Juli Unternaehrer

Chromatin Modifying Enzymes as Modulators of Reprogramming

Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling. Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming, the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.

Influence of Threonine Metabolism on S-Adenosylmethionine and Histone Methylation

SAM, Histones, and Stem Cells Mouse embryonic stem cells require threonine for growth and express large amounts of the enzyme that catalyzes the first step in threonine metabolism. To find out what is so important about threonine in these cells, Shyh-Change et al. (p. 222, published online 1 November; see the Perspective by Sassone-Corsi) monitored changes in metabolism by mass spectrometry in induced pluripotent stem cells. Threonine was required to maintain cellular concentrations of S-adenosylmethionine (SAM), a substrate used for protein methylation. Restriction of threonine inhibited methylation of histones, which is characteristic of chromatin in embryonic stem cells. Thus, altered metabolism of threonine and methionine in stem cells may be linked to epigenetic changes that influence genetic reprogramming and decisions of stem cells to proliferate or differentiate. Unusual threonine metabolism in mouse stem cells influences genetic reprogramming via altered histone methylation. [Also see Perspective by Sassone-Corsi] Threonine is the only amino acid critically required for the pluripotency of mouse embryonic stem cells (mESCs), but the detailed mechanism remains unclear. We found that threonine and S-adenosylmethionine (SAM) metabolism are coupled in pluripotent stem cells, resulting in regulation of histone methylation. Isotope labeling of mESCs revealed that threonine provides a substantial fraction of both the cellular glycine and the acetyl–coenzyme A (CoA) needed for SAM synthesis. Depletion of threonine from the culture medium or threonine dehydrogenase (Tdh) from mESCs decreased accumulation of SAM and decreased trimethylation of histone H3 lysine 4 (H3K4me3), leading to slowed growth and increased differentiation. Thus, abundance of SAM appears to influence H3K4me3, providing a possible mechanism by which modulation of a metabolic pathway might influence stem cell fate.

Distinct Patterns of Membrane Microdomain Partitioning in Th1 and Th2 Cells

Here we show that activated Th1 and Th2 cells have distinct patterns of membrane compartmentalization into lipid rafts. TCR complex members are recruited efficiently to rafts and aggregate with rafts at the site of MHC/peptide contact in Th1 cells but not Th2 cells. TCR/raft association in Th1 cells is deficient in the absence of CD4, suggesting that CD4 aids recruitment of the TCR to rafts. We show differential utilization of rafts in Th1 and Th2 cells by cholesterol depletion studies, which alters calcium signaling in Th1 but not Th2 cells. Furthermore, Th2 cells have a decreased ability to respond to low-affinity peptide stimulation. These studies indicate that components of membrane microdomains are differentially regulated in functionally distinct CD4 T cells.

Induced pluripotent stem cells for modelling human diseases

Research into the pathophysiological mechanisms of human disease and the development of targeted therapies have been hindered by a lack of predictive disease models that can be experimentally manipulated in vitro. This review describes the current state of modelling human diseases with the use of human induced pluripotent stem (iPS) cell lines. To date, a variety of neurodegenerative diseases, haematopoietic disorders, metabolic conditions and cardiovascular pathologies have been captured in a Petri dish through reprogramming of patient cells into iPS cells followed by directed differentiation of disease-relevant cells and tissues. However, realizing the true promise of iPS cells for advancing our basic understanding of disease and ultimately providing novel cell-based therapies will require more refined protocols for generating the highly specialized cells affected by disease, coupled with strategies for drug discovery and cell transplantation.

The Epithelial-Mesenchymal Transition Factor SNAIL Paradoxically Enhances Reprogramming

Summary Reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) entails a mesenchymal to epithelial transition (MET). While attempting to dissect the mechanism of MET during reprogramming, we observed that knockdown (KD) of the epithelial-to-mesenchymal transition (EMT) factor SNAI1 (SNAIL) paradoxically reduced, while overexpression enhanced, reprogramming efficiency in human cells and in mouse cells, depending on strain. We observed nuclear localization of SNAI1 at an early stage of fibroblast reprogramming and using mouse fibroblasts expressing a knockin SNAI1-YFP reporter found cells expressing SNAI1 reprogrammed at higher efficiency. We further demonstrated that SNAI1 binds the let-7 promoter, which may play a role in reduced expression of let-7 microRNAs, enforced expression of which, early in the reprogramming process, compromises efficiency. Our data reveal an unexpected role for the EMT factor SNAI1 in reprogramming somatic cells to pluripotency.

A nontranscriptional role for Oct4 in the regulation of mitotic entry

Significance Embryonic stem cells and induced pluripotent stem cells have abbreviated cell cycles. To achieve this rapid proliferation, several molecular safeguards that normally distinguish healthy from transformed cells are altered. Understanding how these pluripotent stem cells balance the demands of their unique cell cycles against the need to maintain a stable genome is critical to unlocking their great promise for regenerative medicine. Here, we demonstrate that Oct4 (octamer-binding transcription factor 4), a transcription factor required to maintain pluripotency, inhibits the activation of cyclin-dependent kinase (Cdk) 1, the master regulator of mitosis, and delays mitotic entry in a nontranscriptional manner. To our knowledge, our study is the first demonstration of a nontranscriptional function of the pluripotency regulator Oct4. Rapid progression through the cell cycle and a very short G1 phase are defining characteristics of embryonic stem cells. This distinct cell cycle is driven by a positive feedback loop involving Rb inactivation and reduced oscillations of cyclins and cyclin-dependent kinase (Cdk) activity. In this setting, we inquired how ES cells avoid the potentially deleterious consequences of premature mitotic entry. We found that the pluripotency transcription factor Oct4 (octamer-binding transcription factor 4) plays an unappreciated role in the ES cell cycle by forming a complex with cyclin–Cdk1 and inhibiting Cdk1 activation. Ectopic expression of Oct4 or a mutant lacking transcriptional activity recapitulated delayed mitotic entry in HeLa cells. Reduction of Oct4 levels in ES cells accelerated G2 progression, which led to increased chromosomal missegregation and apoptosis. Our data demonstrate an unexpected nontranscriptional function of Oct4 in the regulation of mitotic entry.

Erratum: Donor cell type can influence the epigenome and differentiation potential of human induced pluripotent stem cells (Nature Biotechnology (2011) 29 (1117-1119))

579 Bruce L. Booth is with Atlas Venture, Cambridge, Massachusetts, USA, and Bijan Salehizadeh is at Highland Capital Partners, Menlo Park, California, USA. Both authors focus on early stage healthcare and life sciences investments. Both also blog about healthcare and venture capital at http://www.LifeSciVC. com and http://www.thebij.com, respectively. e-mail: bruce@atlasventure.com (Fig. 1a). This is substantially more than all technology venture sectors. By comparison, the returns also differ considerably from the S&P 500’s 4% return aggregate over the decade, although the venture IRRs have not accounted for fees. It is worth noting that venture capital certainly did not offer daily liquidity like the major equity indices, which the 2008 credit crisis painfully highlighted. Changing the time period to exclude the 2000–2001 bubble does not fundamentally alter the comparative realized performance between healthcare and technology sectors. Breaking down healthcare into its subsectors, therapeutics companies (pharmaceuticals and biotech) and healthcare services have outperformed medical devices and healthcare software (Fig. 1b). Importantly, the return dynamics have changed considerably over time. In the 1990s, returns in many technology sectors outperformed those of life sciences (Fig. 1c). Even so, most of those sectors saw striking reversals of fortune in the past decade, many with substantial negative percentage changes. Pharmaceuticals was the only subsector to actually see an increase in realized venture capital returns across the two decades. As the old axiom goes, past performance is not a predictor of future returns, but these data certainly suggest that, contrary to widely held perceptions, over the past decade healthcare, and more specifically life sciences investing, was a steady source of overall returns above and beyond other venture capital sectors. Importantly, these data are a look backward at companies that had raised capital and achieved exits from 2000 to 2010 and excludes realizations from technology companies that have gone public in the past several months, such as returns, ‘hit rates’ of winners versus losers and the scalability of deals across sectors.