Epithelial-to-mesenchymal transition (EMT) is a developmental process important for cell fate determination. Fibroblasts, a product of EMT, can be reset into induced pluripotent stem cells (iPSCs) via exogenous transcription factors but the underlying mechanism is unclear. Here we show that the generation of iPSCs from mouse fibroblasts requires a mesenchymal-to-epithelial transition (MET) orchestrated by suppressing pro-EMT signals from the culture medium and activating an epithelial program inside the cells. At the transcriptional level, Sox2/Oct4 suppress the EMT mediator Snail, c-Myc downregulates TGF-beta1 and TGF-beta receptor 2, and Klf4 induces epithelial genes including E-cadherin. Blocking MET impairs the reprogramming of fibroblasts whereas preventing EMT in epithelial cells cultured with serum can produce iPSCs without Klf4 and c-Myc. Our work not only establishes MET as a key cellular mechanism toward induced pluripotency, but also demonstrates iPSC generation as a cooperative process between the defined factors and the extracellular milieu. PAPERCLIP:

Immunoglobulin (Ig) genes are hypermutated in B lymphocytes that are the precursors to memory B cells. The mutations are linked to transcription initiation, but non-Ig promoters are permissible for the mutation process; thus, other genes expressed in mutating B cells may also be subject to somatic hypermutation. Significant mutations were not observed in c-MYC, S14, or alpha-fetoprotein (AFP) genes, but BCL-6 was highly mutated in a large proportion of memory B cells of normal individuals. The mutation pattern was similar to that of Ig genes.

With the ever increasing demand for energy to meet the needs of growth in population and improvement in the living standards in particular in developing countries, the abundant unconventional oil reserves (about 70% of total world oil), such as heavy oil, oil/tar sands and shale oil, are playing an increasingly important role in securing global energy supply. Compared with the conventional reserves unconventional oil reserves are characterized by extremely high viscosity and density, combined with complex chemistry. As a result, petroleum production from unconventional oil reserves is much more difficult and costly with more serious environmental impacts. As a key underpinning science, understanding the interfacial phenomena involved in unconventional petroleum production, such as oil liberation from host rocks, oil-water emulsions and demulsification, is critical for developing novel processes to improve oil production while reducing GHG emission and other environmental impacts at a lower operating cost. In the past decade, significant efforts and advances have been made in applying the principles of interfacial sciences to better understand complex unconventional oil-systems, while many environmental and production challenges remain. In this critical review, the recent research findings and progress in the interfacial sciences related to unconventional petroleum production are critically reviewed. In particular, the chemistry of unconventional oils, liberation mechanisms of oil from host rocks and mechanisms of emulsion stability and destabilization in unconventional oil production systems are discussed in detail. This review also seeks to summarize the current state-of-the-art characterization techniques and brings forward the challenges and opportunities for future research in this important field of physical chemistry and petroleum.

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The activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class-switch recombination of Ig genes. It has been shown that in vitro, AID protein deaminates C in single-stranded DNA or the coding-strand DNA that is being transcribed but not in double-stranded DNA. However, in vivo, both DNA strands are mutated equally during SHM. We show that AID efficiently deaminates C on both DNA strands of a supercoiled plasmid, acting preferentially on SHM hotspot motifs. However, this DNA is not targeted by AID when it is relaxed after treatment with topoisomerase I, and thus, supercoiling plays a crucial role for AID targeting to this DNA. Most of the mutations are in negatively supercoiled regions, suggesting a mechanism of AID targeting in vivo. During transcription the DNA sequences upstream of the elongating RNA polymerase are negatively supercoiled, and this transient change in DNA topology may allow AID to access both DNA strands.A major advance in the study of somatic hypermutation (SHM) and class-switch recombination (CSR) has been the discovery of the activation-induced cytidine deaminase (AID) (1-3), which appears to be the long-sought SHM mutator factor and inducer of CSR (4). Current in vitro data show that AID is a DNA-specific cytidine deaminase that preferentially removes the amino group of cytidine in single-stranded DNA and in the nontranscribed strand when transcription is active (5-11). These findings are consistent with previous experiments in which SHM is linked to transcription (12, 13). However, in vivo, both DNA strands are equally mutated (14). Furthermore, in ung Ϫ/Ϫ mice, where almost all of the C or G mutations are transitions due to unrepaired AID lesions, equal targeting of both strands is confirmed (15). Single-stranded DNA would also occur in vivo as a potential AID target during DNA replication, which would explain why both DNA strands are targeted during SHM and CSR. However, experiments with a cell line that undergoes SHM in culture support the conclusion that AID can act during the G 1 phase of the cell cycle, and therefore, is not restricted to the S phase (16) (S. Gasior and U.S., unpublished data). A nonexclusive third possibility is that DNA topology (e.g., supercoiling) creates an AID-accessible conformation. In vivo, Ig genes are associated in nucleosomes with histones and other chromatin proteins. These associations, as well as the process of transcription, affect the topology of DNA. To test the possibility that supercoiled DNA as it exists in vivo may be a target for AID, we carried out cytidine deamination assays in vitro with AID purified from insect cells (5). The supercoiled target DNA was an Escherichia coli plasmid that had been manipulated to allow bacterial resistance to carbenicillin only when the initiator AUG of an ampicillin resistance (Amp r ) gene was created by AID deamination of an ACG triplet. Materials and MethodsPlasmid Construction. The kanamycin-resistance (Kan r ) gene was inserted into the SacII site in pBluescript KS(II...

Recent studies have boosted our understanding of long noncoding RNAs (lncRNAs) in numerous biological processes, but few have examined their roles in somatic cell reprogramming. Through expression profiling and functional screening, we have identified that the large intergenic noncoding RNA p21 (lincRNA-p21) impairs reprogramming. Notably, lincRNA-p21 is induced by p53 but does not promote apoptosis or cell senescence in reprogramming. Instead, lincRNA-p21 associates with the H3K9 methyltransferase SETDB1 and the maintenance DNA methyltransferase DNMT1, which is facilitated by the RNA-binding protein HNRNPK. Consequently, lincRNA-p21 prevents reprogramming by sustaining H3K9me3 and/or CpG methylation at pluripotency gene promoters. Our results provide insight into the role of lncRNAs in reprogramming and establish a novel link between p53 and heterochromatin regulation.

Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by activation-induced cytosine deaminase (AID). The uracil, and potentially neighboring bases, are processed by error-prone base excision repair and mismatch repair. Deficiencies in Ung, Msh2, or Msh6 affect SHM and CSR. To determine whether Msh2/Msh6 complexes which recognize single-base mismatches and loops were the only mismatch-recognition complexes required for SHM and CSR, we analyzed these processes in Msh6−/−Ung−/− mice. SHM and CSR were affected in the same degree and fashion as in Msh2−/−Ung−/− mice; mutations were mostly C,G transitions and CSR was greatly reduced, making Msh2/Msh3 contributions unlikely. Inactivating Ung alone reduced mutations from A and T, suggesting that, depending on the DNA sequence, varying proportions of A,T mutations arise by error-prone long-patch base excision repair. Further, in Msh6−/−Ung−/− mice the 5′ end and the 3′ region of Ig genes was spared from mutations as in wild-type mice, confirming that AID does not act in these regions. Finally, because in the absence of both Ung and Msh6, transition mutations from C and G likely are “footprints” of AID, the data show that the activity of AID is restricted drastically in vivo compared with AID in cell-free assays.

Oncogenic transcription factors are known to mediate the conversion of somatic cells to tumour or induced pluripotent stem cells (iPSCs). Here we report c-Jun as a barrier for iPSC formation. c-Jun is expressed by and required for the proliferation of mouse embryonic fibroblasts (MEFs), but not mouse embryonic stem cells (mESCs). Consistently, c-Jun is induced during mESC differentiation, drives mESCs towards the endoderm lineage and completely blocks the generation of iPSCs from MEFs. Mechanistically, c-Jun activates mesenchymal-related genes, broadly suppresses the pluripotent ones, and derails the obligatory mesenchymal to epithelial transition during reprogramming. Furthermore, inhibition of c-Jun by shRNA, dominant-negative c-Jun or Jdp2 enhances reprogramming and replaces Oct4 among the Yamanaka factors. Finally, Jdp2 anchors 5 non-Yamanaka factors (Id1, Jhdm1b, Lrh1, Sall4 and Glis1) to reprogram MEFs into iPSCs. Our studies reveal c-Jun as a guardian of somatic cell fate and its suppression opens the gate to pluripotency.