[Show abstract][Hide abstract] ABSTRACT: One of the two X chromosomes in female mammals is inactivated by the noncoding Xist RNA. In mice, X chromosome inactivation (XCI) is regulated by the antisense RNA Tsix that represses Xist on the active X chromosome. In the absence of Tsix, PRC2-mediated histone H3 lysine 27 tri-methylation (H3K27me3) is established over the Xist promoter. Simultaneous disruption of Tsix and PRC2 lead to derepression of Xist and in turn silencing of the single X chromosome in male embryonic stem cells. Here we identify histone H3 lysine 36 tri-methylation
(H3K36me3) as a modification that is recruited by Tsix co-transcriptionally and extends over the Xist promoter. Reduction of H3K36me3 by expression of a mutated histone H3.3 with a substitution of methionine for lysine at position
36 causes a significant derepression of Xist. Moreover depletion of the H3K36 methylase Setd2 leads to upregulation of Xist suggesting H3K36me3 as a modification that contributes to the mechanism of Tsix function in regulating XCI. Furthermore we find that reduction of H3K36me3 does not facilitate an increase in H3K27me3 over
the Xist promoter indicating that additional mechanisms exist by which Tsix blocks PRC2 recruitment to the Xist promoter.
[Show abstract][Hide abstract] ABSTRACT: Mouse parthenogenetic haploid embryonic stem cells (ESCs) are pluripotent cells generated from chemically activated oocytes. Haploid ESCs provide an opportunity to study the effect of genetic alterations because of their hemizygotic characteristics. However, their further application for the selection of unique phenotypes remains limited since ideal reporters to monitor biological processes such as cell differentiation are missing. Here, we report the application of CRISPR/Cas9-mediated knock-in of a reporter cassette, which does not disrupt endogenous target genes in mouse haploid ESCs. We first validated the system by inserting the P2A-Venus reporter cassette into the housekeeping gene locus. In addition to the conventional strategy using the Cas9 nuclease, we employed the Cas9 nickase and truncated sgRNAs to reduce off-target mutagenesis. These strategies induce targeted insertions with an efficiency that correlated with sgRNA guiding activity. We also engineered the neural marker gene Sox1 locus and verified the precise insertion of the P2A-Venus reporter cassette and its functionality by monitoring neural differentiation. Our data demonstrate the successful application of the CRISPR/Cas9-mediated knock-in system for establishing haploid knock-in ESC lines carrying gene specific reporters. Genetically modified haploid ESCs have potential for applications in forward genetic screening of developmental pathways.
[Show abstract][Hide abstract] ABSTRACT: Most animal genomes are diploid, and mammalian development depends on specific adaptations that have evolved secondary to diploidy. Genomic imprinting and dosage compensation restrict haploid development to early embryos. Recently, haploid mammalian development has been reinvestigated since the establishment of haploid embryonic stem cells (ESCs) from mouse embryos. Haploid cells possess one copy of each gene, facilitating the generation of loss-of-function mutations in a single step. Recessive mutations can then be assessed in forward genetic screens. Applications of haploid mammalian cell systems in screens have been illustrated in several recent publications. Haploid ESCs are characterized by a wide developmental potential and can contribute to chimeric embryos and mice. Different strategies for introducing genetic modifications from haploid ESCs into the mouse germline have been further developed. Haploid ESCs therefore introduce new possibilities in mammalian genetics and could offer an unprecedented tool for genome exploration in the future.
Annual Review of Cell and Developmental Biology 10/2014; 30(1):705-22. DOI:10.1146/annurev-cellbio-100913-012920 · 16.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Haploid mouse embryonic stem cells are a handy tool to investigate loss of function mutations. Furthermore, these cells can contribute to chimeric embryos and can be used to introduce genetic modifications into mouse germ lines. Nevertheless, haploid cell cultures have to be maintained by flow sorting on a regular basis due to spontaneous diploidization. Here we present physical differences between haploid and diploid cells, which can be used in applied cell sorting of haploid cell cultures.
[Show abstract][Hide abstract] ABSTRACT: Haploid genetics holds great promise for understanding genome evolution and function. Much of the work on haploid genetics has previously been limited to microbes, but possibilities now extend to animal species, including mammals. Whereas haploid animals were described decades ago, only very recent advances in culture techniques have facilitated haploid embryonic stem cell derivation in mammals. This article examines the potential use of haploid cells and puts haploid animal cells into a historical and biological context. Application of haploid cells in genetic screening holds promise for advancing the genetic exploration of mammalian genomes.
Development 04/2014; 141(7):1423-1426. DOI:10.1242/dev.102202 · 6.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During X chromosome inactivation (XCI), the Polycomb Repressive Complex 2 (PRC2) is thought to participate in the early maintenance of the inactive state. Although Xist RNA is essential for the recruitment of PRC2 to the X chromosome, the precise mechanism remains unclear. Here, we demonstrate that the PRC2 cofactor Jarid2 is an important mediator of Xist-induced PRC2 targeting. The region containing the conserved B and F repeats of Xist is critical for Jarid2 recruitment via its unique N-terminal domain. Xist-induced Jarid2 recruitment occurs chromosome-wide independently of a functional PRC2 complex, unlike at other parts of the genome, such as CG-rich regions, where Jarid2 and PRC2 binding are interdependent. Conversely, we show that Jarid2 loss prevents efficient PRC2 and H3K27me3 enrichment to Xist-coated chromatin. Jarid2 thus represents an important intermediate between PRC2 and Xist RNA for the initial targeting of the PRC2 complex to the X chromosome during onset of XCI.
[Show abstract][Hide abstract] ABSTRACT: Sequencing projects have revealed the information of many animal genomes and thereby enabled the exploration of genome evolution. Insights into how genomes have been repeatedly modified provide a basis for understanding evolutionary innovation and the ever increasing complexity of animal developmental programs. Animal genomes are diploid in most cases, suggesting that redundant information in two copies of the genome increases evolutionary fitness. Genomes are well adapted to a diploid state. Changes of ploidy can be accommodated early in development but they rarely permit successful development into adulthood. In mammals, epigenetic mechanisms including imprinting and X inactivation restrict haploid development. These restrictions are relaxed in an early phase of development suggesting that dosage regulation appears less critical. Here we review the recent literature on haploid genomes and dosage effects and try to embed recent findings in an evolutionary perspective.
[Show abstract][Hide abstract] ABSTRACT: Mammalian genomes contain several billion base pairs of DNA that are packaged in chromatin fibres. At selected gene loci, cohesin complexes have been proposed to arrange these fibres into higher-order structures, but how important this function is for determining overall chromosome architecture and how the process is regulated are not well understood. Using conditional mutagenesis in the mouse, here we show that depletion of the cohesin-associated protein Wapl stably locks cohesin on DNA, leads to clustering of cohesin in axial structures, and causes chromatin condensation in interphase chromosomes. These findings reveal that the stability of cohesin-DNA interactions is an important determinant of chromatin structure, and indicate that cohesin has an architectural role in interphase chromosome territories. Furthermore, we show that regulation of cohesin-DNA interactions by Wapl is important for embryonic development, expression of genes such as c-myc (also known as Myc), and cell cycle progression. In mitosis, Wapl-mediated release of cohesin from DNA is essential for proper chromosome segregation and protects cohesin from cleavage by the protease separase, thus enabling mitotic exit in the presence of functional cohesin complexes.
[Show abstract][Hide abstract] ABSTRACT: Spreading of dosage compensation over the X chromosome in Drosophila males requires the noncoding roX1 and roX2 RNAs. In this issue, Ilik et al. (2013) and Maenner et al. (2013) show that these RNAs contain discrete binding sites that are remodeled during assembly of the dosage compensation complex.
[Show abstract][Hide abstract] ABSTRACT: Tumor progression is associated with invasiveness and metastatic potential. The special AT-rich binding protein 1 (SATB1) has been identified as a key factor in the progression of breast cancer cells to a malignant phenotype and is associated with progression of human tumors. In normal development, SATB1 coordinates gene expression of progenitor cells by functioning as a genome organizer. In contrast to progenitor and tumor cells, SATB1 expression in nontransformed cells is not compatible with proliferation. Here we show that SATB1 expression in mouse embryonic fibroblasts induces cell cycle arrest and senescence that is associated with elevated p16 protein levels. Deletion of p16 overcomes the SATB1-induced senescence. We further provide evidence for an interaction of SATB1 with the retinoblastoma (RB)/E2F pathway downstream of p16. A combined deletion of the RB proteins, RB, p107 and p130 (triple-mutant; TM), prevents SATB1-induced G1 arrest, which is restored upon the reintroduction of RB into SATB1-expressing TM fibroblasts. SATB1 interacts with the E2F/RB complex and regulates the cyclin E promoter in an E2F-dependent manner. These findings demonstrate that p16 and the RB/E2F pathway are critical for SATB1-induced cell cycle arrest. In the absence of p16, SATB1 causes anchorage-independent growth and invasive phenotype in fibroblasts. Our data illustrate that p16 mutations collaborate with the oncogenic activity of SATB1. Consistent with our finding, a literature survey shows that deletion of p16 is generally associated with SATB1 expressing human cell lines and tumors.Oncogene advance online publication, 20 May 2013; doi:10.1038/onc.2013.158.
[Show abstract][Hide abstract] ABSTRACT: Background
Histone deacetylase inhibitors (HDACi) cause histone hyperacetylation and H3K4 hypermethylation in various cell types. They find clinical application as anti-epileptics and chemotherapeutic agents, but the pathways through which they operate remain unclear. Surprisingly, changes in gene expression caused by HDACi are often limited in extent and can be positive or negative. Here we have explored the ability of the clinically important HDACi valproic acid (VPA) to alter histone modification and gene expression, both globally and at specific genes, in mouse embryonic stem (ES) cells.
Microarray expression analysis of ES cells exposed to VPA (1 mM, 8 h), showed that only 2.4% of genes showed a significant, >1.5-fold transcriptional change. Of these, 33% were down-regulated. There was no correlation between gene expression and VPA-induced changes in histone acetylation or H3K4 methylation at gene promoters, which were usually minimal. In contrast, all Hoxb genes showed increased levels of H3K9ac after exposure to VPA, but much less change in other modifications showing bulk increases. VPA-induced changes were lost within 24 h of inhibitor removal. VPA significantly increased the low transcription of Hoxb4 and Hoxb7, but not other Hoxb genes. Expression of Hoxb genes increased in ES cells lacking functional Polycomb silencing complexes PRC1 and PRC2. Surprisingly, VPA caused no further increase in Hoxb transcription in these cells, except for Hoxb1, whose expression increased several fold. Retinoic acid (RA) increased transcription of all Hoxb genes in differentiating ES cells within 24 h, but thereafter transcription remained the same, increased progressively or fell progressively in a locus-specific manner.
Hoxb genes in ES cells are unusual in being sensitive to VPA, with effects on both cluster-wide and locus-specific processes. VPA increases H3K9ac at all Hoxb loci but significantly overrides PRC-mediated silencing only at Hoxb4 and Hoxb7. Hoxb1 is the only Hoxb gene that is further up-regulated by VPA in PRC-deficient cells. Our results demonstrate that VPA can exert both cluster-wide and locus-specific effects on Hoxb regulation.
[Show abstract][Hide abstract] ABSTRACT: Vitamin C is an antioxidant that maintains the activity of iron and α-ketoglutarate-dependent dioxygenases. Despite these enzymes being implicated in a wide range of biological pathways, vitamin C is rarely included in common cell culture media. Recent studies show that reprogramming of pluripotent stem cells is enhanced when vitamin C is present, thereby illustrating previous limitations in reprogramming cultures. Here, we summarize understanding of dioxygenase function in reprogramming and epigenetic regulation. The available data suggest a link between dioxygenase function and stem cell differentiation, which is exposed to environmental influence and is relevant for human disease.
[Show abstract][Hide abstract] ABSTRACT: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.
[Show abstract][Hide abstract] ABSTRACT: The specialized cell types of tissues and organs are generated during development and are replenished over lifetime though the process of differentiation. During differentiation the characteristics and identity of cells are changed to meet their functional requirements. Differentiated cells then faithfully maintain their characteristic gene expression patterns. On the molecular level transcription factors have a key role in instructing specific gene expression programs. They act together with chromatin regulators which stabilize expression patterns. Current evidence indicates that epigenetic mechanisms are essential for maintaining stable cell identities. Conversely, the disruption of chromatin regulators is associated with disease and cellular transformation. In mammals, a large number of chromatin regulators have been identified. The Polycomb group complexes and the DNA methylation system have been widely studied in development. Other chromatin regulators remain to be explored. This chapter focuses on recent advances in understanding epigenetic regulation in embryonic and adult stem cells in mammals. The available data illustrate that several chromatin regulators control key lineage specific genes. Different epigenetic systems potentially could provide stability and guard against loss or mutation of individual components. Recent experiments also suggest intervals in cell differentiation and development when new epigenetic patterns are established. Epigenetic patterns have been observed to change at a progenitor state after stem cells commit to differentiation. This finding is consistent with a role of epigenetic regulation in stabilizing expression patterns after their establishment by transcription factors. However, the available data also suggest that additional, presently unidentified, chromatin regulatory mechanisms exist. Identification of these mechanism is an important aim for future research to obtain a more complete framework for understanding stem cell differentiation during tissue homeostasis.
Advances in Experimental Medicine and Biology 01/2013; 786:307-28. DOI:10.1007/978-94-007-6621-1_17 · 1.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this issue, Nechanitzky et al. question the role of SATB proteins in conferring cellular competence to respond to Xist. Here, Wutz and Agrelo respond, discussing ways to reconcile the available data. Both groups agree that multiple redundant factors may contribute in parallel.
[Show abstract][Hide abstract] ABSTRACT: In mammals, one of the two X chromosomes of female cells is inactivated for dosage compensation between the sexes. X chromosome inactivation is initiated in early embryos by the noncoding Xist RNA. Subsequent chromatin modifications on the inactive X chromosome (Xi) lead to a remarkable stability of gene repression in somatic cell lineages. In mice, reactivation of genes on the Xi accompanies the establishment of pluripotent cells of the female blastocyst and the development of primordial germ cells. Xi reactivation also occurs when pluripotency is established during the reprogramming of somatic cells to induced pluripotent stem cells. The mechanism of Xi reactivation has attracted increasing interest for studying changes in epigenetic patterns and for improving methods of cell reprogramming. Here, we review recent advances in the understanding of Xi reactivation during development and reprogramming and illustrate potential clinical applications.
Cellular and Molecular Life Sciences CMLS 09/2012; 70(14). DOI:10.1007/s00018-012-1174-3 · 5.81 Impact Factor