strongly support the ‘‘overlap’’ hypothesis. (reviewed in ref. 1). Lamins interact with diverse partners, Like Ce-emerin, Ce-MAN1 binds directly to Ce-lamin and including membrane proteins emerin and LAP2␤ as well as Ce-BAF in vitro. Although Ce-MAN1 itself was revealed to be histones, transcription factors, and other proteins (reviewed in essential, we found that partial reduction of Ce-MAN1 was lethal ref. 1). This structural network of lamins and associated proteins in cells that completely lacked emerin, demonstrating that is collectively termed the nuclear ‘‘lamina.’’ In metazoan cells, emerin and MAN1 have overlapping functions essential for cell the is essential to maintain the shape and division. integrity of the nucleus and for DNA replication, RNA tran- Materials and Methods scription, chromatin organization, cell cycle regulation, cell Antibodies and Indirect Immunofluorescence Staining of C. elegans. development and differentiation, nuclear migration, and apo- C. elegans (N2) embryos, larvae, and adults were fixed and ptosis (reviewed in refs. 1–4). prepared for indirect immunofluorescence staining as described Specific mutations in nuclear lamina genes cause a wide range (22) by using the following polyclonal antisera: rat anti-Ce- of heritable human diseases, termed (reviewed in MAN1 serum 3597, rat anti-Ce-emerin serum 3598, mouse refs. 5–8). The best-studied is Emery–Dreifuss anti-Ce-emerin serum 3272, and rat anti-UNC-84 serum 3595, muscular dystrophy (EDMD), characterized by early contrac- which were used at a 1:100 dilution (13, 22, 24). Affinity-purified tures of the Achilles, elbow, and neck tendons, progressive rabbit anti-Ce-lamin antibodies were used at a 1:400 dilution muscle wasting, and conduction defects in the heart (9, 10). The (20). Rat serum 3778 against Ce-BAF was generated through X linked form of EDMD is caused by loss of emerin (11), an Covance Research Products (Denver, PA) by using a keyhole integral protein of the nuclear inner membrane. The autosomal limpet hemocyanin-conjugated synthetic peptide (Boston Bio- dominant form of EDMD is caused by missense (and other) Molecules, Woburn, MA; see ref. 13) comprising Ce-BAF mutations in LMNA, the encoding A type lamins (7). Thus, EDMD can result from relatively subtle changes in lamin A filaments or from the loss of a specific protein (emerin) that Abbreviations: EDMD, Emery–Dreifuss muscular dystrophy; BAF, barrier-to-autointegra- binds lamin A. tion factor; dsRNA, double-stranded RNA; RNAi, RNA-mediated interference; DAPI, 4Ј,6- Emerin contains a LEM domain, the defining Ϸ40-residue diamidino-2-phenylindole. motif shared by a family of nuclear proteins that includes LAP2, §K.L.W. and Y.G. contributed equally to this work. emerin, MAN1, lem-3, and otefin (8, 12–15). A newly identified ¶To whom correspondence should be addressed. E-mail: [email protected].

4598–4603 ͉ PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0730821100 Downloaded by guest on September 25, 2021 residues 28–41 plus one cysteine for conjugation (PTYGTKLT- DAGFDKC). Rat anti-BAF was used at a 1:100 dilution. Anti- bodies specific for phosphohistone H3 were from Upstate Group (Waltham, MA). mAb mAb414, which recognizes a subset of nucleoporins in C. elegans (13), was purchased from Babco (Richmond, CA). All secondary antibodies were purchased from The Jackson Laboratory. Double labeling of Ce-MAN1 and Ce-lamin in C. elegans embryos by using immunogold TEM was performed exactly as described (25), with rat anti-Ce-MAN1 serum 3597 diluted 1:30 and rabbit polyclonal anti-Ce-lamin serum diluted 1:10.

RNA-Mediated Interference (RNAi) Experiments. Double-stranded RNA (dsRNA) corresponding to Ce-emerin and Ce-lamin were synthesized as described (refs. 22 and 20, respectively). dsRNA corresponding to Ce-MAN1 residues 147–385 (see Fig. 1) was synthesized by using plasmid pJKL503.1. The dsRNA (0.1–1 ␮g͞␮l) was injected into both gonads of N2 hermaphrodites as described (20, 22, 24). From 12 to 60 h after injection, adults and embryos were either examined for viability as described (20) or fixed and prepared for indirect immunofluorescence staining as described (13).

Ce-MAN1 Expression, Synthesis of [35S]Cysteine͞Methionine-Labeled Proteins, and Blot Overlay Assays. Blot overlay assays were done essentially as described (19). All constructs were verified by sequencing. Each Ce-MAN1 construct was transformed into Escherichia coli strain BL21 (DE3). Transformed cells contain- ing each plasmid were grown to an OD600 of 0.6, and Ce-MAN1 expression was induced by 0.4 mM isopropyl ␤-D-thiogalactoside for 4 h. Cells were pelleted 5 min at 14,000 ϫ g and resuspended in 2ϫ SDS sample buffer. Proteins from unfractionated bacterial lysates were separated on 10% SDS͞PAGE gels, transferred to nitrocellulose membranes (Schleicher & Schuell), and blocked for1hinPBScontaining0.1% Tween 20 5% nonfat dry milk. The expressed proteins had the expected mobility in SDS͞PAGE assays (data not shown). The T7 promoters on expression vectors pET7a (for Ce-lamin) and pET15b (for Ce-BAF) were used to Fig. 1. Localization of lamin and MAN1 in C. elegans and MAN1-binding 35 ͞ drive synthesis of [ S]cysteine methionine-labeled Ce-lamin interactions in vitro.(A) Indirect immunofluorescence double-staining of L1 and Ce-BAF proteins by using the TNT Quick Coupled Tran- larvae for endogenous lamin and MAN1; anterior is upwards. (Bar ϭ 10 ␮m.) scription͞Translation System (Promega), following the manu- (B) Immunogold TEM colocalization of lamin and MAN1 on chromatin in facturer’s protocol. Proteins were transcribed͞translated indi- detergent-extracted nuclei. C. elegans embryos were treated with 0.5% Triton vidually for 90 min at 30°C. Protein lysates from bacteria X-100 to remove nuclear membranes and then immunogold-labeled with expressing recombinant Ce-MAN1 polypeptides were resolved rabbit antibodies against the rod and tail domains of lamin, plus rat serum ͞ 3597 against the C terminus of MAN1. Secondary antibodies were conjugated by SDS PAGE, transferred to PVDF, and washed twice in blot ϭ rinse buffer (10 mM Tris⅐HCl, pH 7.4͞150 mM NaCl͞1mM with 12 nm gold (lamin) or 6 nm gold (MAN1), respectively. (Bar 150 nm.) (C) ͞ Nuclear envelope localization of MAN1 depends on lamin. Lamin-deficient EDTA 0.1% Tween 20) for 5 min at 22–24°C. These blots then [lmn-1(RNAi)] embryos were double-stained by indirect immunofluorescence ␮ 35 ͞ were incubated overnight at 4°C with 20 Ci of [ S]cysteine for endogenous lamin and MAN1. (Bar ϭ 10 ␮m.) (D) Recombinant MAN1 methionine-labeled probe protein (either Ce-BAF or Ce-lamin) fragments tested by blot overlay for binding to lamin and BAF. (Upper) diluted 1:200 into blot rinse buffer containing 0.1% FCS (final Diagram of C. elegans full-length MAN1 protein (500 residues) and recombi- volume, 10 ml), washed twice in blot rinse buffer, dried for 2 h nant fragments MAN-N (residues 1–333) and MAN-C (residues 400–500). at 22–24°C, and exposed to Hyperfilm (Amersham Pharmacia). Shaded boxes indicate the N-terminal LEM domain (gray) and two transmem- brane spans (black). (Lower) Bacterial lysates containing either MAN-N (N) or Results MAN-C (C) were separated on SDS gels, blotted, and probed with in vitro 35 35 Double-immunofluorescence staining of C. elegans with anti- synthesized [ S]lamin or [ S]BAF (Left and Right, respectively). bodies against the endogenous Ce-lamin and Ce-MAN1 showed that they colocalized at the nuclear periphery throughout em- MAN1 (e.g., see figure 3B in ref. 22, which was performed at the bryonic (13), larval (Fig. 1A), and adult development (data not same time). We assume that Ce-MAN1 protein in the lmn- shown). We concluded that Ce-MAN1 is expressed in all cell CELL BIOLOGY types examined and may be ubiquitous in C. elegans. By immu- 1(RNAi) embryos had diffused into the ER. However, the nogold electron microscopy, a C-terminal epitope of Ce-MAN1 background staining was too high to rule out the alternative localized near the nuclear inner membrane (data not shown) and possibility that Ce-MAN1 protein becomes unstable and is remained associated with peripheral chromatin and Ce-lamin in degraded in cells that lack Ce-lamin. To determine whether detergent-extracted nuclei (Fig. 1B; Ce-MAN1, 6 nm gold; Ce-MAN1 bound directly to Ce-lamin, we used [35S]Ce-lamin to Ce-lamin, 12 nm gold). Consistent with a Ce-lamin-dependent probe blots of protein lysates from bacteria that expressed either localization, Ce-MAN1 was undetectable at the nuclear enve- the N-terminal (residues 1–333; MAN-N) or C-terminal (resi- lope when lmn-1 expression was reduced, whereas control dues 400–500; MAN-C) nucleoplasmic domains of Ce-MAN1 embryos had normal overlapping staining of both lamin and (Fig. 1D). [35S]Ce-lamin bound selectively to the N-terminal

Liu et al. PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 ͉ 4599 Downloaded by guest on September 25, 2021 shown). Nonetheless, this 85–90% reduction of Ce-MAN1 pro- tein caused 15% embryonic lethality (n ϭ 200), with most dead embryos arresting after the 2-fold stage (data not shown). 4Ј,6-Diamidino-2-phenylindole (DAPI) staining revealed that in a few (Ͻ1%) early lem-2(RNAi) embryos, pairs of daughter cells were connected by a thin ‘‘bridge’’ of chromatin (Fig. 2E). Interestingly, indirect immunofluorescence staining suggested that Ce-lamin localized correctly around the chromatin masses, except at the sites of chromatin bridges (Fig. 2F). However, the majority of lem-2(RNAi) embryos had normal mitosis and de- veloped into normal fertile adults. This low-penetrance chro- mosome segregation defect was unlikely to be due to mislocal- ization of other nuclear envelope proteins, because we saw no effects on Ce-lamin, Ce-emerin, or UNC84. Instead, the embry- onic lethality and chromosome segregation defects seen in lem-2(RNAi) embryos, which retained 10–15% Ce-MAN1 pro- tein, suggested that Ce-MAN1 might be an essential component of the nuclear envelope, with roles in cell division or early development.

Ce-MAN1 and Ce-emerin Have Overlapping Functions. We attributed the incomplete penetrance of lethality in lem-2(RNAi) em- bryos to two possible causes: residual Ce-MAN1 protein at the nuclear envelope or functional overlap with another pro- tein(s). Note that these possibilities are not mutually exclusive. As mentioned above, Ce-emerin is the only other nuclear membrane-embedded LEM protein in C. elegans (13) and is nonessential (22). To test the hypothesis that Ce-emerin provides functional backup for Ce-MAN1 in C. elegans,wedid double-RNAi experiments to reduce or eliminate both pro- teins. The results were striking, with 100% embryonic lethality Fig. 2. Immunofluorescence staining of lem-2(RNAi) embryos. (A–D) by the 100-cell stage in lem-2(RNAi); emr-1(RNAi) embryos Double-staining of lem-2(RNAi) embryos by indirect immunofluorescence laid 12–36 h after injection of dsRNA. Immunostaining of for endogenous lamin (B and D), plus either MAN1 (serum 3268) (A)or these dead and dying embryos showed complete loss of emerin (serum 3272) (C). (E and F) DAPI and anti-lamin staining of a Ce-emerin protein (Fig. 3 A and B) and Ϸ90% reduction lem-2(RNAi) embryo at the four-cell stage shows a lamin signal surrounding of Ce-MAN1 protein (Fig. 3 C and D). Thus, in the absence of the segregated chromatin (right) but not the anaphase-bridged chromatin (left). (Bars ϭ 10 ␮m.) Ce-emerin, lowering the levels of Ce-MAN1 caused a com- plete arrest of embryonic development. We concluded that Ce-MAN1 and Ce-emerin perform at least one overlapping fragment, not the C-terminal fragment. Parallel blots were essential function in C. elegans. probed with [35S]Ce-BAF to determine whether Ce-BAF binds the LEM domain of Ce-MAN1, as predicted (14, 15). Ce-BAF The lem-2(RNAi); emr-1(RNAi) Embryos Show a cut Phenotype. To bound the N-terminal fragment as expected but, surprisingly, understand the essential overlapping functions of Ce-MAN1 also bound weakly to the C-terminal fragment, which has no and Ce-emerin in C. elegans, we further characterized the defined LEM domain (Fig. 1D). The presence of a second phenotypes of lem-2(RNAi); emr-1(RNAi) embryos, which died Ce-BAF-binding region in Ce-MAN1, although not currently much earlier than embryos reduced for Ce-MAN1 alone. DAPI staining of double-RNAi embryos at the stages when understood, strengthened our conclusion that Ce-BAF interac- they arrested (Ͻ100 cells) showed that more than 50% of the tion was important for Ce-MAN1 function (see below). In nuclei examined had abnormally condensed chromatin (Fig. summary, these biochemical results showed that Ce-MAN1 3E, arrow; n ϭ 30 embryos). This condensed chromatin interacts with two of emerin’s conserved partners, Ce-lamin phenotype probably was not the result of anaphase chromatin and Ce-BAF, thus supporting the idea that Ce-MAN1 and bridges, because nuclei with condensed chromatin were ob- Ce-emerin have overlapping functions at the inner nuclear served even at the one-cell stage (data not shown). Differential membrane. interference contrast time-lapse microscopy was used to follow the fate of nuclei and chromatin in lem-2(RNAi); emr-1(RNAi) Ce-MAN1 Is Essential for Viability. To test the in vivo function of embryos. This analysis showed that unlike loss of Ce-lamin, Ce-MAN1, which is encoded by the lem-2 gene, we used which destabilizes nuclear shape (20), the loss of both Ce- dsRNA-mediated RNAi (26). The Ce-MAN1 protein was very emerin and Ce-MAN1 did not affect nuclear shape. Thus, at stable. We could obtain embryos with significantly reduced, but least some lamina functions were still normal. Microtubule not eliminated, Ce-MAN1 epitopes. Even after injecting high patterns as determined by immunofluorescence also appeared concentrations of lem-2 dsRNA, residual (slightly punctate) normal (data not shown). The most striking phenotype in nuclear envelope staining of Ce-MAN1 was still detectable in lem-2(RNAi); emr-1(RNAi) embryos was anaphase chromatin lem-2(RNAi) embryos (Figs. 2A and 3D). Reduced Ce-MAN1 bridges, which were present as early as the first nuclear had no effect on the localization of Ce-lamin (Fig. 2A), Ce- divisions (Fig. 3F, arrow). The differential interference con- emerin (Fig. 2C), or an unrelated nuclear membrane protein, trast time-lapse analysis showed that these anaphase bridges UNC84 (data not shown). Quantitative analysis (20) revealed a eventually were torn apart; the resulting daughter cells then 6- to 10-fold decrease in the fluorescence intensity of Ce-MAN1 progressed into the next cell cycle and formed more anaphase in lem-2(RNAi) embryos, relative to control embryos (data not bridges (see movie at www.mbg.cornell.edu/liu/liu.html). The

4600 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0730821100 Liu et al. Downloaded by guest on September 25, 2021 Fig. 3. Phenotypes of lem-2(RNAi); emr-1(RNAi) embryos, which arrest by the 100-cell stage. Shown are embryos from mothers injected with dsRNAs against both MAN1 (lem-2) and emerin (emr-1); embryos were stained with DAPI alone (E and F) or DAPI plus antibodies against endogenous emerin (A and B), MAN1 (C and D), or lamin (I and J). Arrow in E indicates unusually condensed chromatin. Arrow in F indicates the anaphase-bridged chromatin in a DAPI-stained four-cell lem-2(RNAi); emr-1(RNAi) embryo. (G and G) Corresponding differential interference contrast microscopy (G) and merged differential interference contrast microscopy͞DAPI (H) images of an embryo in which cytokinesis occurred while daughter cells were still connected by a dense anaphase bridge. (I and J) An embryo stained for endogenous lamin (J); lamin is detected at the nuclear periphery of the two segregated chromatin masses, but not around the anaphase-bridged chromatin. Anaphase-bridged chromatin in an emr-1(RNAi); lem-2(RNAi) embryo stained with DAPI (K) and antibodies specific for a mitosis-specific phospho- histone H3 epitope (L). Arrows in K and L indicate the anaphase-bridged chromatin; arrowheads indicate a late prophase nucleus. Staining is shown for DNA (M), lamin (N), and endogenous BAF (O) in an uninjected control embryo and for DAPI (P), lamin (Q), and endogenous BAF (R)inanemr-1(RNAi); lem-2(RNAi) embryo. (Bars ϭ 10 ␮m.)

formation of anaphase bridges apparently delayed, but did not in its ability to biochemically exit from mitosis and segregate. block, cytokinesis (arrows in Fig. 3 G– I and movie at To our knowledge, this phenotype has not been reported www.mbg.cornell.edu/liu/papers.html). previously. When cells containing anaphase-bridged were immunostained for endogenous Ce-lamin (20), Ce-lamin was Ce-BAF Is Redistributed in the lem-2(RNAi); emr-1(RNAi) Embryos. In present throughout the two daughter nuclei but appeared Xenopus tissue culture cells, most BAF is found in the nucleus absent from anaphase-bridged chromatin (Fig. 3 I and J; see and enriched at the nuclear envelope but is also detectable in low also Fig. 2F). In C. elegans, Ce-lamin protein is completely amounts in cytoplasm (28). If membrane-anchored LEM pro- absent from the spindle envelope only during mid- to late teins were responsible for concentrating BAF near the nuclear anaphase (13). We therefore hypothesized that the chromatin envelope, we predicted that reduction of both Ce-MAN1 and found in daughter nuclei had properly exited from mitosis and Ce-emerin in C. elegans would mislocalize Ce-BAF. We there- initiated nuclear envelope formation, whereas the anaphase- fore immunolocalized endogenous Ce-BAF in lem-2(RNAi); bridged chromatin remained ‘‘mitotic.’’ To test this idea, we emr-1(RNAi) and control (uninjected) embryos. In wild-type and immunostained cells containing anaphase bridges by using an lem-2(RNAi) interphase cells, Ce-BAF staining was enriched CELL BIOLOGY antibody specific for phosphorylated serine 10 on histone H3, near the peripheral lamins (Fig. 3 M–O), consistent with BAF which is specific for mitotic chromatin (reviewed in ref. 27). localization in vertebrate cells (28). However in lem-2(RNAi); Prophase nuclei stained positively for phosphohistone H3, emr-1(RNAi) embryos, Ce-BAF had an abnormally even distri- verifying this marker (Fig. 3 K and L, arrowhead). Supporting bution on the segregated chromatin (possibly reflecting its our model, staining for phosphohistone H3 was positive on continued binding to DNA, lem-3, or other partners) but was anaphase-bridged chromatin and negative for the correspond- undetectable on the anaphase-bridged chromatin (Fig. 3 P–R, ing nuclear chromatin (Fig. 3 K and L, arrows). Thus, for cells arrow). We concluded that Ce-BAF enrichment at the nuclear that lacked both Ce-MAN1 and Ce-emerin, we concluded that envelope depends on its interactions with membrane-anchored anaphase-bridged chromatin was fundamentally compromised LEM proteins.

Liu et al. PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 ͉ 4601 Downloaded by guest on September 25, 2021 Discussion elongation phase of DNA replication (1). However, lamins and In C. elegans, MAN1 shares many features with emerin (22). LEM proteins are also implicated in transcriptional regulation: Both proteins are expressed ubiquitously throughout develop- disruption of the lamin filament network blocks polymerase ment, both have lamin-dependent nuclear envelope localization, II-dependent mRNA transcription (37), and two different and both interact directly with lamin and BAF. However, in stark LEM proteins (LAP2␤ and emerin) can bind directly to the contrast to emerin, which is not essential (22), MAN1 appears to same transcriptional repressor (31, 38). It is worth noting that be essential for viability in C. elegans. This finding raises the reductions in both Ce-MAN1 and Ce-emerin cause embryos to possibility that MAN1 also may be essential in humans. In C. die at the same stage (Ϸ100 cells) as embryos that either lost elegans, reducing the level of MAN1 protein by Ϸ90% caused their RNA polymerase (through RNAi; ref. 39) or were treated 15% of embryos to die, primarily in late stages of embryonic with ␣-amanitin, a potent inhibitor of RNA polymerase (40). development. We speculate that complete loss of MAN1 might Although we did not test transcription activity, our findings are cause lethality at an earlier stage. However, it was highly consistent with the possibility that LEM domain proteins fortuitous that the RNAi method yielded only partial loss (90%) might influence . Obviously, the mechanisms of MAN1 protein; this made possible our discovery that the by which ‘‘mitotically arrested’’ anaphase-bridged chromo- complete elimination of emerin in MAN1-reduced cells was somes result from loss of Ce-MAN1 and Ce-emerin will be lethal at a much earlier (Ϸ100-cell) stage and was fully penetrant important topics for future work. (100% of embryos). This ‘‘enhanced lethality’’ demonstrates The anaphase-bridged chromatin in cells with reduced conclusively that LEM domain proteins are essential for cell MAN1 and emerin had an unexpected characteristic: this division and that emerin has at least one significant function in chromatin retained a mitosis-specific phosphohistone epitope C. elegans that overlaps with MAN1 and prevents the death of even though segregated chromatin in the same cell apparently MAN1-reduced cells. had progressed into G1 phase. Because this aspect of the C. elegans cells with reduced levels of both MAN1 and phenotype was novel, we do not know whether it arises ͞ emerin had an ‘‘anaphase bridge cut’’ phenotype (29). Poten- uniquely from the loss of LEM protein function. The an- ͞ tially similar anaphase bridge cut phenotypes were reported aphase-bridged chromatin also failed to recruit lamins or BAF, previously in C. elegans embryos with reduced lamin (20) or further suggesting that it remained biochemically mitotic. reduced BAF (ref. 21; M.S-T. and K.L.W., unpublished re- These results add to previous evidence that BAF binding to sults). We have shown that both Ce-MAN1 and Ce-emerin can chromatin is mitotically regulated (32). We conclude that LEM interact with Ce-lamin and BAF, that lamins are required to proteins are required for critical (but still unknown) events localize both Ce-emerin and Ce-MAN1 at the nuclear enve- before mitosis that enable chromosome segregation. Thus, the lope, and that proper localization is critical for their function phenotypes seen in emerin͞MAN1 double-RNAi embryos (this work; refs. 22 and 30). This suggests that MAN1 and could arise from a combination of causes, including disrupted emerin are components of an interconnected network of BAF function and the loss of activities that depend directly on lamin- and BAF-binding proteins required for the fundamen- MAN1 and emerin. This functional overlap normally may tal workings of the nucleus. Consistent with this hypothesis, protect many human tissues against the loss of emerin and human BAF, emerin, and lamin interact with nanomolar to could provide a key to identifying the ‘‘lost’’ or uncompensated micromolar affinities and form stable three-way complexes in functions that produce EDMD. vitro (31). Furthermore, BAF appears to be essential in human cells to incorporate emerin, LAP2␤, and A type lamins, but not J.L. thanks Alejandro Sanchez (Carnegie Institution of Washington) for B type lamins, into assembling nuclei (32). Why disrupting this the phospho-H3 antibodies and is grateful to Andy Fire (Carnegie network would lead to the anaphase-bridging phenotype cur- Institution of Washington), in whose lab part of this work was done. This rently is unknown. Many causes, including defects in DNA work was funded by grants from the USA–Israel Binational Science replication (33), topoisomerase II (34), or the spindle check- Foundation, Israel Science Foundation, and Austrian Bank (to Y.G.), point (e.g., securin; refs. 35 and 36), could lead to the the National Institutes of Health (GM64535 to K.L.W.), and the Cornell anaphase-bridging phenotype. Lamins are required for the University new faculty startup fund (to J.L.).

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