Perinuclear Anchoring of H3K9-Methylated Chromatin Stabilizes Induced Cell Fate in C. elegans Embryos.

Interphase chromatin is organized in distinct nuclear sub-compartments, reflecting its degree of compaction and transcriptional status. In Caenorhabditis elegans embryos, H3K9 methylation is necessary to silence and to anchor repeat-rich heterochromatin at the nuclear periphery. In a screen for perinuclear anchors of heterochromatin, we identified a previously uncharacterized C. elegans chromodomain protein, CEC-4. CEC-4 binds preferentially mono-, di-, or tri-methylated H3K9 and localizes at the nuclear envelope independently of H3K9 methylation and nuclear lamin. CEC-4 is necessary for endogenous heterochromatin anchoring, but not for transcriptional repression, in contrast to other known H3K9 methyl-binders in worms, which mediate gene repression but not perinuclear anchoring. When we ectopically induce a muscle differentiation program in embryos, cec-4 mutants fail to commit fully to muscle cell fate. This suggests that perinuclear sequestration of chromatin during development helps restrict cell differentiation programs by stabilizing commitment to a specific cell fate. PAPERCLIP.

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.

Correlation of antigen expression with epigenetic modifications after rAAV delivery of a human factor IX variant in mice and rhesus macaques.

We investigated long-term human coagulation factor IX (huFIX) expression of a novel variant when delivered into mice and rhesus macaques and compared transduction efficiencies using two different adeno-associated virus (AAV) capsids. In hemophilic mice injected with KP1-packaged recombinant AAV (rAAV) expressing the hyperactive FIX variant specific activity plasma levels were 10-fold or 2-fold enhanced when compared with wild-type or Padua huFIX injected mice, respectively. In rhesus macaques AAV-LK03 capsid outperformed AAV-KP1 in terms of antigen expression and liver transduction. Two animals from each group showed sustained low-level huFIX expression at 3 months after administration, while one animal from each group lost huFIX mRNA and protein expression over time, despite comparable vector copies. We investigated whether epigenetic differences in the vector episomes could explain this loss of transcription. Cut&Tag analysis revealed lower levels of activating histone marks in the two animals that lost expression. When comparing rAAV genome associated histone modifications in rhesus macaques with those in mice injected with the same vector, the activating histone marks were starkly decreased in macaque-derived episomes. Differential epigenetic marking of AAV genomes may explain different expression profiles in mice and rhesus macaques, as well as the wide dose response variation observed in primates in both preclinical and human clinical trials.

On TADs and LADs: Spatial Control Over Gene Expression.

The combinatorial action of transcription factors drives cell-type-specific gene expression patterns. However, transcription factor binding and gene regulation occur in the context of chromatin, which modulates DNA accessibility. High-resolution chromatin interaction maps have defined units of chromatin that are in spatial proximity, called topologically associated domains (TADs). TADs can be further classified based on expression activity, replication timing, or the histone marks or non-histone proteins associated with them. Independently, other chromatin domains have been defined by their likelihood to interact with non-DNA structures, such as the nuclear lamina. Lamina-associated domains (LADs) correlate with low gene expression and late replication timing. TADs and LADs have recently been evaluated with respect to cell-type-specific gene expression. The results shed light on the relevance of these forms of chromatin organization for transcriptional regulation, and address specifically how chromatin sequestration influences cell fate decisions during organismal development.

Histones and histone modifications in perinuclear chromatin anchoring: from yeast to man.

It is striking that within a eukaryotic nucleus, the genome can assume specific spatiotemporal distributions that correlate with the cell's functional states. Cell identity itself is determined by distinct sets of genes that are expressed at a given time. On the level of the individual gene, there is a strong correlation between transcriptional activity and associated histone modifications. Histone modifications act by influencing the recruitment of non-histone proteins and by determining the level of chromatin compaction, transcription factor binding, and transcription elongation. Accumulating evidence also shows that the subnuclear position of a gene or domain correlates with its expression status. Thus, the question arises whether this spatial organization results from or determines a gene's chromatin status. Although the association of a promoter with the inner nuclear membrane (INM) is neither necessary nor sufficient for repression, the perinuclear sequestration of heterochromatin is nonetheless conserved from yeast to man. How does subnuclear localization influence gene expression? Recent work argues that the common denominator between genome organization and gene expression is the modification of histones and in some cases of histone variants. This provides an important link between local chromatin structure and long-range genome organization in interphase cells. In this review, we will evaluate how histones contribute to the latter, and discuss how this might help to regulate genes crucial for cell differentiation.

Mechanisms of heterochromatin subnuclear localization.

Transcriptionally repressed heterochromatin becomes the dominant form of chromatin in most terminally differentiated cells. Moreover, in most cells, at least one class of heterochromatin is positioned adjacent to the nuclear lamina. Recent approaches have addressed the mechanism of heterochromatin localization, in order to determine whether spatial segregation contributes to gene repression. Findings in worms and human cells confirm a role for histone H3K9 methylation in heterochromatin positioning, identifying a modification that is also necessary for gene repression of worm transgenic arrays. These pathways appear to be conserved, although mutations in mammalian cells have weaker effects, possibly due to redundancy in positioning mechanisms. We propose a general model in which perinuclear anchoring is linked to an epigenetic propagation of the heterochromatic state, through histone modification.

The AAV capsid can influence the epigenetic marking of rAAV delivered episomal genomes in a species dependent manner.

Recombinant adeno-associated viral vectors (rAAVs) are among the most commonly used vehicles for in vivo based gene therapies. However, it is hard to predict which AAV capsid will provide the most robust expression in human subjects due to the observed discordance in vector-mediated transduction between species. In our study, we use a primate specific capsid, AAV-LK03, to demonstrate that the limitation of this capsid towards transduction of mouse cells is unrelated to cell entry and nuclear transport but rather due to depleted histone H3 chemical modifications related to active transcription, namely H3K4me3 and H3K27ac, on the vector DNA itself. A single-amino acid insertion into the AAV-LK03 capsid enables efficient transduction and the accumulation of active-related epigenetic marks on the vector chromatin in mouse without compromising transduction efficiency in human cells. Our study suggests that the capsid protein itself is involved in driving the epigenetic status of the vector genome, most likely during the process of uncoating. Programming viral chromatin states by capsid design may enable facile DNA transduction between vector and host species and ultimately lead to rational selection of AAV capsids for use in humans.

Dynamics of expression of ARID1A and ARID1B subunits in mouse embryos and in cells during the cell cycle.

The mammalian SWI/SNF chromatin remodeling complexes play essential roles in cell cycle control through the transcriptional regulation of cell-cycle-specific genes. These complexes depend on the energy of ATP hydrolysis provided by the BRG1 or BRM catalytic subunit. They contain seven or more noncatalytic subunits, some being constitutive components, with others having paralogs that assemble in a combinatory manner producing different SWI/SNF-related complexes with specific functions. ARID1A and ARID1B are mutually exclusive subunits of the BAF complex. The specific presence of these subunits in the complex has been demonstrated to determine whether SWI/SNF functions as a corepressor (ARID1A) or as a coactivator (ARID1B) of the cell cycle genes. Our aim has been to analyze the relevance of the ARID1 subunits in development. We have compared the patterns of expression of these two genes through various mouse embryonic stages. Arid1a is expressed widely and intensively, whereas Arid1b is poorly transcribed and expressed in selected regions. Moreover, ARID1A and ARID1B present different kinetics of expression in the cell cycle. ARID1A accumulates in G0 and is downregulated throughout the cell cycle phases but is completely eliminated during mitosis, whereas ARID1B is expressed at comparable levels at all phases, even during mitosis. These kinetics probably affect the incorporation patterns of the ARID1 proteins to the complex and hence modulate SWI/SNF activity during proliferation and arrest.

Mechanism of chromatin segregation to the nuclear periphery in C. elegans embryos.

In eukaryotic organisms, gene regulation occurs in the context of chromatin. In the interphase nucleus, euchromatin and heterochromatin occupy distinct space during cell differentiation, with heterochromatin becoming enriched at the nuclear and nucleolar peripheries. This organization is thought to fine-tune gene expression. To elucidate the mechanisms that govern this level of genome organization, screens were carried out in C. elegans which monitored the loss of heterochromatin sequestration at the nuclear periphery. This led to the identification of a novel chromodomain protein, CEC-4 (Caenorhabditis elegans chromodomain protein 4) that mediates the anchoring of H3K9 methylation-bearing chromatin at the nuclear periphery in early to mid-stage embryos. Surprisingly, the loss of CEC-4 does not derepress genes found in heterochromatic domains, nor does it affect differentiation under standard laboratory conditions. On the other hand, CEC-4 contributes to the efficiency with which muscle differentiation is induced following ectopic expression of the master regulator, HLH-1. This is one of the first phenotypes specifically attributed to the ablation of heterochromatin anchoring.

The formation and sequestration of heterochromatin during development: delivered on 7 September 2012 at the 37th FEBS Congress in Sevilla, Spain.

Chromatin is not randomly positioned in the nucleus, but is distributed in subdomains based on its degree of compaction and transcriptional status. Recent studies have shed light on the logic of chromatin distribution, showing that tissue-specific promoters drive distinct patterns of gene positioning during cell-type differentiation. In addition, the sequestration of heterochromatin at the nuclear envelope has been found to depend on lamin and lamin-associated proteins. On the chromatin side, H3K9 monomethylation, dimethylation and trimethylation were shown to be the critical signals for perinuclear anchoring in worm embryonic nuclei. Downregulation of an equivalent histone methyltransferase, G9a, in human cells has a similar effect. In worms, the sequestration of the terminal methyltransferase by repressed chromatin may facilitate the propagation of a heterochromatin compartment, much as the sequestration of the silent information regulatory complex does at telomeric foci in budding yeast. These results argue for conserved logic in eukaryotic nuclear organization.

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.