We mapped Polycomb-associated H3K27 trimethylation (H3K27me3) and Trithorax-associated H3K4 trimethylation (H3K4me3) across the whole genome in human embryonic stem (ES) cells. The vast majority of H3K27me3 colocalized on genes modified with H3K4me3. These commodified genes displayed low expression levels and were enriched in developmental function. Another significant set of genes lacked both modifications and was also expressed at low levels in ES cells but was enriched for gene function in physiological responses rather than development. Commodified genes could change expression levels rapidly during differentiation, but so could a substantial number of genes in other modification categories. SOX2, POU5F1, and NANOG, pluripotency-associated genes, shifted from modification by H3K4me3 alone to colocalization of both modifications as they were repressed during differentiation. Our results demonstrate that H3K27me3 modifications change during early differentiation, both relieving existing repressive domains and imparting new ones, and that colocalization with H3K4me3 is not restricted to pluripotent cells.

Several extrinsic signals such as LIF, BMP and Wnt can support the self-renewal and pluripotency of embryonic stem (ES) cells through regulating the "pluripotent genes." A unique homeobox transcription factor, Nanog, is one of the key downstream effectors of these signals. Elevated level of Nanog can maintain the mouse ES cell self-renewal independent of LIF and enable human ES cell growth without feeder cells. In addition to the external signal pathways, intrinsic transcription factors such as FoxD3, P53 and Oct4 are also involved in regulating the expression of Nanog. Functionally, Nanog works together with other key pluripotent factors such as Oct4 and Sox2 to control a set of target genes that have important functions in ES cell pluripotency. These key factors form a regulatory network to support or limit each other's expression level, which maintains the properties of ES cells.

Mammalian cell totipotency is a subject that has fascinated scientists for generations. A long lasting question whether some of the somatic cells retains totipotency was answered by the cloning of Dolly at the end of the 20th century. The dawn of the 21 st has brought forward great expectations in harnessing the power of totipotentcy in medicine. Through stem cell biology, it is possible to generate any parts of the human body by stem cell engineering. Considerable resources will be devoted to harness the untapped potentials of stem cells in the foreseeable future which may transform medicine as we know today. At the molecular level, totipotency has been linked to a singular transcription factor and its expression appears to define whether a cell should be totipotent. Named Oct4, it can activate or repress the expression of various genes. Curiously, very little is known about Oct4 beyond its ability to regulate gene expression. The mechanism by which Oct4 specifies totipotency remains entirely unresolved. In this review, we summarize the structure and function of Oct4 and address issues related to Oct4 function in maintaining totipotency or pluripotency of embryonic stem cells.

Embryonic stem (ES) cells possess the ability to renew themselves while maintaining the capacity to differentiate into virtually all cell types of the body. Current evidence suggests that ES cells maintain their pluripotent state by expressing a battery of transcription factors including Oct4 and Nanog. However, little is known about how ES cells maintain the expression of these pluripotent factors in ES cells. Here we present evidence that Oct4, Nanog, and FoxD3 form a negative feedback loop to maintain their expression in pluripotent ES cells. First, Oct4 maintains Nanog activity by directly activating its promoter at sub-steady-state concentration but repressing it at or above steady-state levels. On the other hand, FoxD3 behaves as a positive activator of Nanog to counter the repressive effect of Oct4. The expression of Oct4 is activated by FoxD3 and Nanog but repressed by Oct4 itself, thus, exerting an important negative feedback loop to limit its own activity. Indeed, overexpression of either FoxD3 or Nanog in ES cells failed to increase the concentration of Oct4 beyond the steady-state concentration, whereas knocking down either FoxD3 or Nanog reduces the expression of Oct4 in ES cells. Finally, overexpression of Oct4 or Nanog failed to compensate the loss of Nanog or Oct4, respectively, suggesting that both are required for ES self-renewal and pluripotency. Our results suggest the FoxD3-Nanog-Oct4 loop anchors an interdependent network of transcription factors that regulate stem cell pluripotency.

Background: Gene editing in human patient-specific iPSCs is critical for regenerative medicine. Results: Nonintegrating ␤-thalassemia iPSCs corrected by TALENs display undetectable off targets and can be differentiated into erythroblasts expressing normal ␤-globin. Conclusion: TALENs can be used for HBB correction efficiently in ␤-thalassemia iPSCs with different types. Significance: Our study extends TALENs for gene correction in patient-specific iPSCs and may have applications in cell therapy.

Embryonic stem cells are pluripotent progenitors for virtually all cell types in our body and thus possess unlimited therapeutic potentials for regenerative medicine. NANOG, an NK-2 type homeodomain gene, has been proposed to play a key role in maintaining stem cell pluripotency presumably by regulating the expression of genes critical to stem cell renewal and differentiation. Here, we provide the evidence that NANOG behaves as a transcription activator with two unusually strong activation domains embedded in its C terminus. First, we identified these two transactivators by employing the Gal4-DNA binding domain fusion and reporter system and named them WR and CD2. Whereas CD2 contains no obvious structural motif, the WR or Trp repeat contains 10 pentapeptide repeats starting with a Trp in each unit. Substitution of Trp with Ala in each repeat completely abolished its activity, whereas mutations at the conserved Ser, Gln, and Asn had relatively minor or no effect on WR activity. We then validated the activities of WR and CD2 in NANOG by constructing a reporter plasmid bearing five NANOG binding sites. Deletion of both WR and CD2 from NANOG completely eliminated its transactivation function. Paradoxically, whereas the removal of CD2 reduced NANOG activity by ϳ30 -70%, the removal of WR not only did not diminish but actually enhanced its activity by ϳ50 -100% depending on the cell lines analyzed. These data suggest that either WR or CD2 is sufficient for NANOG to function as a transactivator.

Polycomb repressive complex 2 and the epigenetic mark that it deposits, H3K27me3, are evolutionarily conserved and play critical roles in development and cancer. However, their roles in cell fate decisions in early embryonic development remain poorly understood. Here we report that knockout of polycomb repressive complex 2 genes in human embryonic stem cells causes pluripotency loss and spontaneous differentiation toward a meso-endoderm fate, owing to de-repression of BMP signalling. Moreover, human embryonic stem cells with deletion of EZH1 or EZH2 fail to differentiate into ectoderm lineages. We further show that polycomb repressive complex 2-deficient mouse embryonic stem cells also release Bmp4 but retain their pluripotency. However, when converted into a primed state, they undergo spontaneous differentiation similar to that of hESCs. In contrast, polycomb repressive complex 2 is dispensable for pluripotency when human embryonic stem cells are converted into the naive state. Our studies reveal both lineage- and pluripotent state-specific roles of polycomb repressive complex 2 in cell fate decisions.

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