A hyperdynamic H3.3 nucleosome marks promoter regions in pluripotent embryonic stem cells.

Histone variants and their chaperones are key regulators of eukaryotic transcription, and are critical for normal development. The histone variant H3.3 has been shown to play important roles in pluripotency and differentiation, and although its genome-wide patterns have been investigated, little is known about the role of its dynamic turnover in transcriptional regulation. To elucidate the role of H3.3 dynamics in embryonic stem cell (ESC) biology, we generated mouse ESC lines carrying a single copy of a doxycycline (Dox)-inducible HA-tagged version of H3.3 and monitored the rate of H3.3 incorporation by ChIP-seq at varying time points following Dox induction, before and after RA-induced differentiation. Comparing H3.3 turnover profiles in ESCs and RA-treated cells, we identified a hyperdynamic H3.3-containing nucleosome at the -1 position in promoters of genes expressed in ESCs. This dynamic nucleosome is restricted and shifted downstream into the +1 position following differentiation. We suggest that histone turnover dynamics provides an additional mechanism involved in expression regulation, and that a hyperdynamic -1 nucleosome marks promoters in ESCs. Our data provide evidence for regional regulation of H3.3 turnover in ESC promoters, and calls for testing, in high resolution, the dynamic behavior of additional histone variants and other structural chromatin proteins.

HP1 is involved in regulating the global impact of DNA methylation on alternative splicing.

The global impact of DNA methylation on alternative splicing is largely unknown. Using a genome-wide approach in wild-type and methylation-deficient embryonic stem cells, we found that DNA methylation can either enhance or silence exon recognition and affects the splicing of more than 20% of alternative exons. These exons are characterized by distinct genetic and epigenetic signatures. Alternative splicing regulation of a subset of these exons can be explained by heterochromatin protein 1 (HP1), which silences or enhances exon recognition in a position-dependent manner. We constructed an experimental system using site-specific targeting of a methylated/unmethylated gene and demonstrate a direct causal relationship between DNA methylation and alternative splicing. HP1 regulates this gene's alternative splicing in a methylation-dependent manner by recruiting splicing factors to its methylated form. Our results demonstrate DNA methylation's significant global influence on mRNA splicing and identify a specific mechanism of splicing regulation mediated by HP1.

Multilayered chromatin analysis reveals E2f, Smad and Zfx as transcriptional regulators of histones.

Histones, the building blocks of eukaryotic chromatin, are essential for genome packaging, function and regulation. However, little is known about their transcriptional regulation. Here we conducted a comprehensive computational analysis, based on chromatin immunoprecipitation-sequencing and -microarray analysis (ChIP-seq and ChIP-chip) data of over 50 transcription factors and histone modifications in mouse embryonic stem cells. Enrichment scores supported by gene expression data from gene knockout studies identified E2f1 and E2f4 as master regulators of histone genes, CTCF and Zfx as repressors of core and linker histones, respectively, and Smad1, Smad2, YY1 and Ep300 as restricted or cell type-specific regulators. We propose that histone gene regulation is substantially more complex than previously thought, and that a combination of factors orchestrate histone gene regulation, from strict synchronization with S phase to targeted regulation of specific histone subtypes.

Histone modifications and lamin A regulate chromatin protein dynamics in early embryonic stem cell differentiation.

Embryonic stem cells are characterized by unique epigenetic features including decondensed chromatin and hyperdynamic association of chromatin proteins with chromatin. Here we investigate the potential mechanisms that regulate chromatin plasticity in embryonic stem cells. Using epigenetic drugs and mutant embryonic stem cells lacking various chromatin proteins, we find that histone acetylation, G9a-mediated histone H3 lysine 9 (H3K9) methylation and lamin A expression, all affect chromatin protein dynamics. Histone acetylation controls, almost exclusively, euchromatin protein dynamics; lamin A expression regulates heterochromatin protein dynamics, and G9a regulates both euchromatin and heterochromatin protein dynamics. In contrast, we find that DNA methylation and nucleosome repeat length have little or no effect on chromatin-binding protein dynamics in embryonic stem cells. Altered chromatin dynamics associates with perturbed embryonic stem cell differentiation. Together, these data provide mechanistic insights into the epigenetic pathways that are responsible for chromatin plasticity in embryonic stem cells, and indicate that the genome's epigenetic state modulates chromatin plasticity and differentiation potential of embryonic stem cells.

Chromatin plasticity in pluripotent cells.

ESCs (embryonic stem cells), derived from the blastocyst stage embryo, are characterized by an indefinite ability for self-renewal as well as pluripotency, enabling them to differentiate into all cell types of the three germ layers. In the undifferentiated state, ESCs display a global promiscuous transcriptional programme which is restricted gradually upon differentiation. Supporting transcriptional promiscuity, chromatin in pluripotent cells is more 'plastic' or 'open', with decondensed heterochromatin architecture, enrichment of active histone modifications, and a hyperdynamic association of chromatin proteins with chromatin. During ESC differentiation, nuclear architecture and chromatin undergo substantial changes. Heterochromatin foci appear smaller, more numerous and more condensed in the differentiated state, the nuclear lamina becomes more defined and chromatin protein dynamics becomes restricted. In the present chapter we discuss chromatin plasticity and epigenetics and the mechanisms that regulate the various chromatin states, which are currently a central theme in the studies of stem cell maintenance and differentiation, and which will no doubt assist in delineating the secrets of pluripotency and self-renewal.

Stem cells do play with dice: a statistical physics view of transcription.

Embryonic stem cells display wide-spread pervasive transcriptional output. Here, we propose that multiple simultaneous transcriptional states underlay pluripotency.

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.

Invertebrate lamins.

Lamins are the main component of the nuclear lamina and considered to be the ancestors of all intermediate filament proteins. They are localized mainly at the nuclear periphery where they form protein complexes with integral proteins of the nuclear inner membrane, transcriptional regulators, histones and chromatin modifiers. Studying lamins in invertebrate species has unique advantages including the smaller number of lamin genes in the invertebrate genomes and powerful genetic analyses in Caenorhabditis elegans and Drosophila melanogaster. These simpler nuclear lamina systems allow direct analyses of their structure and functions. Here we give an overview of recent advances in the field of invertebrate nuclear lamins with special emphasis on their evolution, assembly and functions.

Nuclear morphology: when round kernels do the Charleston.

New studies in Drosophila have identified a novel nuclear envelope protein with a farnesyl moiety, termed Kugelkern/Charleston, that helps regulate the size, shape and position of cellular blastoderm nuclei.