Induced pluripotent stem cells as a next-generation biomedical interface.

Recent advances in DNA sequencing technologies and subsequent progress in genome-wide association study (GWAS) are rapidly changing the landscape of human diseases. Our knowledge on disease-gene linkage has been exponentially growing, and soon we will obtain complete maps of SNPs and mutations linked to nearly all major disease conditions. These studies will undoubtedly lead us to a more comprehensive understanding of how multiple genetic modifications link to human pathobiology. But what comes next after we discover these genetic linkages? To truly understand the mechanisms of how polygenic modifications identified through GWAS lead to disease conditions, we need an experimental interface to study their pathobiological effects. In this study, induced pluripotent stem cells (iPSCs), retaining all the genetic information from patients, will likely serve as a powerful resource. Indeed, pioneering studies have demonstrated that disease-specific iPSCs are useful for understanding disease mechanisms. Moreover, iPSC-derived cells, when recapitulating some disease phenotypes in vitro, can be a fast track screening tool for drug discovery. Further, with GWAS information, iPSCs will become a valuable tool to predict drug efficacy and toxicity for individuals, thus promoting personalized medicine. In this review, we will discuss how patient-specific iPSCs will become a powerful biomedical interface in clinical translational research.

Differential CpG island methylation of murine adenine nucleotide translocase genes.

Adenine nucleotide translocase (Ant) mediates the exchange of ADP and ATP across the inner mitochondrial membrane in eukaryotes. Mice possess three distinct but highly homologous Ant isoforms, encoded by independent genes, whose transcription depends upon tissue type. Ant1 is expressed selectively in heart and skeletal muscles, Ant2 is ubiquitously expressed in most tissues but lower in skeletal muscle and testis, while Ant4 is exclusively expressed in the testis. Of interest, each of these Ant genes contains CpG islands in their proximal promoter regions. We investigated the methylation status of the three Ant genes in various tissues with active and inactive transcription. In contrast to the Ant4 gene in which CpG island methylation is essential for gene repression, the CpG islands of Ant1 and Ant2 are hypomethylated regardless of the gene expression status throughout the tissues of male mice. Despite the tissue specific expression profile of Ant1, CpG methylation is unlikely involved in the regulation of the gene. Consistent with these findings, addition of a CpG-demethylating agent, 5-aza-2'-deoxycitidine, to fibroblasts increased the expression of Ant4 but not Ant1 or Ant2 genes. This study provides insight regarding the differential regulation of Ant isoforms in mammals, whereby both the Ant1 and Ant2 genes are capable of expression, but the Ant4 gene is completely repressed throughout somatic tissues. To the best of our knowledge, this is a first example to clearly demonstrate a differential usage of CpG island methylation within a family of genes.

A heterogeneous expression pattern for Nanog in embryonic stem cells.

Nanog is a critical homeodomain factor responsible for maintaining embryonic stem (ES) cell self-renewal and pluripotency. Of interest, Nanog expression is not homogeneous in the conventional culture of murine ES cells. A Nanog-high population expresses markers for pluripotent ES cells, whereas a Nanog-low population expresses markers for primitive endoderm, such as Gata6. Since the inner cell mass of early blastocysts has recently been reported to be heterogeneous in terms of Nanog and Gata6 expression, ES cells appear to closely resemble the developing stage from which they originate. We further demonstrate that Nanog can directly repress Gata6 expression through its binding to the proximal promoter region of the Gata6 gene and that overexpression of Nanog reduces heterogeneity during ES cell maintenance. Interestingly, Nanog heterogeneity does not correlate with the heterogeneous expression of stage-specific embryonic antigen-1, suggesting that multiple but overlapping levels of heterogeneity may exist in ES cells. These findings provide insight into the factors that control ES cell self-renewal and the earliest lineage commitment to primitive endoderm while also suggesting methods to promote homogeneity during ES cell maintenance. Disclosure of potential conflicts of interest is found at the end of this article.

A conserved E2F6-binding element in murine meiosis-specific gene promoters.

During gametogenesis, germ cells must undergo meiosis in order to become viable haploid gametes. Successful completion of this process is dependent upon the expression of genes whose protein products function specifically in meiosis. Failure to express these genes in meiotic cells often results in infertility, whereas aberrant expression in somatic cells may lead to mitotic catastrophe. The mechanisms responsible for regulating the timely expression of meiosis-specific genes have not been fully elucidated. Here we demonstrate that E2F6, a member of the E2F family of transcription factors, is essential for the repression of the newly identified meiosis-specific gene, Slc25a31 (also known as Ant4, Aac4), in somatic cells. This discovery, along with previous studies, prompted us to investigate the role of E2F6 in the regulation of meiosis-specific genes in general. Interestingly, the core E2F6-binding element (TCCCGC) was highly conserved in the proximal promoter regions of 19 out of 24 (79.2%) meiosis-specific genes. This was significantly higher than the frequency found in the promoters of all mouse genes (15.4%). In the absence of E2F6, only a portion of these meiosis-specific genes was derepressed in somatic cells. However, endogenous E2F6 bound to the promoters of these meiosis-specific genes regardless of whether they required E2F6 for their repression in somatic cells. Further, E2F6 overexpression was capable of reducing their transcription. These findings indicate that E2F6 possesses a broad ability to bind to and regulate the meiosis-specific gene population.