PRMT5-mediated histone H4 arginine-3 symmetrical dimethylation marks chromatin at G + C-rich regions of the mouse genome.

Symmetrical dimethylation on arginine-3 of histone H4 (H4R3me2s) has been reported to occur at several repressed genes, but its specific regulation and genomic distribution remained unclear. Here, we show that the type-II protein arginine methyltransferase PRMT5 controls H4R3me2s in mouse embryonic fibroblasts (MEFs). In these differentiated cells, we find that the genome-wide pattern of H4R3me2s is highly similar to that in embryonic stem cells. In both the cell types, H4R3me2s peaks are detected predominantly at G + C-rich regions. Promoters are consistently marked by H4R3me2s, independently of transcriptional activity. Remarkably, H4R3me2s is mono-allelic at imprinting control regions (ICRs), at which it marks the same parental allele as H3K9me3, H4K20me3 and DNA methylation. These repressive chromatin modifications are regulated independently, however, since PRMT5-depletion in MEFs resulted in loss of H4R3me2s, without affecting H3K9me3, H4K20me3 or DNA methylation. Conversely, depletion of ESET (KMT1E) or SUV420H1/H2 (KMT5B/C) affected H3K9me3 and H4K20me3, respectively, without altering H4R3me2s at ICRs. Combined, our data indicate that PRMT5-mediated H4R3me2s uniquely marks the mammalian genome, mostly at G + C-rich regions, and independently from transcriptional activity or chromatin repression. Furthermore, comparative bioinformatics analyses suggest a putative role of PRMT5-mediated H4R3me2s in chromatin configuration in the nucleus.

Generation of cloned mice from adult neurons by direct nuclear transfer.

Whereas cloning mammals by direct somatic cell nuclear transfer has been successful using a wide range of donor cell types, neurons from adult brain remain "unclonable" for unknown reasons. Here, using a combination of two epigenetic approaches, we examined whether neurons from adult mice could be cloned. First, we used a specific antibody to discover cell types with reduced amounts of a repressive histone mark-dimethylated histone H3 lysine 9 (H3K9me2)-and identified CA1 pyramidal cells in the hippocampus and Purkinje cells in the cerebellum as candidates. Second, reconstructed embryos were treated with trichostatin A (TSA), a potent histone deacetylase inhibitor. Using CA1 cells, cloned offspring were obtained at high rates, reaching 10.2% and 4.6% (of embryos transferred) for male and female donors, respectively. Cerebellar Purkinje cell nuclei were too large to maintain their genetic integrity during nuclear transfer, leading to developmental arrest of embryos. However, gene expression analysis using cloned blastocysts corroborated a high rate of genomic reprogrammability of CA1 pyramidal and Purkinje cells. Neurons from the hippocampal dentate gyrus and cerebral cortex, which had higher amounts of H3K9me2, could also be used for producing cloned offspring, but the efficiencies were low. A more thorough analysis revealed that TSA treatment was essential for cloning adult neuronal cells. This study demonstrates, to our knowledge for the first time, that adult neurons can be cloned by nuclear transfer. Furthermore, our data imply that reduced amounts of H3K9me2 and increased histone acetylation appear to act synergistically to improve the development of cloned embryos.

Naive-like conversion overcomes the limited differentiation capacity of induced pluripotent stem cells.

Although induced pluripotent stem (iPS) cells are indistinguishable from ES cells in their expression of pluripotent markers, their differentiation into targeted cells is often limited. Here, we examined whether the limited capacity of iPS cells to differentiate into neural lineage cells could be mitigated by improving their base-line level of pluripotency, i.e. by converting them into the so-called "naive" state. In this study, we used rabbit iPS and ES cells because of the easy availability of both cell types and their typical primed state characters. Repeated passages of the iPS cells permitted their differentiation into early neural cell types (neural stem cells, neurons, and glial astrocytes) with efficiencies similar to ES cells. However, unlike ES cells, their ability to differentiate later into neural cells (oligodendrocytes) was severely compromised. In contrast, after these iPS cells had been converted to a naive-like state, they readily differentiated into mature oligodendrocytes developing characteristic ramified branches, which could not be attained even with ES cells. These results suggest that the naive-like conversion of iPS cells might endow them with a higher differentiation capacity.

Establishment of paternal genomic imprinting in mouse prospermatogonia analyzed by nuclear transfer.

In mice, the establishment of paternal genomic imprinting in male germ cells starts at midgestation, as suggested by DNA methylation analyses of differentially methylated regions (DMRs). However, this information is based on averages from mixed populations of germ cells, and the DNA methylation pattern might not always provide a full representation of imprinting status. To obtain more detailed information on the establishment of paternal imprinting, single prospermatogonia at Embryonic Days 15.5 (E15.5), E16.5, and E17.5 and at Day 0.5 after birth were cloned using nuclear transfer; previous reports suggested that cloned embryos reflected the donor's genomic imprinting status. Then, the resultant fetuses (E9.5) were analyzed for the DNA methylation pattern of three paternal DMRs (IG-DMR, H19 DMR, and Rasgrf1 DMR) and the expression pattern of imprinted genes therein. The overall data indicated that establishment of genomic imprinting in all paternally imprinted regions was completed by E17.5, following a short intermediate period at E16.5. Furthermore, comparison between the methylation status of DMRs and the expression profiles of imprinted genes suggested that methylation of the IG-DMR, but not the H19 DMR, solely governed the control of its imprinted gene cluster. The Rasgrf1 DMR seemed to be imprinted later than the other two genes. We also found that the methylation status of the Gtl2 DMR, the secondary DMR that acquires DNA methylation after fertilization, was likely to follow the methylation status of the upstream IG-DMR. Thus, the systematic analyses of prospermatogonium-derived embryos provided additional important information on the establishment of paternal imprinting.

Endomucin, a CD34-like sialomucin, marks hematopoietic stem cells throughout development.

To detect as yet unidentified cell-surface molecules specific to hematopoietic stem cells (HSCs), a modified signal sequence trap was successfully applied to mouse bone marrow (BM) CD34(-)c-Kit(+)Sca-1(+)Lin(-) (CD34(-)KSL) HSCs. One of the identified molecules, Endomucin, is an endothelial sialomucin closely related to CD34. High-level expression of Endomucin was confined to the BM KSL HSCs and progenitor cells, and, importantly, long-term repopulating (LTR)-HSCs were exclusively present in the Endomucin(+)CD34(-)KSL population. Notably, in the yolk sac, Endomucin expression separated multipotential hematopoietic cells from committed erythroid progenitors in the cell fraction positive for CD41, an early embryonic hematopoietic marker. Furthermore, developing HSCs in the intraembryonic aorta-gonad-mesonephros (AGM) region were highly enriched in the CD45(-)CD41(+)Endomucin(+) fraction at day 10.5 of gestation (E10.5) and in the CD45(+)CD41(+)Endomucin(+) fraction at E11.5. Detailed analyses of these fractions uncovered drastic changes in their BM repopulating capacities as well as in vitro cytokine responsiveness within this narrow time frame. Our findings establish Endomucin as a novel cell-surface marker for LTR-HSCs throughout development and provide a powerful tool in understanding HSC ontogeny.

Genetic evidence for Dnmt3a-dependent imprinting during oocyte growth obtained by conditional knockout with Zp3-Cre and complete exclusion of Dnmt3b by chimera formation.

In the male and female germ-lines of mice, both of the two de novo DNA methyltransferases Dnmt3a and Dnmt3b are expressed. By the conditional knockout experiments using the Tnap-Cre gene, we previously showed that deletion of Dnmt3a in primordial germ cells disrupts paternal and maternal imprinting, however, Dnmt3b mutants did not show any defect. Here, we have knocked out Dnmt3a after birth in growing oocytes by using the Zp3-Cre gene and obtained genetic evidence that de novo methylation by Dnmt3a during the oocyte growth stage is indispensable for maternal imprinting. We also carried out DNA methylation analysis in the mutant oocytes and embryos and found that hypomethylation of imprinted genes in Dnmt3a-deficient oocytes was directly inherited to the embryos, but repetitive elements were re-methylated during development. Furthermore, we show that Dnmt3b-deficient cells can contribute to the male and female germ-lines in chimeric mice and can produce normal progeny, establishing that Dnmt3b is dispensable for mouse gametogenesis and imprinting. Finally, Dnmt3-related protein Dnmt3L is not only essential for methylation of imprinted genes but also enhances de novo methylation of repetitive elements in growing oocytes.

Essential and instructive roles of GATA factors in eosinophil development.

GATA transcription factors are major regulators of hematopoietic and immune system. Among GATA factors, GATA-1, GATA-2, and GATA-3 play crucial roles in the development of erythroid cells, hematopoietic stem, and progenitor cells, and T helper type 2 (Th2) cells, respectively. A high level of GATA-1 and GATA-2 expression has been observed in eosinophils, but their roles in eosinophil development remain uncertain both in vitro and in vivo. Here we show that enforced expression of GATA-1 in human primary myeloid progenitor cells completely switches myeloid cell fate into eosinophils. Expression of GATA-1 exclusively promotes development and terminal maturation of eosinophils. Functional domain analyses revealed that the COOH-terminal finger is essential for this capacity while the other domains are dispensable. Importantly, GATA-1-deficient mice failed to develop eosinophil progenitors in the fetal liver. On the other hand, GATA-2 also showed instructive capacity comparable to GATA-1 in vitro and efficiently compensated for GATA-1 deficiency in terms of eosinophil development in vivo, indicating that proper accumulation of GATA factors is critical for eosinophil development. Taken together, our findings establish essential and instructive roles of GATA factors in eosinophil development. GATA-1 and GATA-2 could be novel molecular targets for therapeutic approaches to allergic inflammation.

Reciprocal roles for CCAAT/enhancer binding protein (C/EBP) and PU.1 transcription factors in Langerhans cell commitment.

Myeloid progenitor cells give rise to a variety of progenies including dendritic cells. However, the mechanism controlling the diversification of myeloid progenitors into each progeny is largely unknown. PU.1 and CCAAT/enhancing binding protein (C/EBP) family transcription factors have been characterized as key regulators for the development and function of the myeloid system. However, the roles of C/EBP transcription factors have not been fully identified because of functional redundancy among family members. Using high titer--retroviral infection, we demonstrate that a dominant-negative C/EBP completely blocked the granulocyte--macrophage commitment of human myeloid progenitors. Alternatively, Langerhans cell (LC) commitment was markedly facilitated in the absence of tumor necrosis factor (TNF)alpha, a strong inducer of LC development, whereas expression of wild-type C/EBP in myeloid progenitors promoted granulocytic differentiation, and completely inhibited TNFalpha-dependent LC development. On the other hand, expression of wild-type PU.1 in myeloid progenitors triggered LC development in the absence of TNFalpha, and its instructive effect was canceled by coexpressed C/EBP. Our findings establish reciprocal roles for C/EBP and PU.1 in LC development, and provide new insight into the molecular mechanism of LC development, which has not yet been well characterized.

Regulation of DNA methylation activity through Dnmt3L promoter methylation by Dnmt3 enzymes in embryonic development.

The genomic DNA is methylated by de novo methyltransferases Dnmt3a and Dnmt3b during early embryonic development. The establishment of appropriate methylation patterns depends on a fine regulation of the methyltransferase activity. The activity of both enzymes increases in the presence of Dnmt3L, a Dnmt3a/3b-like protein. However, it is unclear how the function of Dnmt3L is regulated. We found here that the expression of Dnmt3L is controlled via its promoter methylation during embryonic development. Genetic studies showed that Dnmt3a, Dnmt3b and Dnmt3L are all involved in the methylation of the Dnmt3L promoter. Disruption of both Dnmt3a and Dnmt3b genes in mouse rendered the Dnmt3L promoter devoid of methylation, causing incomplete repression of the Dnmt3L transcription in embryonic stem cells and embryos. Disruption of either Dnmt3a or Dnmt3b led to reduced methylation and increased transcription of Dnmt3L, but severe hypomethylation occurred only when Dnmt3b was deficient. Consistent with the major contribution of Dnmt3b in the Dnmt3L promoter methylation, methylation of Dnmt3L was significantly reduced in mouse models of the human ICF syndrome carrying point mutations in Dnmt3b. Interestingly, Dnmt3L also contributes to the methylation of its own promoter in embryonic development. We thus propose an auto-regulatory mechanism for the control of DNA methylation activity whereby the activity of the Dnmt3L promoter is epigenetically modulated by the methylation machinery including Dnmt3L itself. Insufficient methylation of the DNMT3L promoter during embryonic development due to deficiency in DNMT3B might be implicated in the pathogenesis of the ICF syndrome.

Maternal DNMT3A-dependent de novo methylation of the paternal genome inhibits gene expression in the early embryo.

De novo DNA methylation (DNAme) during mammalian spermatogenesis yields a densely methylated genome, with the exception of CpG islands (CGIs), which are hypomethylated in sperm. While the paternal genome undergoes widespread DNAme loss before the first S-phase following fertilization, recent mass spectrometry analysis revealed that the zygotic paternal genome is paradoxically also subject to a low level of de novo DNAme. However, the loci involved, and impact on transcription were not addressed. Here, we employ allele-specific analysis of whole-genome bisulphite sequencing data and show that a number of genomic regions, including several dozen CGI promoters, are de novo methylated on the paternal genome by the 2-cell stage. A subset of these promoters maintains DNAme through development to the blastocyst stage. Consistent with paternal DNAme acquisition, many of these loci are hypermethylated in androgenetic blastocysts but hypomethylated in parthenogenetic blastocysts. Paternal DNAme acquisition is lost following maternal deletion of Dnmt3a, with a subset of promoters, which are normally transcribed from the paternal allele in blastocysts, being prematurely transcribed at the 4-cell stage in maternal Dnmt3a knockout embryos. These observations uncover a role for maternal DNMT3A activity in post-fertilization epigenetic reprogramming and transcriptional silencing of the paternal genome.

Dynamic transition of Dnmt3b expression in mouse pre- and early post-implantation embryos.

The de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns in mouse development. Dnmt3b is more highly expressed in early developmental stages than Dnmt3a, and is thought to have an important role in the epigenetic gene regulation during early embryogenesis. Previous reports suggest that Dnmt3b is expressed preferentially in the embryonic lineage, but less in the extra-embryonic lineage, in early post-implantation embryos. However, it is unclear when this lineage-specific differential expression is established. Here we demonstrate that Dnmt3b shows a dynamic expression change during pre- and early post-implantation development. Contrary to the expectation, Dnmt3b is preferentially expressed in the trophectoderm rather than the inner cell mass at the mid blastocyst stage. Subsequently, the spatial Dnmt3b expression gradually changes during pre- and early post-implantation development, and finally Dnmt3b expression is settled in the embryonic lineage at the epiblast stage. The findings are consistent with the role for Dnmt3b in cell-lineage specification and the creation of lineage-specific DNA methylation patterns.

The Fundamental Characteristics of a Translational Scientist.

Translational science is defined as the field of investigation focused on understanding the scientific and operational principles underlying each step of the translational process. Further development of the field is advanced by describing the key desirable characteristics of individuals who seek to uncover these principles to increase the efficiency and efficacy of translation. The members of Translation Together, a newly launched international collaborative effort to advance translational innovation, present here a consensus representation of the fundamental characteristics of a translational scientist. We invite all stakeholders to contribute in the ongoing efforts to develop the field and educate the next generation of translational scientists.

De novo DNA methylation independent establishment of maternal imprint on X chromosome in mouse oocytes.

Mouse blastocyst stage embryo stained for histone H3 lysine-27 trimethylation (red) and DNA (blue). H3K27me3 marks the inactive X chromosome. The study by Chiba et al. in this issue suggests that de novo DNA methyltransferases are dispensable for setting the imprint on the maternally-derived X chromsome in growing oocytes. See Chiba et al. in this issue.

De novo DNA methylation independent establishment of maternal imprint on X chromosome in mouse oocytes.

In female mouse embryos, the paternal X chromosome (Xp) is preferentially inactivated during preimplantation development and trophoblast differentiation. This imprinted X-chromosome inactivation (XCI) is partly due to an activating imprint on the maternal X chromosome (Xm), which is set during oocyte growth. However, the nature of this imprint is unknown. DNA methylation is one candidate, and therefore we examined whether disruptions of the two de novo DNA methyltransferases in growing oocytes affect imprinted XCI. We found that accumulation of histone H3 lysine-27 trimethylation, a hallmark of XCI, occurs normally on the Xp, and not on the Xm, in female blastocysts developed from the mutant oocytes. Furthermore, the allelic expression patterns of X-linked genes including Xist and Tsix were unchanged in preimplantation embryos and also in the trophoblast. These results show that a maternal disruption of the DNA methyltransferases has no effect on imprinted XCI and argue that de novo DNA methylation is dispensable for Xm imprinting. This underscores the difference between imprinted XCI and autosomal imprinting.

Insertion of an imprinted insulator into the IgH locus reveals developmentally regulated, transcription-dependent control of V(D)J recombination.

The assembly of antigen receptor loci requires a developmentally regulated and lineage-specific recombination between variable (V), diversity (D), and joining (J) segments through V(D)J recombination. The process is regulated by accessibility control elements, including promoters, insulators, and enhancers. The IgH locus undergoes two recombination steps, D-J(H) and then V(H)-DJ(H), but it is unclear how the availability of the DJ(H) substrate could influence the subsequent V(H)-DJ(H) recombination step. The Eµ enhancer plays a critical role in V(D)J recombination and controls a set of sense and antisense transcripts. We epigenetically perturbed the early events at the IgH locus by inserting the imprinting control region (ICR) of the Igf2/H19 locus or a transcriptional insulator devoid of the imprinting function upstream of the Eµ enhancer. The insertions recapitulated the main epigenetic features of their endogenous counterparts, including differential DNA methylation and binding of CTCF/cohesins. Whereas the D-J(H) recombination step was unaffected, both the insulator insertions led to a severe impairment of V(H)-DJ(H) recombination. Strikingly, the inhibition of V(H)-DJ(H) recombination correlated consistently with a strong reduction of DJ(H) transcription and incomplete demethylation. Thus, developmentally regulated transcription following D-J(H) recombination emerges as an important mechanism through which the Eµ enhancer controls V(H)-DJ(H) recombination.

ICR noncoding RNA expression controls imprinting and DNA replication at the Dlk1-Dio3 domain.

Imprinted genes play essential roles in development, and their allelic expression is mediated by imprinting control regions (ICRs). The Dlk1-Dio3 locus is among the few imprinted domains controlled by a paternally methylated ICR. The unmethylated maternal copy activates imprinted expression early in development through an unknown mechanism. We find that in mouse embryonic stem cells (ESCs) and in blastocysts, this function is linked to maternal, bidirectional expression of noncoding RNAs (ncRNAs) from the ICR. Disruption of ICR ncRNA expression in ESCs affected gene expression in cis, led to acquisition of aberrant histone and DNA methylation, delayed replication timing along the domain on the maternal chromosome, and changed its subnuclear localization. The epigenetic alterations persisted during differentiation and affected the neurogenic potential of the stem cells. Our data indicate that monoallelic expression at an ICR of enhancer RNA-like ncRNAs controls imprinted gene expression, epigenetic maintenance processes, and DNA replication in embryonic cells.

Somatic donor cell type correlates with embryonic, but not extra-embryonic, gene expression in postimplantation cloned embryos.

The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (P<1.0 × 10(-26)). In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1-5% per embryos transferred in our laboratory), because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.

Genomic imprinting and human disease.

In many epigenetic phenomena, covalent modifications on DNA and chromatin mediate somatically heritable patterns of gene expression. Genomic imprinting is a classical example of epigenetic regulation in mammals. To date, more than 100 imprinted genes have been identified in humans and mice. Many of these are involved in foetal growth and deve lopment, others control behaviour. Mono-allelic expression of imprinted genes depends on whether the gene is inherited from the mother or the father. This remarkable pattern of expression is controlled by specialized sequence elements called ICRs (imprinting control regions). ICRs are marked by DNA methylation on one of the two parental alleles. These allelic marks originate from either the maternal or the paternal germ line. Perturbation of the allelic DNA methylation at ICRs is causally involved in several human diseases, including the Beckwith-Wiedemann and Silver-Russell syndromes, associated with aberrant foetal growth. Perturbed imprinted gene expression is also implicated in the neuro-developmental disorders Prader-Willi syndrome and Angelman syndrome. Embryo culture and human-assisted reproduction procedures can increase the occurrence of imprinting-related disorders. Recent research shows that, besides DNA methylation, covalent histone modifications and non-histone proteins also contribute to imprinting regulation. The involvement of imprinting in specific human pathologies (and in cancer) emphasizes the need to further explore the underlying molecular mechanisms.