Nick Gilbert

Severe Global DNA Hypomethylation Blocks Differentiation and Induces Histone Hyperacetylation in Embryonic Stem Cells

ABSTRACT It has been reported that DNA methyltransferase 1-deficient (Dnmt1−/−) embryonic stem (ES) cells are hypomethylated (20% CpG methylation) and die through apoptosis when induced to differentiate. Here, we show that Dnmt[3a−/−,3b−/−] ES cells with just 0.6% of their CpG dinucleotides behave differently: the majority of cells within the culture are partially or completely blocked in their ability to initiate differentiation, remaining viable while retaining the stem cell characteristics of alkaline phosphatase and Oct4 expression. Restoration of DNA methylation levels rescues these defects. Severely hypomethylated Dnmt[3a−/−,3b−/−] ES cells have increased histone acetylation levels, and those cells that can differentiate aberrantly express extraembryonic markers of differentiation. Dnmt[3a−/−,3b−/−] ES cells with >10% CpG methylation are able to terminally differentiate, whereas Dnmt1−/− ES cells with 20% of the CpG methylated cannot differentiate. This demonstrates that successful terminal differentiation is not dependent simply on adequate methylation levels. There is an absolute requirement that the methylation be delivered by the maintenance enzyme Dnmt1.

The role of chromatin structure in regulating the expression of clustered genes

Much of what we know about the chromatin-based mechanisms that regulate gene expression in mammals has come from the study of what are, paradoxically, atypical genes. These are clusters of structurally and/or functionally related genes that are coordinately regulated during development, or between different cell types. Can unravelling the mechanisms of gene regulation at these gene clusters help us to understand how other genes are controlled? Moreover, can it explain why there is clustering of apparently unrelated genes in mammalian genomes?

Chromatin Organization in the Mammalian Nucleus

Mammalian cells package their DNA into chromatin and arrange it in the nucleus as chromosomes. In interphase cells chromosomes are organized in a radial distribution with the most gene-dense chromosomes toward the center of the nucleus. Gene transcription, replication, and repair are influenced by the underlying chromatin architecture, which in turn is affected by the formation of chromosome territories. This arrangement in the nucleus presumably facilitates cellular functions to occur in an efficient and ordered fashion and exploring the link between transcription and nuclear organization will be an exciting area of further research.

Formation of facultative heterochromatin in the absence of HP1

Facultative heterochromatin is a cytological manifestation of epigenetic mechanisms that regulate gene expression. Constitutive heterochromatin is marked by distinctive histone H3 methylation and the presence of HP1 proteins, but the chromatin modifications of facultative heterochromatin are less clear. We have examined histone modifications and HP1 in the facultative heterochromatin of nucleated erythrocytes and show that mouse and chicken erythrocytes have different mechanisms of heterochromatin formation. Mouse embryonic erythrocytes have abundant HP1, increased tri‐methylation of H3 at K9 and loss of H3 tri‐methylation at K27. In contrast, we show that HP1 proteins are lost during the differentiation of chicken erythrocytes, and that H3 tri‐methylation at both K9 and K27 is reduced. This coincides with the appearance of the variant linker histone H5. HP1s are also absent from erythrocytes of Xenopus and zebrafish. Our data show that in the same cell lineage there are different mechanisms for forming facultative heterochromatin in vertebrates. To our knowledge, this is the first report of cell types that lack HP1s and that have gross changes in the levels of histone modifications.

DNA METHYLATION AFFECTS NUCLEAR ORGANIZATION, HISTONE MODIFICATIONS, AND LINKER HISTONE BINDING BUT NOT CHROMATIN COMPACTION

DNA methylation has been implicated in chromatin condensation and nuclear organization, especially at sites of constitutive heterochromatin. How this is mediated has not been clear. In this study, using mutant mouse embryonic stem cells completely lacking in DNA methylation, we show that DNA methylation affects nuclear organization and nucleosome structure but not chromatin compaction. In the absence of DNA methylation, there is increased nuclear clustering of pericentric heterochromatin and extensive changes in primary chromatin structure. Global levels of histone H3 methylation and acetylation are altered, and there is a decrease in the mobility of linker histones. However, the compaction of both bulk chromatin and heterochromatin, as assayed by nuclease digestion and sucrose gradient sedimentation, is unaltered by the loss of DNA methylation. This study shows how the complete loss of a major epigenetic mark can have an impact on unexpected levels of chromatin structure and nuclear organization and provides evidence for a novel link between DNA methylation and linker histones in the regulation of chromatin structure.

cGAS surveillance of micronuclei links genome instability to innate immunity

DNA is strictly compartmentalized within the nucleus to prevent autoimmunity; despite this, cyclic GMP–AMP synthase (cGAS), a cytosolic sensor of double-stranded DNA, is activated in autoinflammatory disorders and by DNA damage. Precisely how cellular DNA gains access to the cytoplasm remains to be determined. Here, we report that cGAS localizes to micronuclei arising from genome instability in a mouse model of monogenic autoinflammation, after exogenous DNA damage and spontaneously in human cancer cells. Such micronuclei occur after mis-segregation of DNA during cell division and consist of chromatin surrounded by its own nuclear membrane. Breakdown of the micronuclear envelope, a process associated with chromothripsis, leads to rapid accumulation of cGAS, providing a mechanism by which self-DNA becomes exposed to the cytosol. cGAS is activated by chromatin, and consistent with a mitotic origin, micronuclei formation and the proinflammatory response following DNA damage are cell-cycle dependent. By combining live-cell laser microdissection with single cell transcriptomics, we establish that interferon-stimulated gene expression is induced in micronucleated cells. We therefore conclude that micronuclei represent an important source of immunostimulatory DNA. As micronuclei formed from lagging chromosomes also activate this pathway, recognition of micronuclei by cGAS may act as a cell-intrinsic immune surveillance mechanism that detects a range of neoplasia-inducing processes.

Distinctive higher-order chromatin structure at mammalian centromeres

The structure of the higher-order chromatin fiber has not been defined in detail. We have used a novel approach based on sucrose gradient centrifugation to compare the conformation of centromeric satellite DNA-containing higher-order chromatin fibers with bulk chromatin fibers obtained from the same mouse fibroblast cells. Our data show that chromatin fibers derived from the centromeric domain of a chromosome exist in a more condensed structure than bulk chromatin whereas pericentromeric chromatin fibers have an intermediate conformation. From the standpoint of current models, our data are interpreted to suggest that satellite chromatin adopts a regular helical conformation compatible with the canonical 30-nm chromatin fiber whereas bulk chromatin fibers appear less regularly folded and are perhaps intermittently interrupted by deformations. This distinctive conformation of the higher-order chromatin fiber in the centromeric domain of the mammalian chromosome could play a role in the formation of heterochromatin and in the determination of centromere identity.

Nuclear organization of centromeric domains is not perturbed by inhibition of histone deacetylases

It is well established that modification of lysines in histone molecules correlates with gene expression and chromatin structure. It is not known whether this operates entirely at a local level, e.g. through the recruitment of specific proteins, or whether histone modifications might impact on more long-range aspects of chromatin organization. There is a distinctive organization of chromatin within the nucleus and the chromatin at the nuclear periphery of mammalian cells appears to be hypoacetylated. Previously it had been suggested that inhibition of histone deacetylases by TSA causes a gross remodeling of nuclear structure, specifically the recruitment of centromeric heterochromatin to the nuclear periphery. Here, we have quantified the nuclear organization of histone modifications and the localization of centromeric domains in human cells before and after TSA treatment. TSA alters the nuclear distribution of histone acetylation, but not that of histone methylation. TSA elevates levels of histone acetylation at the nuclear periphery but we see no alteration in the position of centromeric domains in the nuclei of treated cells. We conclude that the distinctive nuclear localization of centromeric domains is independent of histone acetylation.

DNA methylation in mouse embryonic stem cells and development.

Mammalian development is associated with considerable changes in global DNA methylation levels at times of genomic reprogramming. Normal DNA methylation is essential for development but, despite considerable advances in our understanding of the DNA methyltransferases, the reason that development fails when DNA methylation is deficient remains unclear. Furthermore, although much is known about the enzymes that cause DNA methylation, comparatively little is known about the mechanisms or significance of active demethylation in early development. In this review, we discuss the roles of the various DNA methyltransferases and their likely functions in development.

Supercoiling in DNA and chromatin

Supercoiling is a fundamental property of DNA and chromatin. It is modulated by polymerase and topoisomerase activities and, through regulated constraint, by DNA/chromatin binding proteins. As a non-covalent and elusive topological modification, supercoiling has proved intractable to research despite being a crucial regulator of nuclear structure and function. Recent studies have improved our understanding of the formation, regulation and organisation of supercoiling domains in vivo, and reinforce the prospect that the propagation of supercoiling can influence local and global chromatin structure. However, to further our understanding the development of new experimental tools and models are required to better dissect the mechanics of this key topological regulator.