DNA synthesis is required for reprogramming mediated by stem cell fusion.

Embryonic stem cells (ESCs) can instruct the conversion of differentiated cells toward pluripotency following cell-to-cell fusion by a mechanism that is rapid but poorly understood. Here, we used centrifugal elutriation to enrich for mouse ESCs at sequential stages of the cell cycle and showed that ESCs in S/G2 phases have an enhanced capacity to dominantly reprogram lymphocytes and fibroblasts in heterokaryon and hybrid assays. Reprogramming success was associated with an ability to induce precocious nucleotide incorporation within the somatic partner nuclei in heterokaryons. BrdU pulse-labeling experiments revealed that virtually all successfully reprogrammed somatic nuclei, identified on the basis of Oct4 re-expression, had undergone DNA synthesis within 24 hr of fusion with ESCs. This was essential for successful reprogramming because drugs that inhibited DNA polymerase activity effectively blocked pluripotent conversion. These data indicate that nucleotide incorporation is an early and critical event in the epigenetic reprogramming of somatic cells in experimental ESC-heterokaryons.

Different roles for Tet1 and Tet2 proteins in reprogramming-mediated erasure of imprints induced by EGC fusion.

Genomic imprinting directs the allele-specific marking and expression of loci according to their parental origin. Differential DNA methylation at imprinted control regions (ICRs) is established in gametes and, although largely preserved through development, can be experimentally reset by fusing somatic cells with embryonic germ cell (EGC) lines. Here, we show that the Ten-Eleven Translocation proteins Tet1 and Tet2 participate in the efficient erasure of imprints in this model system. The fusion of B cells with EGCs initiates pluripotent reprogramming, in which rapid re-expression of Oct4 is accompanied by an accumulation of 5-hydroxymethylcytosine (5hmC) at several ICRs. Tet2 was required for the efficient reprogramming capacity of EGCs, whereas Tet1 was necessary to induce 5-methylcytosine oxidation specifically at ICRs. These data show that the Tet1 and Tet2 proteins have discrete roles in cell-fusion-mediated pluripotent reprogramming and imprint erasure in somatic cells.

Embryonic stem cell-derived hemangioblasts remain epigenetically plastic and require PRC1 to prevent neural gene expression.

Many lineage-specific developmental regulator genes are transcriptionally primed in embryonic stem (ES) cells; RNA Pol(II) is bound at their promoters but is prevented from productive elongation by the activity of polycomb repressive complexes (PRC) 1 and 2. This epigenetically poised state is thought to enable ES cells to rapidly execute multiple differentiation programs and is recognized by a simultaneous enrichment for trimethylation of lysine 4 and trimethylation of lysine 27 of histone H3 (bivalent chromatin) across promoter regions. Here we show that the chromatin profile of this important cohort of genes is progressively modified as ES cells differentiate toward blood-forming precursors. Surprisingly however, neural specifying genes, such as Nkx2-2, Nkx2-9, and Sox1, remain bivalent and primed even in committed hemangioblasts, as conditional deletion of PRC1 results in overt and inappropriate expression of neural genes in hemangioblasts. These data reinforce the importance of PRC1 for normal hematopoietic differentiation and reveal an unexpected epigenetic plasticity of mesoderm-committed hemangioblasts.

Structural but not functional conservation of an immune molecule: a tachylectin-like gene in Hydractinia.

Tachylectin-related proteins are a recently characterized group of pattern recognition molecules, functioning in the innate immunity of various animals, from the ancient sponges to vertebrates. Tachylectins are characterized by six internal tandem repeats forming beta-propeller domains. We have identified and characterized a tachylectin-related gene in the colonial marine hydroid, Hydractinia echinata. The predicted gene product, termed CTRN, contained an N-terminal signal peptide and had a well-conserved tachylectin-like structure. RT-PCR analyses revealed only post-metamorphic expression while no mRNA was detected during embryonic development or in planula larvae. Exposure of colonies to LPS under conditions known to activate an immune response in Hydractinia did not result in upregulation of the gene. In situ hybridization analysis of metamorphosed animals detected CTRN transcripts only in a small subpopulation of neurons and their precursor cells, localized in a ring-like structure around the mouth of polyps. The same ring-like structure of CTRN expressing neurons was also observed in young polyp buds, predicting the position of the future mouth. This type of expression pattern can hardly be attributed to an immune-relevant gene. Thus, despite high structural similarity to tachylectins, this cnidarian member of this group seems to be an exception to all other tachylectins identified so far as it seems to have no function in cnidarian innate immunity.

Jarid2 Coordinates Nanog Expression and PCP/Wnt Signaling Required for Efficient ESC Differentiation and Early Embryo Development.

Jarid2 is part of the Polycomb Repressor complex 2 (PRC2) responsible for genome-wide H3K27me3 deposition. Unlike other PRC2-deficient embryonic stem cells (ESCs), however, Jarid2-deficient ESCs show a severe differentiation block, altered colony morphology, and distinctive patterns of deregulated gene expression. Here, we show that Jarid2(-/-) ESCs express constitutively high levels of Nanog but reduced PCP signaling components Wnt9a, Prickle1, and Fzd2 and lowered ß-catenin activity. Depletion of Wnt9a/Prickle1/Fzd2 from wild-type ESCs or overexpression of Nanog largely phenocopies these cellular defects. Co-culture of Jarid2(-/-) with wild-type ESCs restores variable Nanog expression and ß-catenin activity and can partially rescue the differentiation block of mutant cells. In addition, we show that ESCs lacking Jarid2 or Wnt9a/Prickle1/Fzd2 or overexpressing Nanog induce multiple ICM formation when injected into normal E3.5 blastocysts. These data describe a previously unrecognized role for Jarid2 in regulating a core pluripotency and Wnt/PCP signaling circuit that is important for ESC differentiation and for pre-implantation development.

Atypical heterochromatin organization and replication are rapidly acquired by somatic cells following fusion-mediated reprogramming by mouse ESCs.

We recently reported that mouse embryonic stem cells (ESCs) in S/G 2 are more efficient at reprogramming somatic cells than ESCs at other stages of the cell cycle. We also provided evidence that DNA replication is induced in the nuclei of somatic partners upon fusion with ESC partners, and showed that this was critical for their conversion toward a pluripotent state. (1) Here we have used counterflow centrifugal elutriation to enrich for ESCs at different cell cycle phases, so as to examine in detail the properties of S/G 2 phase cells. This revealed that the replication and organization of DAPI-intense heterochromatin in ESCs is unusual in two respects. First, replication of heterochromatin occurred earlier during S phase and was associated with precocious H3S10 phosphorylation. Second, heterochromatin protein 1 α (HP1α), which invariably marks DAPI-intense and H3K9me3-enriched pericentromeric domains in mouse somatic cells, (2) was not necessarily associated with these H3K9me3-enriched domains in undifferentiated ESCs. These data, which complement recent replication timing (3) and electron spectroscopic imaging (ESI) analyses, (4) suggest that heterochromatin is atypical in ESCs. Interestingly, as these unusual features were rapidly acquired by somatic nuclei upon ESC fusion-mediated reprogramming, our results suggest that fundamental changes in cell cycle structure and heterochromatin dynamics may be important for conferring pluripotency.

Reprogramming somatic cells towards pluripotency by cellular fusion.

Pluripotent cells arise within the inner cell mass (ICM) of mammals and have the potential to generate all cell types of the adult organism through a process of commitment and ordered differentiation. Despite many decades of investigation, the mechanisms that guide and stabilise cell fate choice as well as those that can be engineered to promote its reversal, remain only partially resolved. Reprogramming of somatic cells towards a pluripotent-like state can be achieved by several different experimental routes including nuclear transfer, the supply of a defined cocktail of transcription factors, or by fusing somatic cells with a pluripotent stem cell partner. These approaches have been used to demonstrate the remarkable intrinsic epigenetic plasticity of many terminally differentiated cell types, as well as to define the factors that are required for pluripotent conversion. In this review we summarise some recent advances using cell fusion-based studies to better understand the basis of pluripotency and the epigenetic mechanisms that promote cell type inter-conversion.

Using heterokaryons to understand pluripotency and reprogramming.

Reprogramming differentiated cells towards pluripotency can be achieved by different experimental strategies including the forced expression of specific 'inducers' and nuclear transfer. While these offer unparalleled opportunities to generate stem cells and advance disease modelling, the relatively low levels of successful reprogramming achieved (1-2%) makes a direct analysis of the molecular events associated with productive reprogramming very challenging. The generation of transient heterokaryons between human differentiated cells (such as lymphocytes or fibroblasts) and mouse pluripotent stem cell lines results in a much higher frequency of successful conversion (15% SSEA4 expressing cells) and provides an alternative approach to study early events during reprogramming. Under these conditions, differentiated nuclei undergo a series of remodelling events before initiating human pluripotent gene expression and silencing differentiation-associated genes. When combined with genetic or RNAi-based approaches and high-throughput screens, heterokaryon studies can provide important new insights into the factors and mechanisms required to reprogramme unipotent cells towards pluripotency.

The transcriptome of the colonial marine hydroid Hydractinia echinata.

An increasing amount of expressed sequence tag (EST) and genomic data, predominantly for the cnidarians Acropora, Hydra and Nematostella, reveals that cnidarians have a high genomic complexity, despite being one of the morphologically simplest multicellular animals. Considering the diversity of cnidarians, we performed an EST project on the hydroid Hydractinia echinata, to contribute towards a broader coverage of this phylum. After random sequencing of almost 9000 clones, EST characterization revealed a broad diversity in gene content. Corroborating observations in other cnidarians, Hydractinia sequences exhibited a higher sequence similarity to vertebrates than to ecdysozoan invertebrates. A significant number of sequences were hitherto undescribed in metazoans, suggesting that these may be either cnidarian innovations or ancient genes lost in the bilaterian genomes analysed so far. However, we cannot rule out some degree of contamination from commensal bacteria. The identification of unique Hydractinia sequences emphasizes that the acquired genomic information generated so far is not large enough to be representative of the highly diverse cnidarian phylum. Finally, a database was created to store all the acquired information (http://www.mchips.org/hydractinia_echinata.html).