Multiple mechanisms deregulate EZH2 and histone H3 lysine 27 epigenetic changes in myeloid malignancies.

Polycomb repressive complex 2 (PRC2) is involved in trimethylation of histone H3 lysine 27 (H3K27), chromatin condensation and transcriptional repression. The silencing function of PRC2 complex is mostly attributed to its intrinsic activity for methylating H3K27. Unlike in B-cell lymphomas, enhancer of zeste homolog 2 (EZH2) mutations in myeloid malignancies are inactivating/hypomorphic. When we assessed the mutational status in myeloid malignancies (N=469 cases examined), we found EZH2 and EED/SUZ12 mutations in 8% and 3.3% of cases, respectively. In addition to mutant cases, reduced EZH2 expression was also found in 78% cases with hemizygous deletion (-7/del7q cases involving EZH2 locus) and 41% of cases with diploid chromosome 7, most interestingly cases with spliceosomal mutations (U2AF1/SRSF2 mutations; 63% of cases). EZH2 mutations were characterized by decreased H3K27 trimethylation and increased chromatin relaxation at specific gene loci accompanied by higher transcriptional activity. One of the major downstream target is HOX gene family, involved in the regulation of stem cell self-renewal. HOXA9 was found to be overexpressed in cases with decreased EZH2 expression either by EZH2/spliceosomal mutations or because of -7/del7q. In summary, our results suggest that loss of gene repression through a variety of mutations resulting in reduced H3K27 trimethylation may contribute to leukemogenesis.

Cell growth inhibition by the multifunctional multivalent zinc-finger factor CTCF.

The 11-zinc finger protein CCTC-binding factor (CTCF) employs different sets of zinc fingers to form distinct complexes with varying CTCF- target sequences (CTSs) that mediate the repression or activation of gene expression and the creation of hormone-responsive gene silencers and of diverse vertebrate enhancer-blocking elements (chromatin insulators). To determine how these varying effects would integrate in vivo, we engineered a variety of expression systems to study effects of CTCF on cell growth. Here we show that ectopic expression of CTCF in many cell types inhibits cell clonogenicity by causing profound growth retardation without apoptosis. In asynchronous cultures, the cell-cycle profile of CTCF-expressing cells remained unaltered, which suggested that progression through the cycle was slowed at multiple points. Although conditionally induced CTCF caused the S-phase block, CTCF can also arrest cell division. Viable CTCF-expressing cells could be maintained without dividing for several days. While MYC is the well-characterized CTCF target, the inhibitory effects of CTCF on cell growth could not be ascribed solely to repression of MYC, suggesting that additional CTS-driven genes involved in growth-regulatory circuits, such as p19ARF, are likely to contribute to CTCF-induced growth arrest. These findings indicate that CTCF may regulate cell-cycle progression at multiple steps within the cycle, and add to the growing evidence for the function of CTCF as a tumor suppressor gene.

Scheduled perturbation in DNA during in vitro differentiation of mouse embryo-derived cells.

Studies of sister chromatid exchanges (SCE) and recombination rate of certain minisatellite DNAs have demonstrated that their levels are considerably higher during the preimplantation stage than in latest developmental stages of embryos. It appeared likely that single-strand DNA breaks (SSB) may be relevant to both events during early development. With this in mind, we estimated SSB during in vitro retinoic acid (RA)-induced and spontaneous differentiation of mouse teratocarcinoma (EC) and embryonic stem (ES) cells. Using the method of nucleoid sedimentation and single-cell DNA electrophoresis, we have observed a dramatic increase in the SSB during the first 2-4 mitoses after beginning of differentiation of EC cells, followed by a gradual return to the basal level characteristic of undifferentiated cells. The increase in the SSB was manifested as the appearance of mass nucleoids with slow sedimentation rates, as well as the low-weight mass fragments in DNA patterns of most cells. We concluded that not less than half of genomic DNA has been nicked at the early steps of differentiation. The decrease in SSB level was observed in spite of continuing differentiation, as judged by embryonic antigens and morphological criteria. Also, the increase in the SCE level coincided with that of SSB, possibly being its consequence. The scheduled "surge" of SSB may be the earliest event in commencing differentiation at steps without a phenotypic manifestation.

Embryonic stem cells derived from morulae, inner cell mass, and blastocysts of mink: comparisons of their pluripotencies.

A characterization of cell lines that we derived from morulae (three lines), blastocysts (two lines), and the inner cell mass (ICM) is given. The karyotype of all the lines was normal; the genotype of four lines was XX, and four lines were genotypically XY. The pluripotencies and commitment status of the derived lines were estimated. First, there were not less than two-thirds of cells in the populations of the lines derived from morulae and the ICM with both Xs active; 70-100% of cells of the blastocyst-derived lines had one of the Xs in an inactive state. The activity of glucose-6-phosphate dehydrogenase (G6PD) in the lines (genotype XX) derived from morulae and ICM was found to be twofold higher than in lines with genotype XY, and G6PD activity was the same in the blastocyst-derived XX lines and XY lines. Second, when injected intraperitoneally into athymic mice, morulae- and ICM-derived cells gave rise to simple and complex embryoid bodies (EB) resembling to typical "cystic" mouse EBs. Third, when injected subcutaneously to athymic mice, the ICM- or morula-derived cells gave rise to typical teratomas containing derivatives of the three germ layers and components of organogenesis. Comparisons of cell lines of different derivations demonstrated that the pluripotencies of the ES cells derived from morulae or the ICM are higher than those of blastocyst derivation.

Isolation and cultivation of blastocyst-derived stem cell lines from American mink (Mustela vison).

Ten embryonic stem (ES) cell lines from mink blastocysts were isolated and characterized. All the lines had a normal diploid karyotype; of the ten lines studied, five had the XX and five had the XY constitution. Testing of the pluripotency of the ES-like cells demonstrated that 1) among four lines of genotype XX, and X was late-replicating in three; both Xs were active in about one-third of cells of line MES8, and analysis of glucose-6-phosphate dehydrogenase revealed no dosage compensation for the X-linked gene; 2) when cultured in suspension, the majority of lines were capable of forming "simple" embryoid bodies (EB), and two only showed the capacity for forming "cystic" multilayer EBs. However, formation of ectoderm or foci of yolk sac hematopoiesis, a feature of mouse ES cells, was not observed in the "cystic" EB; 3) when cultured as a monolayer without feeder, the ES cells differentiated into either vimentin-positive fibroblast-like cells or cytokeratin-positive epithelial-like cells (less frequently); neural cells appeared in two lines; 4) when injected into athymic mice, only one of the four tested lines gave rise to tumors. These were fibrosarcomas composed of fibroblast-like cells, with an admixture of smooth muscular elements and stray islets of epithelial tissue; (5) when the ES cells of line MES1 were injected into 102 blastocyst cavities and subsequently transplanted into foster mothers, we obtained 30 offspring. Analysis of the biochemical markers and coat color did not demonstrate the presence of chimaeras among offspring. Thus the cell lines derived from mink blastocysts are true ES cells. However, their pluripotential capacities are restricted.