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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2003 Apr 8;100(8):4598–4603. doi: 10.1073/pnas.0730821100

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

Jun Liu *, Kenneth K Lee †, Miriam Segura-Totten †, Ester Neufeld ‡, Katherine L Wilson †,§, Yosef Gruenbaum ‡,§,
PMCID: PMC153601  PMID: 12684533

Abstract

Emerin and MAN1 are LEM domain-containing integral membrane proteins of the vertebrate nuclear envelope. The function of MAN1 is unknown, whereas emerin is known to interact with nuclear lamins, barrier-to-autointegration factor (BAF), nesprin-1α, and a transcription repressor. Mutations in emerin cause X-linked recessive Emery–Dreifuss muscular dystrophy. Emerin and MAN1 homologs are both conserved in Caenorhabditis elegans, but loss of Ce-emerin has no detectable phenotype. We therefore used C. elegans to test the hypothesis that Ce-MAN1 overlaps functionally with Ce-emerin. Supporting this model, Ce-MAN1 interacted directly with Ce-lamin and Ce-BAF in vitro and required Ce-lamin for its nuclear envelope localization. Interestingly, RNA interference-mediated removal of ≈90% of Ce-MAN1 was lethal to ≈15% of embryos. However, in the absence of Ce-emerin, ≈90% reduction of Ce-MAN1 was lethal to all embryos by the 100-cell stage, with a phenotype involving repeated cycles of anaphase chromosome bridging and cytokinesis [“cell untimely torn” (cut) phenotype]. Immunostaining showed that the anaphase-bridged chromatin specifically retained a mitosis-specific phosphohistone H3 epitope and failed to recruit detectable Ce-lamin or Ce-BAF. These findings show that LEM domain proteins are essential for cell division and that Ce-emerin and Ce-MAN1 share at least one and possibly multiple overlapping functions, which may be relevant to Emery–Dreifuss muscular dystrophy.

Lamins form stable networks of filaments located near the inner membrane of the nucleus and in the nuclear interior (reviewed in ref. 1). Lamins interact with diverse partners, including membrane proteins emerin and LAP2β as well as histones, transcription factors, and other proteins (reviewed in ref. 1). This structural network of lamins and associated proteins is collectively termed the nuclear “lamina.” In metazoan cells, the nuclear lamina is essential to maintain the shape and integrity of the nucleus and for DNA replication, RNA transcription, chromatin organization, cell cycle regulation, cell development and differentiation, nuclear migration, and apoptosis (reviewed in refs. 1–4).

Specific mutations in nuclear lamina genes cause a wide range of heritable human diseases, termed laminopathies (reviewed in refs. 5–8). The best-studied laminopathy is Emery–Dreifuss muscular dystrophy (EDMD), characterized by early contractures of the Achilles, elbow, and neck tendons, progressive muscle wasting, and conduction defects in the heart (9, 10). The X linked form of EDMD is caused by loss of emerin (11), an integral protein of the nuclear inner membrane. The autosomal dominant form of EDMD is caused by missense (and other) mutations in LMNA, the gene 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 binds lamin A.

Emerin contains a LEM domain, the defining ≈40-residue motif shared by a family of nuclear proteins that includes LAP2, emerin, MAN1, lem-3, and otefin (8, 12–15). A newly identified family member, named SANE (16), has not yet been localized definitively. The LEM domains of LAP2 and emerin mediate their direct binding to a chromatin protein named barrier-to-autointegration factor (BAF) (reviewed in ref. 17). All LEM proteins tested (LAP2α, LAP2β, and emerin) also have a separate domain that confers direct binding to A or B type lamins (3, 18, 19).

Only three LEM proteins are conserved in Caenorhabditis elegans: Ce-MAN1 and Ce-emerin, which have a transmembrane domain, and lem-3, which does not (2, 13). The small number of LEM proteins in C. elegans and the presence of conserved genes encoding BAF and one B type lamin facilitate the study of their functions and interactions in vivo. Reducing the level of either lamin or BAF homologs in C. elegans causes abnormal nuclear structure, catastrophic exit from mitosis (chromosome missegregation and anaphase chromosome bridging), and early embryonic lethality (20, 21). In contrast, elimination of emerin has no detectable phenotype in C. elegans (22). In humans, emerin is expressed in nearly all tissues, but the null phenotype is restricted to skeletal muscles, cardiac function, and major tendons (23), suggesting that the unaffected tissues may express protein(s) that overlap functionally with emerin.

MAN1 and emerin are the only integral membrane LEM proteins in C. elegans, with similar biochemical extraction properties and cell cycle dynamics (13). We therefore used C. elegans to determine whether MAN1 has any functional overlap with emerin. Our results strongly support the “overlap” hypothesis. Like Ce-emerin, Ce-MAN1 binds directly to Ce-lamin and Ce-BAF in vitro. Although Ce-MAN1 itself was revealed to be essential, we found that partial reduction of Ce-MAN1 was lethal in cells that completely lacked emerin, demonstrating that emerin and MAN1 have overlapping functions essential for cell division.

Materials and Methods

Antibodies and Indirect Immunofluorescence Staining of C. elegans.

C. elegans (N2) embryos, larvae, and adults were fixed and prepared for indirect immunofluorescence staining as described (22) by using the following polyclonal antisera: rat anti-Ce-MAN1 serum 3597, rat anti-Ce-emerin serum 3598, mouse anti-Ce-emerin serum 3272, and rat anti-UNC-84 serum 3595, which were used at a 1:100 dilution (13, 22, 24). Affinity-purified rabbit anti-Ce-lamin antibodies were used at a 1:400 dilution (20). Rat serum 3778 against Ce-BAF was generated through Covance Research Products (Denver, PA) by using a keyhole limpet hemocyanin-conjugated synthetic peptide (Boston BioMolecules, Woburn, MA; see ref. 13) comprising Ce-BAF residues 28–41 plus one cysteine for conjugation (PTYGTKLTDAGFDKC). Rat anti-BAF was used at a 1:100 dilution. Antibodies 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 μg/μl) 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).

Figure 1.

Figure 1

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Localization of lamin and MAN1 in C. elegans and MAN1-binding interactions in vitro. (A) Indirect immunofluorescence double-staining of L1 larvae for endogenous lamin and MAN1; anterior is upwards. (Bar = 10 μm.) (B) Immunogold TEM colocalization of lamin and MAN1 on chromatin in detergent-extracted nuclei. C. elegans embryos were treated with 0.5% Triton X-100 to remove nuclear membranes and then immunogold-labeled with rabbit antibodies against the rod and tail domains of lamin, plus rat serum 3597 against the C terminus of MAN1. Secondary antibodies were conjugated 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 [lmn-1(RNAi)] embryos were double-stained by indirect immunofluorescence for endogenous lamin and MAN1. (Bar = 10 μm.) (D) Recombinant MAN1 fragments tested by blot overlay for binding to lamin and BAF. (Upper) Diagram of C. elegans full-length MAN1 protein (500 residues) and recombinant fragments MAN-N (residues 1–333) and MAN-C (residues 400–500). Shaded boxes indicate the N-terminal LEM domain (gray) and two transmembrane spans (black). (Lower) Bacterial lysates containing either MAN-N (N) or MAN-C (C) were separated on SDS gels, blotted, and probed with in vitro synthesized [35S]lamin or [35S]BAF (Left and Right, respectively).

Ce-MAN1 Expression, Synthesis of [35S]Cysteine/Methionine-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 containing each plasmid were grown to an OD600 of 0.6, and Ce-MAN1 expression was induced by 0.4 mM isopropyl β-d-thiogalactoside for 4 h. Cells were pelleted 5 min at 14,000 × g and resuspended in 2× SDS sample buffer. Proteins from unfractionated bacterial lysates were separated on 10% SDS/PAGE gels, transferred to nitrocellulose membranes (Schleicher & Schuell), and blocked for 1 h in PBS containing 0.1% Tween 20 5% nonfat dry milk. The expressed proteins had the expected mobility in SDS/PAGE assays (data not shown). The T7 promoters on expression vectors pET7a (for Ce-lamin) and pET15b (for Ce-BAF) were used to drive synthesis of [35S]cysteine/methionine-labeled Ce-lamin and Ce-BAF proteins by using the TNT Quick Coupled Transcription/Translation System (Promega), following the manufacturer's protocol. Proteins were transcribed/translated individually for 90 min at 30°C. Protein lysates from bacteria expressing recombinant Ce-MAN1 polypeptides were resolved by SDS/PAGE, transferred to PVDF, and washed twice in blot rinse buffer (10 mM Tris⋅HCl, pH 7.4/150 mM NaCl/1 mM EDTA/0.1% Tween 20) for 5 min at 22–24°C. These blots then were incubated overnight at 4°C with 20 μCi of [35S]cysteine/methionine-labeled probe protein (either Ce-BAF or Ce-lamin) diluted 1:200 into blot rinse buffer containing 0.1% FCS (final volume, 10 ml), washed twice in blot rinse buffer, dried for 2 h at 22–24°C, and exposed to Hyperfilm (Amersham Pharmacia).

Results

Double-immunofluorescence staining of C. elegans with antibodies against the endogenous Ce-lamin and Ce-MAN1 showed that they colocalized at the nuclear periphery throughout embryonic (13), larval (Fig. 1A), and adult development (data not shown). We concluded that Ce-MAN1 is expressed in all cell types examined and may be ubiquitous in C. elegans. By immunogold electron microscopy, a C-terminal epitope of Ce-MAN1 localized near the nuclear inner membrane (data not shown) and remained associated with peripheral chromatin and Ce-lamin in detergent-extracted nuclei (Fig. 1B; Ce-MAN1, 6 nm gold; Ce-lamin, 12 nm gold). Consistent with a Ce-lamin-dependent localization, Ce-MAN1 was undetectable at the nuclear envelope when lmn-1 expression was reduced, whereas control embryos had normal overlapping staining of both lamin and MAN1 (e.g., see figure 3B in ref. 22, which was performed at the same time). We assume that Ce-MAN1 protein in the lmn-1(RNAi) embryos had diffused into the ER. However, the background staining was too high to rule out the alternative possibility that Ce-MAN1 protein becomes unstable and is degraded in cells that lack Ce-lamin. To determine whether Ce-MAN1 bound directly to Ce-lamin, we used [35S]Ce-lamin to probe blots of protein lysates from bacteria that expressed either the N-terminal (residues 1–333; MAN-N) or C-terminal (residues 400–500; MAN-C) nucleoplasmic domains of Ce-MAN1 (Fig. 1D). [35S]Ce-lamin bound selectively to the N-terminal fragment, not the C-terminal fragment. Parallel blots were probed with [35S]Ce-BAF to determine whether Ce-BAF binds the LEM domain of Ce-MAN1, as predicted (14, 15). Ce-BAF bound the N-terminal fragment as expected but, surprisingly, also bound weakly to the C-terminal fragment, which has no defined LEM domain (Fig. 1D). The presence of a second Ce-BAF-binding region in Ce-MAN1, although not currently understood, strengthened our conclusion that Ce-BAF interaction was important for Ce-MAN1 function (see below). In summary, these biochemical results showed that Ce-MAN1 interacts with two of emerin's conserved partners, Ce-lamin and Ce-BAF, thus supporting the idea that Ce-MAN1 and Ce-emerin have overlapping functions at the inner nuclear membrane.

Ce-MAN1 Is Essential for Viability.

To test the in vivo function of Ce-MAN1, which is encoded by the lem-2 gene, we used dsRNA-mediated RNAi (26). The Ce-MAN1 protein was very stable. We could obtain embryos with significantly reduced, but not eliminated, Ce-MAN1 epitopes. Even after injecting high concentrations of lem-2 dsRNA, residual (slightly punctate) nuclear envelope staining of Ce-MAN1 was still detectable in lem-2(RNAi) embryos (Figs. 2A and 3D). Reduced Ce-MAN1 had no effect on the localization of Ce-lamin (Fig. 2A), Ce-emerin (Fig. 2C), or an unrelated nuclear membrane protein, UNC84 (data not shown). Quantitative analysis (20) revealed a 6- to 10-fold decrease in the fluorescence intensity of Ce-MAN1 in lem-2(RNAi) embryos, relative to control embryos (data not shown). Nonetheless, this 85–90% reduction of Ce-MAN1 protein caused 15% embryonic lethality (n = 200), with most dead embryos arresting after the 2-fold stage (data not shown). 4′,6-Diamidino-2-phenylindole (DAPI) staining revealed that in a few (<1%) 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 developed into normal fertile adults. This low-penetrance chromosome segregation defect was unlikely to be due to mislocalization of other nuclear envelope proteins, because we saw no effects on Ce-lamin, Ce-emerin, or UNC84. Instead, the embryonic lethality and chromosome segregation defects seen in lem-2(RNAi) embryos, which retained 10–15% Ce-MAN1 protein, suggested that Ce-MAN1 might be an essential component of the nuclear envelope, with roles in cell division or early development.

Figure 2.

Figure 2

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Immunofluorescence staining of lem-2(RNAi) embryos. (A–D) Double-staining of lem-2(RNAi) embryos by indirect immunofluorescence for endogenous lamin (B and D), plus either MAN1 (serum 3268) (A) or emerin (serum 3272) (C). (E and F) DAPI and anti-lamin staining of a lem-2(RNAi) embryo at the four-cell stage shows a lamin signal surrounding the segregated chromatin (right) but not the anaphase-bridged chromatin (left). (Bars = 10 μm.)

Figure 3.

Figure 3

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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 microscopy/DAPI (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 phosphohistone 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) in an emr-1(RNAi); lem-2(RNAi) embryo. (Bars = 10 μm.)

Ce-MAN1 and Ce-emerin Have Overlapping Functions.

We attributed the incomplete penetrance of lethality in lem-2(RNAi) embryos to two possible causes: residual Ce-MAN1 protein at the nuclear envelope or functional overlap with another protein(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, we did double-RNAi experiments to reduce or eliminate both proteins. The results were striking, with 100% embryonic lethality by the 100-cell stage in lem-2(RNAi); emr-1(RNAi) embryos laid 12–36 h after injection of dsRNA. Immunostaining of these dead and dying embryos showed complete loss of Ce-emerin protein (Fig. 3 A and B) and ≈90% reduction of Ce-MAN1 protein (Fig. 3 C and D). Thus, in the absence of Ce-emerin, lowering the levels of Ce-MAN1 caused a complete arrest of embryonic development. We concluded that Ce-MAN1 and Ce-emerin perform at least one overlapping essential function in C. elegans.

The lem-2(RNAi); emr-1(RNAi) Embryos Show a cut Phenotype.

To understand the essential overlapping functions of Ce-MAN1 and Ce-emerin in C. elegans, we further characterized the phenotypes of lem-2(RNAi); emr-1(RNAi) embryos, which died much earlier than embryos reduced for Ce-MAN1 alone. DAPI staining of double-RNAi embryos at the stages when they arrested (<100 cells) showed that more than 50% of the nuclei examined had abnormally condensed chromatin (Fig. 3E, arrow; n = 30 embryos). This condensed chromatin phenotype probably was not the result of anaphase chromatin bridges, because nuclei with condensed chromatin were observed even at the one-cell stage (data not shown). Differential interference contrast time-lapse microscopy was used to follow the fate of nuclei and chromatin in lem-2(RNAi); emr-1(RNAi) embryos. This analysis showed that unlike loss of Ce-lamin, which destabilizes nuclear shape (20), the loss of both Ce-emerin and Ce-MAN1 did not affect nuclear shape. Thus, at least some lamina functions were still normal. Microtubule patterns as determined by immunofluorescence also appeared normal (data not shown). The most striking phenotype in lem-2(RNAi); emr-1(RNAi) embryos was anaphase chromatin bridges, which were present as early as the first nuclear divisions (Fig. 3F, arrow). The differential interference contrast time-lapse analysis showed that these anaphase bridges eventually were torn apart; the resulting daughter cells then progressed into the next cell cycle and formed more anaphase bridges (see movie at www.mbg.cornell.edu/liu/liu.html). The formation of anaphase bridges apparently delayed, but did not block, cytokinesis (arrows in Fig. 3 G– I and movie at www.mbg.cornell.edu/liu/papers.html).

When cells containing anaphase-bridged chromosomes were immunostained for endogenous Ce-lamin (20), Ce-lamin was present throughout the two daughter nuclei but appeared absent from anaphase-bridged chromatin (Fig. 3 I and J; see also Fig. 2F). In C. elegans, Ce-lamin protein is completely absent from the spindle envelope only during mid- to late anaphase (13). We therefore hypothesized that the chromatin found in daughter nuclei had properly exited from mitosis and initiated nuclear envelope formation, whereas the anaphase-bridged chromatin remained “mitotic.” To test this idea, we immunostained cells containing anaphase bridges by using an antibody specific for phosphorylated serine 10 on histone H3, which is specific for mitotic chromatin (reviewed in ref. 27). Prophase nuclei stained positively for phosphohistone H3, verifying this marker (Fig. 3 K and L, arrowhead). Supporting our model, staining for phosphohistone H3 was positive on anaphase-bridged chromatin and negative for the corresponding nuclear chromatin (Fig. 3 K and L, arrows). Thus, for cells that lacked both Ce-MAN1 and Ce-emerin, we concluded that anaphase-bridged chromatin was fundamentally compromised in its ability to biochemically exit from mitosis and segregate. To our knowledge, this phenotype has not been reported previously.

Ce-BAF Is Redistributed in the lem-2(RNAi); emr-1(RNAi) Embryos.

In Xenopus tissue culture cells, most BAF is found in the nucleus and enriched at the nuclear envelope but is also detectable in low amounts in cytoplasm (28). If membrane-anchored LEM proteins were responsible for concentrating BAF near the nuclear envelope, we predicted that reduction of both Ce-MAN1 and Ce-emerin in C. elegans would mislocalize Ce-BAF. We therefore immunolocalized endogenous Ce-BAF in lem-2(RNAi); emr-1(RNAi) and control (uninjected) embryos. In wild-type and lem-2(RNAi) interphase cells, Ce-BAF staining was enriched near the peripheral lamins (Fig. 3 M–O), consistent with BAF localization in vertebrate cells (28). However in lem-2(RNAi); emr-1(RNAi) embryos, Ce-BAF had an abnormally even distribution on the segregated chromatin (possibly reflecting its continued binding to DNA, lem-3, or other partners) but was undetectable on the anaphase-bridged chromatin (Fig. 3 P–R, arrow). We concluded that Ce-BAF enrichment at the nuclear envelope depends on its interactions with membrane-anchored LEM proteins.

Discussion

In C. elegans, MAN1 shares many features with emerin (22). Both proteins are expressed ubiquitously throughout development, both have lamin-dependent nuclear envelope localization, and both interact directly with lamin and BAF. However, in stark contrast to emerin, which is not essential (22), MAN1 appears to be essential for viability in C. elegans. This finding raises the possibility that MAN1 also may be essential in humans. In C. elegans, reducing the level of MAN1 protein by ≈90% caused 15% of embryos to die, primarily in late stages of embryonic development. We speculate that complete loss of MAN1 might cause lethality at an earlier stage. However, it was highly fortuitous that the RNAi method yielded only partial loss (90%) of MAN1 protein; this made possible our discovery that the complete elimination of emerin in MAN1-reduced cells was lethal at a much earlier (≈100-cell) stage and was fully penetrant (100% of embryos). This “enhanced lethality” demonstrates conclusively that LEM domain proteins are essential for cell division and that emerin has at least one significant function in C. elegans that overlaps with MAN1 and prevents the death of MAN1-reduced cells.

C. elegans cells with reduced levels of both MAN1 and emerin had an “anaphase bridge/cut” phenotype (29). Potentially similar anaphase bridge/cut phenotypes were reported previously in C. elegans embryos with reduced lamin (20) or reduced BAF (ref. 21; M.S-T. and K.L.W., unpublished results). We have shown that both Ce-MAN1 and Ce-emerin can interact with Ce-lamin and BAF, that lamins are required to localize both Ce-emerin and Ce-MAN1 at the nuclear envelope, and that proper localization is critical for their function (this work; refs. 22 and 30). This suggests that MAN1 and emerin are components of an interconnected network of lamin- and BAF-binding proteins required for the fundamental workings of the nucleus. Consistent with this hypothesis, human BAF, emerin, and lamin interact with nanomolar to micromolar affinities and form stable three-way complexes in vitro (31). Furthermore, BAF appears to be essential in human cells to incorporate emerin, LAP2β, and A type lamins, but not B type lamins, into assembling nuclei (32). Why disrupting this network would lead to the anaphase-bridging phenotype currently is unknown. Many causes, including defects in DNA replication (33), topoisomerase II (34), or the spindle checkpoint (e.g., securin; refs. 35 and 36), could lead to the anaphase-bridging phenotype. Lamins are required for the elongation phase of DNA replication (1). However, lamins and LEM proteins are also implicated in transcriptional regulation: disruption of the lamin filament network blocks polymerase II-dependent mRNA transcription (37), and two different LEM proteins (LAP2β and emerin) can bind directly to the same transcriptional repressor (31, 38). It is worth noting that reductions in both Ce-MAN1 and Ce-emerin cause embryos to die at the same stage (≈100 cells) as embryos that either lost their RNA polymerase (through RNAi; ref. 39) or were treated with α-amanitin, a potent inhibitor of RNA polymerase (40). Although we did not test transcription activity, our findings are consistent with the possibility that LEM domain proteins might influence gene expression. Obviously, the mechanisms by which “mitotically arrested” anaphase-bridged chromosomes result from loss of Ce-MAN1 and Ce-emerin will be important topics for future work.

The anaphase-bridged chromatin in cells with reduced MAN1 and emerin had an unexpected characteristic: this chromatin retained a mitosis-specific phosphohistone epitope even though segregated chromatin in the same cell apparently had progressed into G1 phase. Because this aspect of the phenotype was novel, we do not know whether it arises uniquely from the loss of LEM protein function. The anaphase-bridged chromatin also failed to recruit lamins or BAF, further suggesting that it remained biochemically mitotic. These results add to previous evidence that BAF binding to chromatin is mitotically regulated (32). We conclude that LEM proteins are required for critical (but still unknown) events before mitosis that enable chromosome segregation. Thus, the phenotypes seen in emerin/MAN1 double-RNAi embryos could arise from a combination of causes, including disrupted BAF function and the loss of activities that depend directly on MAN1 and emerin. This functional overlap normally may protect many human tissues against the loss of emerin and could provide a key to identifying the “lost” or uncompensated functions that produce EDMD.

Acknowledgments

J.L. thanks Alejandro Sanchez (Carnegie Institution of Washington) for the phospho-H3 antibodies and is grateful to Andy Fire (Carnegie Institution of Washington), in whose lab part of this work was done. This work was funded by grants from the USA–Israel Binational Science Foundation, Israel Science Foundation, and Austrian Bank (to Y.G.), the National Institutes of Health (GM64535 to K.L.W.), and the Cornell University new faculty startup fund (to J.L.).

Abbreviations

EDMD

Emery–Dreifuss muscular dystrophy

BAF

barrier-to-autointegration factor

dsRNA

double-stranded RNA

RNAi

RNA-mediated interference

DAPI

4′,6-diamidino-2-phenylindole

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