Tatiana V. Cohen

LBR and Lamin A/C Sequentially Tether Peripheral Heterochromatin and Inversely Regulate Differentiation

Eukaryotic cells have a layer of heterochromatin at the nuclear periphery. To investigate mechanisms regulating chromatin distribution, we analyzed heterochromatin organization in different tissues and species, including mice with mutations in the lamin B receptor (Lbr) and lamin A (Lmna) genes that encode nuclear envelope (NE) proteins. We identified LBR- and lamin-A/C-dependent mechanisms tethering heterochromatin to the NE. The two tethers are sequentially used during cellular differentiation and development: first the LBR- and then the lamin-A/C-dependent tether. The absence of both LBR and lamin A/C leads to loss of peripheral heterochromatin and an inverted architecture with heterochromatin localizing to the nuclear interior. Myoblast transcriptome analyses indicated that selective disruption of the LBR- or lamin-A-dependent heterochromatin tethers have opposite effects on muscle gene expression, either increasing or decreasing, respectively. These results show how changes in NE composition contribute to regulating heterochromatin positioning, gene expression, and cellular differentiation during development.

Loss of nucleoplasmic LAP2α–lamin A complexes causes erythroid and epidermal progenitor hyperproliferation

Lamina-associated polypeptide (LAP) 2α is a chromatin-associated protein that binds A-type lamins. Mutations in both LAP2α and A-type lamins are linked to human diseases called laminopathies, but the molecular mechanisms are poorly understood. The A-type lamin–LAP2α complex interacts with and regulates retinoblastoma protein (pRb), but the significance of this interaction in vivo is unknown. Here we address the function of the A-type lamin–LAP2α complex with the use of LAP2α-deficient mice. We show that LAP2α loss causes relocalization of nucleoplasmic A-type lamins to the nuclear envelope and impairs pRb function. This causes inefficient cell-cycle arrest in dense fibroblast cultures and hyperproliferation of epidermal and erythroid progenitor cells in vivo, leading to tissue hyperplasia. Our results support a disease-relevant model in which LAP2α defines A-type lamin localization in the nucleoplasm, which in turn affects pRb-mediated regulation of progenitor cell proliferation and differentiation in highly regenerative tissues.

The nuclear envelope protein MAN1 regulates TGFβ signaling and vasculogenesis in the embryonic yolk sac

MAN1 is an integral protein of the inner nuclear membrane of the nuclear envelope (NE). MAN1 interacts with SMAD transcription factors, which in turn are regulated by the Transforming growth factor beta (TGFβ) superfamily of signaling molecules. To determine the role of MAN1 in mouse development, we used a gene-trap embryonic stem cell clone to derive mice with a functional mutation in MAN1 (Man1GT/GT). Expression of Man1 during early development is initially low but increases at embryonic day 9.5 (E9.5). Coincident with this increase, homozygous gene-trapped Man1 (Man1GT/GT) embryos die by E10.5. Examination of mutant embryos and tetraploid rescue experiments reveals that abnormal yolk-sac vascularization is the probable cause of lethality. We also established embryonic stem cell lines and their differentiated derivatives that are homozygous for the Man1GT allele. Using these lines, we show that the Man1GT allele results in increased phosphorylation, nuclear localization and elevated levels of SMAD transcriptional activity, predominantly of SMAD2/3, which are regulated by the ALK5 signaling pathway. Our studies identify a previously uncharacterized role for an integral nuclear envelope protein in the regulation of yolk-sac angiogenesis by TGFβ signaling and reveal that the NE has an essential role in regulating transcription factor activity during mouse development.

The lamin B receptor under transcriptional control of C/EBPε is required for morphological but not functional maturation of neutrophils

The lamin B receptor (LBR) is an integral nuclear envelope protein that interacts with chromatin and has homology to sterol reductases. Mutations in LBR result in Pelger-Huët anomaly and HEM-Greenberg skeletal dysplasia, whereas in mice Lbr mutations result in ichthyosis. To further understand the function of the LBR and its role in disease, we derived a novel mouse model with a gene-trap insertion into the Lbr locus (Lbr(GT/GT)). Phenotypically, the Lbr(GT/GT) mice are similar to ichthyosis mice. The Lbr(GT/GT) granulocytes lack a mature segmented nucleus and have a block in late maturation. Despite these changes in nuclear morphology, the innate granulocyte immune function in the killing of Staphylococcus aureus bacteria appears to be intact. Granulocyte differentiation requires the transcription factor C/EBPepsilon. We identified C/EBPepsilon binding sites within the Lbr promoter and used EMSAs and luciferase assays to show that Lbr is transcriptionally regulated by C/EBPepsilon. Our findings indicate that the Lbr(GT/GT) mice are a model for Pelger-Huët anomaly and that Lbr, under transcriptional regulation of C/EBPepsilon, is necessary for morphological but not necessarily functional granulocyte maturation.

Defective skeletal muscle growth in lamin A/C-deficient mice is rescued by loss of Lap2α

Mutations in lamin A/C result in a range of tissue-specific disorders collectively called laminopathies. Of these, Emery-Dreifuss and Limb-Girdle muscular dystrophy 1B mainly affect striated muscle. A useful model for understanding both laminopathies and lamin A/C function is the Lmna(-/-) mouse. We found that skeletal muscle growth and muscle satellite (stem) cell proliferation were both reduced in Lmna(-/-) mice. Lamins A and C associate with lamina-associated polypeptide 2 alpha (Lap2α) and the retinoblastoma gene product, pRb, to regulate cell cycle exit. We found Lap2α to be upregulated in Lmna(-/-) myoblasts (MBs). To specifically test the contribution of elevated Lap2α to the phenotype of Lmna(-/-) mice, we generated Lmna(-/-)Lap2α(-/-) mice. Lifespan and body mass were increased in Lmna(-/-)Lap2α(-/-) mice compared with Lmna(-/-). Importantly, the satellite cell proliferation defect was rescued, resulting in improved myogenesis. Lmna(-/-) MBs also exhibited increased levels of Smad2/3, which were abnormally distributed in the cell and failed to respond to TGFβ1 stimulation as in control cells. However, using SIS3 to inhibit signaling via Smad3 reduced cell death and augmented MB fusion. Together, our results show that perturbed Lap2α/pRb and Smad2/3 signaling are important regulatory pathways mediating defective muscle growth in Lmna(-/-) mice, and that inhibition of either pathway alone or in combination can ameliorate this deleterious phenotype.

Role of toll-like receptors in the pathogenesis of dystrophin-deficient skeletal and heart muscle

Although the cause of Duchenne muscular dystrophy (DMD) is known, the specific factors that initiate and perpetuate disease progression are not well understood. We hypothesized that leaky dystrophin-deficient skeletal muscle releases endogenous danger signals (TLR ligands), which bind to Toll-like receptors (TLRs) on muscle and immune cells and activate downstream processes that facilitate degeneration and regeneration in dystrophic skeletal muscle. Here, we demonstrate that dystrophin-deficient mouse muscle cells show increased expression of several cell-surface and endosomal TLRs. In vitro screening identified ssRNA as a relevant endogenous TLR7 ligand. TLR7 activation led to myd88-dependent production of pro-inflammatory cytokines in dystrophin-deficient muscle cells, and cause significant degeneration/regeneration in vivo in mdx mouse muscle. Also, knockout of the central TLR adaptor protein, myd88 in mdx mice significantly improved skeletal and cardiac muscle function. Likewise, proof-of-concept experiments showed that treating young mdx mice with a TLR7/9 antagonist significantly reduced skeletal muscle inflammation and increased muscle force, suggesting that blocking this pathway may have therapeutic potential for DMD.

The LEM Domain Proteins Emerin and LAP2α Are Dispensable for Human Immunodeficiency Virus Type 1 and Murine Leukemia Virus Infections

ABSTRACT The human nuclear envelope proteins emerin and lamina-associated polypeptide 2α (LAP2α) have been proposed to aid in the early replication steps of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). However, whether these factors are essential for HIV-1 or MLV infection has been questioned. Prior studies in which conflicting results were obtained were highly dependent on RNA interference-mediated gene silencing. To shed light on these contradictory results, we examined whether HIV-1 or MLV could infect primary cells from mice deficient for emerin, LAP2α, or both emerin and LAP2α. We observed HIV-1 and MLV infectivity in mouse embryonic fibroblasts (MEFs) from emerin knockout, LAP2α knockout, or emerin and LAP2α double knockout mice to be comparable in infectivity to wild-type littermate-derived MEFs, indicating that both emerin and LAP2α were dispensable for HIV-1 and MLV infection of dividing, primary mouse cells. Because emerin has been suggested to be important for infection of human macrophages by HIV-1, we also examined HIV-1 transduction of macrophages from wild-type mice or knockout mice, but again we did not observe a difference in susceptibility. These findings prompted us to reexamine the role of human emerin in supporting HIV-1 and MLV infection. Notably, both viruses efficiently infected human cells expressing high levels of dominant-negative emerin. We thus conclude that emerin and LAP2α are not required for the early replication of HIV-1 and MLV in mouse or human cells.

Myogenesis in dysferlin-deficient myoblasts is inhibited by an intrinsic inflammatory response.

Limb-girdle muscular dystrophy type 2B results from mutations in dysferlin, a membrane-associated protein involved in cellular membrane repair. Primary myoblast cultures derived from dysferlinopathy patients show reduced myogenic potential, suggesting that dysferlin may regulate myotube fusion and be required for muscle regeneration. These observations contrast with the findings that muscle develops normally in pre-symptomatic dysferlinopathy patients. To better understand the role of dysferlin in myogenesis, we investigated this process in vitro using cells derived from two mouse models of dysferlinopathy: SJL/J and A/J mice. We observed that myotubes derived from dysferlin-deficient muscle were of significantly smaller diameters, contained fewer myonuclei, and displayed reduced myogenic gene expression compared to dysferlin-sufficient cells. Together, these findings suggest that the absence of dysferlin from myoblasts is detrimental to myogenesis. Pro-inflammatory NFκB signaling was upregulated in dysferlin-deficient myotubes; the anti-inflammatory agent celastrol reduced the NFκB activation and improved myogenesis in dysferlin-deficient cultures. The results suggest that decreased myotube fusion in dysferlin deficiency is attributable to intrinsic inflammatory activation and can be improved using anti-inflammatory mediators.

The isolated muscle fibre as a model of disuse atrophy: characterization using PhAct, a method to quantify f-actin

Research into muscle atrophy and hypertrophy is hampered by limitations of the available experimental models. Interpretation of in vivo experiments is confounded by the complexity of the environment while in vitro models are subject to the marked disparities between cultured myotubes and the mature myofibres of living tissues. Here we develop a method (PhAct) based on ex vivo maintenance of the isolated myofibre as a model of disuse atrophy, using standard microscopy equipment and widely available analysis software, to measure f-actin content per myofibre and per nucleus over two weeks of ex vivo maintenance. We characterize the 35% per week atrophy of the isolated myofibre in terms of early changes in gene expression and investigate the effects on loss of muscle mass of modulatory agents, including Myostatin and Follistatin. By tracing the incorporation of a nucleotide analogue we show that the observed atrophy is not associated with loss or replacement of myonuclei. Such a completely controlled investigation can be conducted with the myofibres of a single muscle. With this novel method we can distinguish those features and mechanisms of atrophy and hypertrophy that are intrinsic to the muscle fibre from those that include activities of other tissues and systemic agents.

Tissue-Specific Ablation of the LIF Receptor in the Murine Uterine Epithelium Results in Implantation Failure

The cytokine leukemia inhibitory factor (LIF) is essential for rendering the uterus receptive for blastocyst implantation. In mice, LIF receptor expression (LIFR) is largely restricted to the uterine luminal epithelium (LE). LIF, secreted from the endometrial glands (GEs), binds to the LIFR, activating the Janus kinase–signal transducer and activation of transcription (STAT) 3 (Jak-Stat3) signaling pathway in the LE. JAK-STAT activation converts the LE to a receptive state so that juxtaposed blastocysts begin to implant. To specifically delete the LIFR in the LE, we derived a line of mice in which Cre recombinase was inserted into the endogenous lactoferrin gene (Ltf-Cre). Lactoferrin expression in the LE is induced by E2, and we demonstrate that Cre recombinase activity is restricted to the LE and GE. To determine the requirement of the LIFR in implantation, we derived an additional mouse line carrying a conditional (floxed) Lifrflx/flx gene. Crossing Ltf-Cre mice with Lifrflx/flx mice generated Lifrflx/Δ:LtfCre/+ females that were overtly normal but infertile. Many of these females, despite repeated matings, did not become pregnant. Unimplanted blastocysts were recovered from the Lifrflx/Δ:LtfCre/+ uteri and, when transferred to wild-type recipients, implanted normally, indicating that uterine receptivity rather than the embryo’s competency is compromised. The loss of Lifr results in both the failure for STAT3 to translocate to the LE nuclei and a reduction in the expression of the LIF regulated gene Msx1 that regulates uterine receptivity. These results reveal that uterine expression of the LIFR is essential for embryo implantation and further define the components of the LIF signaling pathway necessary for effective implantation.