Sylvia Erhardt

Epigenetic reprogramming in mouse primordial germ cells

Genome-wide epigenetic reprogramming in mammalian germ cells, zygote and early embryos, plays a crucial role in regulating genome functions at critical stages of development. We show here that mouse primordial germ cells (PGCs) exhibit dynamic changes in epigenetic modifications between days 10.5 and 12.5 post coitum (dpc). First, contrary to previous suggestions, we show that PGCs do indeed acquire genome-wide de novo methylation during early development and migration into the genital ridge. However, following their entry into the genital ridge, there is rapid erasure of DNA methylation of regions within imprinted and non-imprinted loci. For most genes, the erasure commences simultaneously in PGCs in both male and female embryos, which is completed within 1 day of development. Based on the kinetics of this process, we suggest that this is an active demethylation process initiated upon the entry of PGCs into the gonadal anlagen. The timing of reprogramming in PGCs is crucial since it ensures that germ cells of both sexes acquire an equivalent epigenetic state prior to the differentiation of the definitive male and female germ cells in which new parental imprints are established subsequently. Some repetitive elements, however, show incomplete erasure, which may be essential for chromosome stability and for preventing activation of transposons to reduce the risk of germline mutations. Aberrant epigenetic reprogramming in the germ line would cause the inheritance of epimutations that may have consequences for human diseases as suggested by studies on mouse models.

The Polycomb-Group Gene Ezh2 Is Required for Early Mouse Development

ABSTRACT Polycomb-group (Pc-G) genes are required for the stable repression of the homeotic selector genes and other developmentally regulated genes, presumably through the modulation of chromatin domains. Among the Drosophila Pc-G genes,Enhancer of zeste [E(z)] merits special consideration since it represents one of the Pc-G genes most conserved through evolution. In addition, the E(Z) protein family contains the SET domain, which has recently been linked with histone methyltransferase (HMTase) activity. Although E(Z)-related proteins have not (yet) been directly associated with HMTase activity, mammalian Ezh2 is a member of a histone deacetylase complex. To investigate its in vivo function, we generated mice deficient for Ezh2. The Ezh2 null mutation results in lethality at early stages of mouse development. Ezh2 mutant mice either cease developing after implantation or initiate but fail to complete gastrulation. Moreover, Ezh2-deficient blastocysts display an impaired potential for outgrowth, preventing the establishment of Ezh2-null embryonic stem cells. Interestingly, Ezh2 is up-regulated upon fertilization and remains highly expressed at the preimplantation stages of mouse development. Together, these data suggest an essential role forEzh2 during early mouse development and genetically linkEzh2 with eed and YY1, the only other early-acting Pc-G genes.

Genome-wide analysis reveals a cell cycle–dependent mechanism controlling centromere propagation

Centromeres are the structural and functional foundation for kinetochore formation, spindle attachment, and chromosome segregation. In this study, we isolated factors required for centromere propagation using genome-wide RNA interference screening for defects in centromere protein A (CENP-A; centromere identifier [CID]) localization in Drosophila melanogaster. We identified the proteins CAL1 and CENP-C as essential factors for CID assembly at the centromere. CID, CAL1, and CENP-C coimmunoprecipitate and are mutually dependent for centromere localization and function. We also identified the mitotic cyclin A (CYCA) and the anaphase-promoting complex (APC) inhibitor RCA1/Emi1 as regulators of centromere propagation. We show that CYCA is centromere localized and that CYCA and RCA1/Emi1 couple centromere assembly to the cell cycle through regulation of the fizzy-related/CDH1 subunit of the APC. Our findings identify essential components of the epigenetic machinery that ensures proper specification and propagation of the centromere and suggest a mechanism for coordinating centromere inheritance with cell division.

The DEK oncoprotein is a Su(var) that is essential to heterochromatin integrity

Heterochromatin integrity is crucial for genome stability and regulation of gene expression, but the factors involved in mammalian heterochromatin biology are only incompletely understood. Here we identify the oncoprotein DEK, an abundant nuclear protein with a previously enigmatic in vivo function, as a Suppressor of Variegation [Su(var)] that is crucial to global heterochromatin integrity. We show that DEK interacts directly with Heterochromatin Protein 1 α (HP1α) and markedly enhances its binding to trimethylated H3K9 (H3K9me3), which is key for maintaining heterochromatic regions. Loss of Dek in Drosophila leads to a Su(var) phenotype and global reduction in heterochromatin. Thus, these findings show that DEK is a key factor in maintaining the balance between heterochromatin and euchromatin in vivo.

Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

Polycomb-group proteins are involved in silencing processes caused by a transgenic element from the murine imprinted H19/Igf2 region in Drosophila

A subset of autosomal genes undergo genomic imprinting which results in expression from only the paternal or maternal chromosome. While this phenomenon is restricted to mammals and angiosperms, the underlying silencing mechanisms appear to be evolutionarily conserved. A biallelically unmethylated DNaseI hypersensitive region (A6-A4) between the imprinted Igf2 and H19 genes is conserved in humans and mice and functions as a tissue-specific maintenance element for the imprinted growth factor IGF2. In order to analyse A6-A4 for potentially conserved transcriptional maintenance properties, we have generated transgenic Drosophila harbouring the element in a reporter construct. These flies depicted silencing of the reporter genes lacZ and mini-white. The silenced state of the mini-white gene showed variegation and sensitivity to temperature changes. In addition, two members of the conserved Polycomb group, Enhancer of zeste and Posterior sex combs, were needed for repression. Polycomb group proteins are essential for gene silencing during development. Our results indicate that Polycomb group proteins may also be involved in the regulation of mammalian imprinted genes.

No longer a nuisance: long non-coding RNAs join CENP-A in epigenetic centromere regulation

Centromeres represent the basis for kinetochore formation, and are essential for proper chromosome segregation during mitosis. Despite these essential roles, centromeres are not defined by specific DNA sequences, but by epigenetic means. The histone variant CENP-A controls centromere identity epigenetically and is essential for recruiting kinetochore components that attach the chromosomes to the mitotic spindle during mitosis. Recently, a new player in centromere regulation has emerged: long non-coding RNAs transcribed from repetitive regions of centromeric DNA function in regulating centromeres epigenetically. This review summarizes recent findings on the essential roles that transcription, pericentromeric transcripts, and centromere-derived RNAs play in centromere biology.

The E3 Ligase CUL3/RDX Controls Centromere Maintenance by Ubiquitylating and Stabilizing CENP-A in a CAL1-Dependent Manner

Centromeres are defined by the presence of the histone H3 variant CENP-A in a subset of centromeric nucleosomes. CENP-A deposition to centromeres depends on a specialized loading factor from yeast to humans that is called CAL1 in Drosophila. Here, we show that CAL1 directly interacts with RDX, an adaptor for CUL3-mediated ubiquitylation. However, CAL1 is not a substrate of the CUL3/RDX ligase but functions as an additional substrate-specifying factor for the CUL3/RDX-mediated ubiquitylation of CENP-A. Remarkably, ubiquitylation of CENP-A by CUL3/RDX does not trigger its degradation but stabilizes CENP-A and CAL1. Loss of RDX leads to a rapid degradation of CAL1 and CENP-A and to massive chromosome segregation defects during development. Essentially, we identified a proteolysis-independent role of ubiquitin conjugation in centromere regulation that is essential for the maintenance of the centromere-defining protein CENP-A and its loading factor CAL1.

The ABCs of centromeres

Accurate segregation of genetic information during cell division relies on a multiprotein complex called the kinetochore, whose formation requires specialized centromeric chromatin. Two papers in this issue of Nature Cell Biology identify a multitude of new vertebrate kinetochore proteins that provide insight into the link between centromeric chromatin and the kinetochore, and suggest a functional relationship between centromeres and nucleoli during interphase.

The Histone-Fold Protein CHRAC14 Influences Chromatin Composition in Response to DNA Damage

Chromatin reorganization and the incorporation of specific histone modifications during DNA damage response are essential steps for the successful repair of any DNA lesion. Here, we show that the histone-fold protein CHRAC14 plays an essential role in response to DNA damage in Drosophila. Chrac14 mutants are hypersensitive to genotoxic stress and do not activate the G2/M cell-cycle checkpoint after damage induction. Even though the DNA damage repair process is activated in the absence of CHRAC14, lesions are not repaired efficiently. In the absence of CHRAC14, the centromere-specific histone H3 variant CENP-A localizes to sites of DNA damage, causing ectopic kinetochore formation and genome instability. CENP-A and CHRAC14 are able to interact upon damage. Our data suggest that CHRAC14 modulates chromatin composition in response to DNA damage, which is required for efficient DNA damage repair in Drosophila.