Four sets of experiments were conducted to examine the influence of conjugated linoleic acid (CLA) isomers during proliferation and differentiation of cultures of 3T3-L1 preadipocytes using physiological culturing conditions. Cultures treated with either albumin [bovine serum albumin (BSA) vehicle] or linoleic acid (LA) served as controls. For the proliferation study (Expt.1), cells were cultured in media containing a crude mixture of CLA isomers or pure LA at 0, 10, 50, or 200 microM for 4 d. Preadipocyte proliferation (cell number, 3H-thymidine incorporation into DNA) decreased as the level of CLA increased in the cultures. In contrast, LA had no impact on DNA synthesis. In Experiment 2a, postconfluent cultures were grown in media containing a crude mixture of CLA isomers or LA at 0, 10, 50, or 200 microM for the next 6 d. Postconfluent cultures supplemented with 50-200 microM CLA had less triglyceride (TG) and were smaller in size than cultures supplemented with similar amounts of LA. In Experiment 2b, postconfluent cultures supplemented with 200 microM of a crude mixture of CLA isomers or LA were harvested on days 1, 3, 6, or 9. Differences in TG content of cultures supplemented with 200 microM CLA compared to control and LA-supplemented cultures became apparent after 3 d of culture. Experiments 3a and 3b examined whether the fatty acid vehicle (BSA vs. ethanol) or the vitamin E status (+/-0.2 mM alpha-tocopherol) of the cultures altered CLA's impact on preadipocyte TG content. In Experiment 3a, ethanol-treated cultures had more TG than non-ethanol-treated cultures regardless of the fatty acid treatment. In Experiment 3b, cultures treated with 100 microM of either a crude mixture of CLA or the trans-10,cis-12 CLA isomer without supplemental vitamin E for 6 d had less TG than CLA-treated cultures containing vitamin E. In Experiment 4, postconfluent cultures were grown in media containing 100 microM LA or either a crude mixture of CLA isomers or the trans-10,cis-12 CLA isomer for 24-96 h to assess CLA's influence on the cell cycle and indices of apoptosis. Cultures treated with 100 microM CLA for 24-96 h had more apoptotic cells than BSA- or LA-treated cultures. Furthermore, cultures treated for 48 h with CLA had fewer cells in the S-phase than control cultures. The effects of the trans-10,cis-12 CLA isomer were more pronounced than those of the crude mixture of CLA isomers. These data suggest that CLA may exert its antiobesity effects by inhibiting proliferation, attenuating TG content, and/or inducing apoptosis in (pre)adipocytes.

Transcription-factor-induced reprogramming of somatic cells to pluripotency is a very inefficient process, probably due to the existence of important epigenetic barriers that are imposed during differentiation and that contribute to preserving cell identity. In an effort to decipher the molecular nature of these barriers, we followed a genome-wide approach, in which we identified macrohistone variants (macroH2A) as highly expressed in human somatic cells but downregulated after reprogramming to pluripotency, as well as strongly induced during differentiation. Knockdown of macrohistone variants in human keratinocytes increased the efficiency of reprogramming to pluripotency, whereas overexpression had opposite effects. Genome-wide occupancy profiles show that in human keratinocytes, macroH2A.1 preferentially occupies genes that are expressed at low levels and are marked with H3K27me3, including pluripotency-related genes and bivalent developmental regulators. The presence of macroH2A.1 at these genes prevents the regain of H3K4me2 during reprogramming, imposing an additional layer of repression that preserves cell identity.

Human epidermal stem cells transit from a slow cycling to an actively proliferating state to contribute to homeostasis. Both stem cell states differ in their cell cycle profiles but must remain guarded from differentiation and senescence. Here we show that Cbx4, a Polycomb Repressive Complex 1 (PRC1)-associated protein, maintains human epidermal stem cells as slow-cycling and undifferentiated, while protecting them from senescence. Interestingly, abrogating the polycomb activity of Cbx4 impairs its antisenescent function without affecting stem cell differentiation, indicating that differentiation and senescence are independent processes in human epidermis. Conversely, Cbx4 inhibits stem cell activation and differentiation through its SUMO ligase activity. Global transcriptome and chromatin occupancy analyses indicate that Cbx4 regulates modulators of epidermal homeostasis and represses factors such as Ezh2, Dnmt1, and Bmi1 to prevent the active stem cell state. Our results suggest that distinct Polycomb complexes balance epidermal stem cell dormancy and activation, while continually preventing senescence and differentiation.

A series of experiments was conducted using 3T3-L1 preadipocytes as the cell model to determine: (i) whether the triglyceride (TG)-lowering effects of a crude mixture of conjugated linoleic acid (CLA) isomers were due to a specific isomer of CLA and the timing of treatment, (ii) if CLA reduced TG content by inhibiting a key regulator of adipogenesis, (iii) if CLA incorporated into either neutral lipid or phospholipid cell fractions, and (iv) whether the effects of CLA treatment were reversible. Trans-10,cis-12 CLA reduced TG content, whereas the cis-9,trans-11 isomer increased TG content compared to vehicle [bovine serum albumin (BSA)] controls. Treatment with 50 microM trans-10,cis-12 CLA during the entire 6 d of differentiation reduced TG content to a greater extent than treatment during either the first 3 d or last 3 d of differentiation. Trans-10,cis-12 CLA treatment of preadipocyte cultures for 48 h increased peroxisome proliferator activated receptor gamma2 (PPARgamma2) protein expression compared to cultures treated with linoleic acid (LA) or the BSA controls. CLA had no effect on adipose P2 (aP2), a fatty acid-binding protein regulated by PPARgamma2. Both the cis-9,trans-11 and the trans-10,cis-12 isomers of CLA were incorporated into neutral lipids and phospholipids. However, cis-9,trans-11 CLA levels were one- to twofold higher than trans-10,cis-12 CLA levels. Moreover, trans-10,cis-12 CLA treatment reduced cis-11 18:1 concentrations in both neutral lipids and phospholipids while increasing cis-9 18:1 and 18:2 concentrations. Palmitoleic acid (16:1) levels were also lower in the neutral lipid fraction of cultures treated with trans-10,cis-12 CLA. Supplementing trans-10,cis-12 CLA-treated cultures (50 microM) with increasing levels of LA resulted in a dose-dependent increase in TG content compared to cultures treated with 50 microM CLA alone. LA supplementation also prevented some of the morphological changes associated with trans-10,cis-12 CLA treatment as seen with scanning electron microscopy. Treatment with 50 microM trans-10,cis-12 CLA for 6 d decreased PPARgamma2 levels, and supplementation of CLA-treated cultures with LA increased PPARgamma2 levels compared with cultures treated with CLA alone. Taken together, these data indicate that in cultures of 3T3-L1 preadipocytes: (i) trans-10,cis-12 CLA is the TG-lowering isomer of CLA, and its effects are dependent on dose, duration of treatment, and the amount of LA in the cultures; (ii) trans-10,cis-12 CLA treatment alters the monounsaturated fatty acid profile of neutral- and phospholipids of the cultures; and (iii) although acute (2-d) trans-10,cis-12 CLA treatment increased PPARgamma2 protein levels, chronic (6-d) treatment decreased PPARgamma2 levels.

MYC proto-oncogene is a key player in cell homeostasis that is commonly deregulated in human carcinogenesis(1). MYC can either activate or repress target genes by forming a complex with MAX (ref. 2). MYC also exerts MAX-independent functions that are not yet fully characterized(3). Cells possess an intrinsic pathway that can abrogate MYC-MAX dimerization and E-box interaction, by inducing phosphorylation of MYC in a PAK2-dependent manner at three residues located in its helix-loop-helix domain(4). Here we show that these carboxy-terminal phosphorylation events switch MYC from an oncogenic to a tumour-suppressive function. In undifferentiated cells, MYC-MAX is targeted to the promoters of retinoic-acid-responsive genes by its direct interaction with the retinoic acid receptor-α (RARα). MYC-MAX cooperates with RARα to repress genes required for differentiation, in an E-box-independent manner. Conversely, on C-terminal phosphorylation of MYC during differentiation, the complex switches from a repressive to an activating function, by releasing MAX and recruiting transcriptional co-activators. Phospho-MYC synergizes with retinoic acid to eliminate circulating leukaemic cells and to decrease the level of tumour invasion. Our results identify an E-box-independent mechanism for transcriptional regulation by MYC that unveils previously unknown functions for MYC in differentiation. These may be exploited to develop alternative targeted therapies.

Jarid2 is required for the genomic recruitment of the polycomb repressive complex-2 (PRC2) in embryonic stem cells. However, its specific role during late development and adult tissues remains largely uncharacterized. Here, we show that deletion of Jarid2 in mouse epidermis reduces the proliferation and potentiates the differentiation of postnatal epidermal progenitors, without affecting epidermal development. In neonatal epidermis, Jarid2 deficiency reduces H3K27 trimethylation, a chromatin repressive mark, in epidermal differentiation genes previously shown to be targets of the PRC2. However, in adult epidermis Jarid2 depletion does not affect interfollicular epidermal differentiation but results in delayed hair follicle (HF) cycling as a consequence of decreased proliferation of HF stem cells and their progeny. We conclude that Jarid2 is required for the scheduled proliferation of epidermal stem and progenitor cells necessary to maintain epidermal homeostasis.

A fast track "Hot Start" process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community. ETOC: The report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field.

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