Loss of an H3K9me anchor rescues laminopathy-linked changes in nuclear organization and muscle function in an Emery-Dreifuss muscular dystrophy model.

Mutations in the nuclear structural protein lamin A produce rare, tissue-specific diseases called laminopathies. The introduction of a human Emery-Dreifuss muscular dystrophy (EDMD)-inducing mutation into the C. elegans lamin (LMN-Y59C), recapitulates many muscular dystrophy phenotypes, and correlates with hyper-sequestration of a heterochromatic array at the nuclear periphery in muscle cells. Using muscle-specific emerin Dam-ID in worms, we monitored the effects of the mutation on endogenous chromatin. An increased contact with the nuclear periphery along chromosome arms, and an enhanced release of chromosomal centers, coincided with the disease phenotypes of reduced locomotion and compromised sarcomere integrity. The coupling of the LMN-Y59C mutation with the ablation of CEC-4, a chromodomain protein that anchors H3K9-methylated chromatin at the nuclear envelope (NE), suppressed the muscle-associated disease phenotypes. Deletion of cec-4 also rescued LMN-Y59C-linked alterations in chromatin organization and some changes in transcription. Sequences that changed position in the LMN-Y59C mutant, are enriched for E2F (EFL-2)-binding sites, consistent with previous studies suggesting that altered Rb-E2F interaction with lamin A may contribute to muscle dysfunction. In summary, we were able to counteract the dominant muscle-specific defects provoked by LMNA mutation by the ablation of a lamin-associated H3K9me anchor, suggesting a novel therapeutic pathway for EDMD.

Nuclear lamins, diseases and aging.

Nuclear lamins are type V intermediate filament proteins. They are the major building blocks of the peripheral nuclear lamina, a complex meshwork of proteins underlying the inner nuclear membrane. In addition to providing nuclear shape and mechanical stability, they are required for chromatin organization, transcription regulation, DNA replication, nuclear assembly and nuclear positioning. Over the past few years, interest in the lamins has increased because of the identification of at least 12 distinct human diseases associated with mutations in the LMNA gene, which encodes A-type lamins. These diseases, collectively termed laminopathies, affect muscle, adipose, bone, nerve and skin cells and range from muscular dystrophies to accelerated aging.

Laminopathic mutations interfere with the assembly, localization, and dynamics of nuclear lamins.

Lamins are nuclear intermediate filament proteins and the major building blocks of the nuclear lamina. Besides providing nuclear shape and mechanical stability, lamins are required for chromatin organization, transcription regulation, DNA replication, nuclear assembly, nuclear positioning, and apoptosis. Mutations in human lamins cause many different heritable diseases, affecting various tissues and causing early aging. Although many of these mutations result in nuclear deformation, their effects on lamin filament assembly are unknown. Caenorhabditis elegans has a single evolutionarily conserved lamin protein, which can form stable 10-nm-thick filaments in vitro. To gain insight into the molecular basis of lamin filament assembly and the effects of laminopathic mutations on this process, we investigated mutations in conserved residues of the rod and tail domains that are known to cause various laminopathies in human. We show that 8 of 14 mutant lamins present WT-like assembly into filaments or paracrystals, whereas 6 mutants show assembly defects. Correspondingly, expressing these mutants in transgenic animals shows abnormal distribution of Ce-lamin, abnormal nuclear shape or change in lamin mobility. These findings help in understanding the role of individual residues and domains in laminopathy pathology and, eventually, promote the development of therapeutic interventions.

LSM2-8 and XRN-2 contribute to the silencing of H3K27me3-marked genes through targeted RNA decay.

In fission yeast and plants, RNA processing and degradation contribute to heterochromatin silencing, alongside conserved pathways of transcriptional repression. It has not been known whether similar pathways exist in metazoans. Here, we describe a pathway of silencing in Caenorhabditis elegans somatic cells, in which the highly conserved RNA-binding complex LSM2-8 contributes selectively to the repression of heterochromatic reporters and endogenous genes bearing the Polycomb mark, histone H3K27me3. This acts by degrading selected transcripts through the XRN-2 exoribonuclease. Disruption of the LSM2-8 pathway leads to mRNA stabilization. Unlike previously described pathways of heterochromatic RNA degradation, LSM2-8-mediated RNA degradation does not target nor require H3K9 methylation. Intriguingly, loss of this pathway coincides with a localized reduction in H3K27me3 at lsm-8-sensitive loci. We have thus uncovered a mechanism of RNA degradation that selectively contributes to the silencing of a subset of H3K27me3-marked genes, revealing a previously unrecognized layer of post-transcriptional control in metazoan heterochromatin.

A Nuclear RNA Degradation Pathway Helps Silence Polycomb/H3K27me3-Marked Loci in Caenorhabditis elegans.

In fission yeast and plants, RNA-processing pathways contribute to heterochromatin silencing, complementing well-characterized pathways of transcriptional repression. However, it was unclear whether this additional level of regulation occurs in metazoans. In a genetic screen, we uncovered a pathway of silencing in Caenorhabditis elegans somatic cells, whereby the highly conserved, RNA-binding complex LSM2-8 contributes to the repression of heterochromatic reporters and endogenous genes bearing the Polycomb mark H3K27me3. Importantly, the LSM2-8 complex works cooperatively with a 5'-3' exoribonuclease, XRN-2, and disruption of the pathway leads to selective mRNA stabilization. LSM2-8 complex-mediated RNA degradation does not target nor depend on H3K9me2/me3, unlike previously described pathways of heterochromatic RNA degradation. Up-regulation of lsm-8-sensitive loci coincides with a localized drop in H3K27me3 levels in the lsm-8 mutant. Put into the context of epigenetic control of gene expression, it appears that targeted RNA degradation helps repress a subset of H3K27me3-marked genes, revealing an unappreciated layer of regulation for facultative heterochromatin in animals.

Chromatin states and nuclear organization in development--a view from the nuclear lamina.

The spatial distribution of chromatin domains in interphase nuclei changes dramatically during development in multicellular organisms. A crucial question is whether nuclear organization is a cause or a result of differentiation. Genetic perturbation of lamina-heterochromatin interactions is helping to reveal the cross-talk between chromatin states and nuclear organization.

Heterochromatin Protein 1ß (HP1ß) has distinct functions and distinct nuclear distribution in pluripotent versus differentiated cells.

BACKGROUND: Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into every cell type and to self-renew. These characteristics correlate with a distinct nuclear architecture, epigenetic signatures enriched for active chromatin marks and hyperdynamic binding of structural chromatin proteins. Recently, several chromatin-related proteins have been shown to regulate ESC pluripotency and/or differentiation, yet the role of the major heterochromatin proteins in pluripotency is unknown. RESULTS: Here we identify Heterochromatin Protein 1ß (HP1ß) as an essential protein for proper differentiation, and, unexpectedly, for the maintenance of pluripotency in ESCs. In pluripotent and differentiated cells HP1ß is differentially localized and differentially associated with chromatin. Deletion of HP1ß, but not HP1α, in ESCs provokes a loss of the morphological and proliferative characteristics of embryonic pluripotent cells, reduces expression of pluripotency factors and causes aberrant differentiation. However, in differentiated cells, loss of HP1ß has the opposite effect, perturbing maintenance of the differentiation state and facilitating reprogramming to an induced pluripotent state. Microscopy, biochemical fractionation and chromatin immunoprecipitation reveal a diffuse nucleoplasmic distribution, weak association with chromatin and high expression levels for HP1ß in ESCs. The minor fraction of HP1ß that is chromatin-bound in ESCs is enriched within exons, unlike the situation in differentiated cells, where it binds heterochromatic satellite repeats and chromocenters. CONCLUSIONS: We demonstrate an unexpected duality in the role of HP1ß: it is essential in ESCs for maintaining pluripotency, while it is required for proper differentiation in differentiated cells. Thus, HP1ß function both depends on, and regulates, the pluripotent state.

Global epigenetic changes during somatic cell reprogramming to iPS cells.

Embryonic stem cells (ESCs) exhibit unique chromatin features, including a permissive transcriptional program and an open, decondensed chromatin state. Induced pluripotent stem cells (iPSCs), which are very similar to ESCs, hold great promise for therapy and basic research. However, the mechanisms by which reprogramming occurs and the chromatin organization that underlies the reprogramming process are largely unknown. Here we characterize and compare the epigenetic landscapes of partially and fully reprogrammed iPSCs to mouse embryonic fibroblasts (MEFs) and ESCs, which serves as a standard for pluripotency. Using immunofluorescence and biochemical fractionations, we analyzed the levels and distribution of a battery of histone modifications (H3ac, H4ac, H4K5ac, H3K9ac, H3K27ac, H3K4me3, H3K36me2, H3K9me3, H3K27me3, and γH2AX), as well as HP1α and lamin A. We find that fully reprogrammed iPSCs are epigenetically identical to ESCs, and that partially reprogrammed iPSCs are closer to MEFs. Intriguingly, combining both time-course reprogramming experiments and data from the partially reprogrammed iPSCs, we find that heterochromatin reorganization precedes Nanog expression and active histone marking. Together, these data delineate the global epigenetic state of iPSCs in conjunction with their pluripotent state, and demonstrate that heterochromatin precedes euchromatin in reorganization during reprogramming.

H3K9 histone acetylation predicts pluripotency and reprogramming capacity of ES cells.

The pluripotent genome is characterized by unique epigenetic features and a decondensed chromatin conformation. However, the relationship between epigenetic regulation and pluripotency is not altogether clear. Here, using an enhanced MEF/ESC fusion protocol, we compared the reprogramming potency and histone modifications of different embryonic stem cell (ESC) lines (R1, J1, E14, C57BL/6) and found that E14 ESCs are significantly less potent, with significantly reduced H3K9ac levels. Treatment of E14 ESCs with histone deacetylase (HDAC) inhibitors (HDACi) increased H3K9ac levels and restored their reprogramming capacity. Microarray and H3K9ac ChIP-seq analyses, suggested increased extracellular matrix (ECM) activity following HDACi treatment in E14 ESCs. These data suggest that H3K9ac may predict pluripotency and that increasing pluripotency by HDAC inhibition acts through H3K9ac to enhance the activity of target genes involved in ECM production to support pluripotency.

An EDMD mutation in C. elegans lamin blocks muscle-specific gene relocation and compromises muscle integrity.

BACKGROUND: In worms, as in other organisms, many tissue-specific promoters are sequestered at the nuclear periphery when repressed and shift inward when activated. It has remained unresolved, however, whether the association of facultative heterochromatin with the nuclear periphery, or its release, has functional relevance for cell or tissue integrity. RESULTS: Using ablation of the unique lamin gene in C. elegans, we show that lamin is necessary for the perinuclear positioning of heterochromatin. We then express at low levels in otherwise wild-type worms a lamin carrying a point mutation, Y59C, which in humans is linked to an autosomal-dominant form of Emery-Dreifuss muscular dystrophy. Using embryos and differentiated tissues, we track the subnuclear position of integrated heterochromatic arrays and their expression. In LMN-1 Y59C-expressing worms, we see abnormal retention at the nuclear envelope of a gene array bearing a muscle-specific promoter. This correlates with impaired activation of the array-borne myo-3 promoter and altered expression of a number of muscle-specific genes. However, an equivalent array carrying the intestine-specific pha-4 promoter is expressed normally and shifts inward when activated in gut cells of LMN-1 Y59C worms. Remarkably, adult LMN-1 Y59C animals have selectively perturbed body muscle ultrastructure and reduced muscle function. CONCLUSION: Lamin helps sequester heterochromatin at the nuclear envelope, and wild-type lamin permits promoter release following tissue-specific activation. A disease-linked point mutation in lamin impairs muscle-specific reorganization of a heterochromatic array during tissue-specific promoter activation in a dominant manner. This dominance and the correlated muscle dysfunction in LMN-1 Y59C worms phenocopies Emery-Dreifuss muscular dystrophy.

Chromatin plasticity and genome organization in pluripotent embryonic stem cells.

In search of the mechanisms that govern pluripotency and embryonic stem cell (ESC) self-renewal, a growing list of evidence highlights chromatin as a leading factor, controlling ESC maintenance and differentiation. In-depth investigation of chromatin in ESCs revealed distinct features, including DNA methylation, histone modifications, chromatin protein composition and nuclear architecture. Here we review recent literature describing different aspects of chromatin and genome organization in ESCs. The emerging theme seems to support a mechanism maintaining chromatin plasticity in ESCs but without any dramatic changes in the organization and nuclear positioning of chromosomes and gene loci themselves. Plasticity thus seems to be supported more by different mechanisms maintaining an open chromatin state and less by regulating the location of genomic regions.

Specific and conserved sequences in D. melanogaster and C. elegans lamins and histone H2A mediate the attachment of lamins to chromosomes.

The intimate association between nuclear lamins and chromatin is thought to regulate higher order chromatin organization. Previous studies have mapped a region between the rod domain and the Ig fold in the tail domain of Drosophila melanogaster lamin Dm0, which binds chromatin in vitro via the histone H2A/H2B dimer. This region contains an evolutionarily conserved nuclear localization signal (NLS) KRKR, and a sequence composed of the amino acids TRAT. Here we show that binding of lamin Dm0 to chromatin requires both NLS and TRAT sequences. Substituting either of the threonine residues in the TRAT sequence with negatively charged residues decreases the binding of lamin Dm0 to chromatin, indicating that this binding could be regulated by phosphorylation. Both lamin Dm0 and C. elegans Ce-lamin bind directly to histone H2A in vitro and this binding requires the NLS. The amino and carboxyl tail domains of histone H2A are each essential, but not sufficient, for binding to lamin Dm0; only a polypeptide containing both histone H2A tail domains binds efficiently to lamin Dm0. Taken together, these results suggest that specific residues in lamin Dm0 and histone H2A mediate the attachment of the nuclear lamina to chromosomes in vivo, which could have implications on the understanding of laminopathic diseases.

Age-related changes of nuclear architecture in Caenorhabditis elegans.

Mutations in lamins cause premature aging syndromes in humans, including the Hutchinson-Gilford Progeria Syndrome (HGPS) and Atypical Werner Syndrome. It has been shown that HGPS cells in culture undergo age-dependent progressive changes in nuclear architecture. However, it is unknown whether similar changes in nuclear architecture occur during the normal aging process. We have observed that major changes of nuclear architecture accompany Caenorhabditis elegans aging. We found that the nuclear architecture in most nonneuronal cell types undergoes progressive and stochastic age-dependent alterations, such as changes of nuclear shape and loss of peripheral heterochromatin. Furthermore, we show that the rate of these alterations is influenced by the insulin/IGF-1 like signaling pathway and that reducing the level of lamin and lamin-associated LEM domain proteins leads to shortening of lifespan. Our work not only provides evidence for changes of nuclear architecture during the normal aging process of a multicellular organism, but also suggests that HGPS is likely a result of acceleration of the normal aging process. Because the nucleus is vital for many cellular functions, our studies raise the possibility that the nucleus is a prominent focal point for regulating aging.