Lamin B1 is required for mouse development and nuclear integrity Laurent Vergnes*†, Miklo´ sPe´ terfy*†, Martin O. Bergo‡, Stephen G. Young‡§, and Karen Reue*†¶ʈ *Veterans Affairs Greater Los Angeles Healthcare System and Departments of †Medicine and ¶Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA 90073; and ‡The Gladstone Institute of Cardiovascular Disease and §Department of Medicine, University of California, San Francisco, CA 94141 Edited by Francis S. Collins, National Institutes of Health, Bethesda, MD, and approved June 1, 2004 (received for review February 27, 2004) Lamins are key structural components of the nuclear lamina, an expression patterns and different assembly properties, suggest- intermediate filament meshwork that lies beneath the inner nu- ing independent functions. For example, B-type lamins are clear membrane. Lamins play a role in nuclear architecture, DNA expressed throughout development, whereas A-type lamins are replication, and gene expression. Mutations affecting A-type expressed only after commitment of cells to a particular differ- lamins have been associated with a variety of human diseases, entiation pathway (10, 11). Also, A- and B-type lamins exhibit including muscular dystrophy, cardiomyopathy, lipodystrophy, distinct solubility properties during mitosis because of temporal and progeria, but mutations in B-type lamins have never been differences in the association with chromatin, with the associa- identified in humans or in experimental animals. To investigate the tion of B-type lamins preceding that of the A-type lamins (12). in vivo function of lamin B1, the major B-type lamin, we generated Mutations in LMNA produce an intriguingly diverse spectrum mice with an insertional mutation in Lmnb1. The mutation resulted of diseases including muscular dystrophies (Emery–Dreifuss in the synthesis of a mutant lamin B1 protein lacking several key muscular dystrophy, limb-girdle muscular dystrophy type 1B), functional domains, including a portion of the rod domain, the neuropathy (Charcot-Marie-Tooth disease type 2), dilated car- nuclear localization signal, and the CAAX motif (the carboxyl- diomyopathy with conduction system disease, familial partial terminal signal for farnesylation). Homozygous Lmnb1 mutant lipodystrophy (s.c. fat loss and diabetes), mandibuloacral dys- mice survived embryonic development but died at birth with plasia (skeletal malformations and lipodystrophy), atypical defects in lung and bone. Fibroblasts from mutant embryos grew Werner’s syndrome, and Hutchinson–Gilford progeria syn- under standard cell-culture conditions but displayed grossly mis- drome (precocious aging syndromes) (13–17). Many of the shapen nuclei, impaired differentiation, increased polyploidy, and phenotypes associated with LMNA mutations in humans have premature senescence. Thus, the lamin B1 mutant mice provide been observed in mice harboring Lmna mutations. Mice with evidence for a broad and nonredundant function of lamin B1 in null mutations in the Lmna gene develop muscular dystrophy mammalian development. These mutant mice and cell lines derived (18), peripheral neuropathy (19), and cardiomyopathy (20). from them will be useful models for studying the role of the nuclear Gene-targeted mice with mutations leading to multiple Lmna lamina in various cellular processes. mRNA splicing abnormalities exhibit aging-like phenotypes akin to those observed in humans with progeria, such as reduced nuclear envelope ͉ lamins ͉ knockout mice ͉ gene trapping lifespan, osteolytic lesions of bones, dental abnormalities, and cells with misshapen nuclei (21). A deficiency in Zmpste24 (a he nuclear lamina, a protein meshwork that lines the inner metalloproteinase of the endoplasmic reticulum) prevents the Tnuclear membrane, is critical in fundamental cellular pro- processing of prelamin A to mature lamin A, resulting in the cesses, including nuclear organization, chromatin segregation, accumulation of prelamin A within cells (22, 23). Zmpste24- DNA replication, and gene transcription (1–5). The principal deficient mice exhibit multiple phenotypes reminiscent of prog- eria, including hair loss, dental abnormalities, and osteolysis of protein components of the lamina are lamins, which are mem- the clavicle and other bones. bers of the intermediate filament protein family. Like other Although dozens of disease-causing LMNA mutations have intermediate filament proteins, lamins possess an amino- been catalogued, defects in the B-type lamins have never been terminal head domain and a highly conserved central ␣-rod identified. The absence of human disease suggests that the loss domain for polymerization and oligomerization (6). Lamins are, of one of the B-type lamins is either inconsequential or causes however, distinguished from other intermediate filament pro- death early in development. ‘‘Knockdown’’ experiments with teins by a nuclear localization motif. In addition, prelamin A and small interfering RNAs (siRNAs) favor the latter possibility, lamins B1 and B2 contain a carboxyl-terminal CAAX motif that because reduced lamin B1 expression caused cultured cells to triggers a series of posttranslational modifications (farnesylation, stop growing and undergo apoptosis, whereas reduced expres- endoproteolytic trimming of the last three amino acid residues, sion of the A-type lamins had no appreciable effect on cell and methylation of the newly exposed farnesylcysteine) (6). growth (24). Aside from their structural role in the formation of the nuclear To define the importance of lamin B1 in mammals, we lamina, lamins A and C are found in the nucleoplasm adjacent generated Lmnb1 mutant mice. In view of the aforementioned to sites of DNA synthesis and RNA processing, suggesting that siRNA experiments (24), we predicted that the mutant mice these proteins could influence both DNA replication and gene would die early in embryonic development. To our surprise, expression (2, 7, 8). however, the mice survived until birth, albeit with bone and lung In vertebrates, lamins are classified as A or B type, based on abnormalities. Lamin B1-deficient fibroblasts grew under stan- sequence homology, expression pattern, biochemical properties, and localization during mitosis. The A-type lamins, lamins A and C, are synthesized from alternatively spliced transcripts of This paper was submitted directly (Track II) to the PNAS office. LMNA and are expressed in most differentiated cells (9). Abbreviations: ES, embryonic stem; MEF, mouse embryonic fibroblast; ␤-gal, ␤-galactosi- Somatic cells also express two B-type lamins, lamin B1 and lamin dase; dpc, days post coitus. B2, which are encoded by LMNB1 and LMNB2, respectively. ʈTo whom correspondence should be addressed at: 11301 Wilshire Boulevard, Building 113, Although the A- and B-type lamins interact with an overlapping Room 312, Los Angeles, CA 90073. E-mail: [email protected]. set of other nuclear envelope proteins, they exhibit distinct © 2004 by The National Academy of Sciences of the USA 10428–10433 ͉ PNAS ͉ July 13, 2004 ͉ vol. 101 ͉ no. 28 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0401424101 Downloaded by guest on September 23, 2021 dard culture conditions but exhibited misshapen nuclei and growth and differentiation abnormalities. Thus, lamin B1 is required for normal embryonic development and postnatal survival, but the lamin B1 mutation did not lead to lethality at the cellular level. Materials and Methods An Insertional Mutation in Lmnb1. Mouse embryonic stem (ES) cells containing a gene-trap insertion in Lmnb1 (cell line XA130) were obtained from BayGenomics (San Francisco) (25). Inser- tion of the gene-trap vector into Lmnb1 was verified by direct sequencing of cDNA obtained by 5Ј rapid amplification of cDNA ends (26). Chimeric mice were generated by blastocyst micro- injection and crossed with C57BL͞6J mice to create mice carrying the mutant Lmnb1 allele (Lmnb1ϩ/⌬). Offspring were genotyped by PCR of genomic DNA with primers specific for the wild-type Lmnb1 allele (Lmnb1 forward, 5Ј-TCCGTGTCGT- GTGGTAGGAGG-3Ј; Lmnb1 reverse, 5Ј-GCAGGAGGGTT- GGGAAAGCC-3Ј) and for the mutant allele carrying the gene-trap insertion (Lmnb1 forward, as above; vector reverse, 5Ј-CACTCCAACCTCCGCAAACTC-3Ј). Cell Culture and Western Blots. Primary mouse embryonic fibro- blasts (MEFs) were prepared from embryos harvested 14.5 days post coitus (dpc) (27). Cells were maintained at 37°Cin5%CO2 ͞ with DMEM containing 10% FBS, penicillin streptomycin, and Fig. 1. Structure and expression of lamin B1 in gene-trap mice. (A) Structure 2 mM glutamine. Two days after reaching confluence, adipocyte of lamin B1 in wild-type and Lmnb1⌬/⌬ mice. The amino-terminal half of lamin differentiation was induced by adding insulin, dexamethasone, B1 consists primarily of the rod domain (1A, 1B, 2A, and 2B) and flanking isomethylbutylxanthine, and the peroxisome proliferator acti- phosphorylation sites (P-site). The C-terminal half contains the nuclear local- vated receptor ␥ ligand, rosiglitazone (BRL 49653; a gift from ization signal (NLS) and the CAAX motif. In Lmnb1⌬/⌬ cells, a portion of the rod Todd Leff, Wayne State University, Detroit) (28). Karyotype domain and the entire carboxyl-terminal domain are replaced by ␤geo. (B) analysis was performed after treating cells for5hwith0.1␮g͞ml RT-PCR analysis of mRNA expression levels for lamins B1 (5Ј and 3Ј regions), B2, ϩ/ϩ ϩ/⌬ ⌬/⌬ colcemid (Sigma) (27). For Western blot analysis, cellular pro- A, and C in Lmnb1 , Lmnb1 , and Lmnb1 MEFs from two embryos