We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation 1. EPSCs had enriched molecular signatures of blastomeres and possessed the developmental potency for all embryonic and extraembryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras, and produce primordial germ celllike cells in vitro. Under similar conditions, human ESCs and iPSCs can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Significantly, trophoblast stem cell-like cells can be generated from both human and porcine EPSCs. Our pathwayinhibition paradigm thus opens a new avenue for generating mammalian pluripotent stem cells, and EPSCs present an unique cellular platform for translational research in biotechnology and regenerative medicine.
Selective macroautophagy is an important protective mechanism against diverse cellular stresses. In contrast to the well-characterized starvation-induced autophagy, the regulation of selective autophagy is largely unknown. Here, we demonstrate that Huntingtin, the Huntington’s disease gene product, functions as a scaffold protein for selective macroautophagy but it is dispensable for nonselective macroautophagy. In Drosophila, Huntingtin genetically interacts with autophagy pathway components. In mammalian cells, Huntingtin physically interacts with the autophagy cargo receptor p62 to facilitate its association with the integral autophagosome component LC3 and with lysine-63-linked ubiquitin-modified substrates. Maximal activation of selective autophagy during stress is attained by the ability of Huntingtin to bind ULK1, a kinase that initiates autophagy, which releases ULK1 from negative regulation via mTOR. Our data uncover an important physiological function of Huntingtin and provide a missing link in the activation of selective macroautophagy in metazoans.
Phosphorylation provides an important mechanism by which transcription factor activity is regulated. Estrogen receptor ␣ (ER␣) is phosphorylated on multiple sites, and stimulation of a number of growth factor receptors and/or protein kinases leads to ligand-independent and/or synergistic increase in transcriptional activation by ER␣ in the presence of estrogen. Here we show that ER␣ is phosphorylated by protein kinase A (PKA) on serine-236 within the DNA binding domain. Mutation of serine-236 to glutamic acid prevents DNA binding by inhibiting dimerization by ER␣, whereas mutation to alanine has little effect on DNA binding or dimerization. Estrogen receptor ␣ (ER␣) mediates the effects of estrogens on cell growth and differentiation. ER␣ is a member of a superfamily of transcription factors that includes receptors for steroid (androgen, ecdysone, glucocorticoid, mineralocorticoid, estrogen, and progesterone) and thyroid hormones; vitamin D 3 ; retinoic acid; and peroxisome proliferator-, farnesoid-, and arachidonic acid-activated receptors, as well as "orphan" receptors for which no ligands have as yet been identified. These receptors are characterized by highly conserved DNA and ligand-binding domains (LBDs) and regulate transcription by binding to cis-acting enhancer elements in promoters of responsive genes as monomers or as homo-or heterodimers (12,13,55,56,83). Comparison of the amino acid sequences of ER␣ from different species shows that the sequences can be divided into six regions, A to F, on the basis of differing amino acid sequence homologies (49). This division can be extended to all other members of the nuclear receptor superfamily. Region C encodes two zinc fingers comprising the DNA-binding domain (DBD). A region N-terminal to the DBD (regions A/B) contains a transcription activation function (AF-1) which can act in a ligand-independent manner when isolated from the LBD. The LBD (region E) contains a ligand-dependent transcription activation function (AF-2). AF-1 and AF-2 activate transcription independently and synergistically and act in a promoter-and cell-specific manner (14, 50-52, 79, 84). Antiestrogens such as tamoxifen and ICI 164,384 antagonize the effects of estrogens by competing with estrogen for binding to ER␣. Tamoxifen or its derivative 4-hydroxytamoxifen is a partial antagonist; it inhibits transcriptional activation by AF-2 but enables transcription through AF-1 (14, 58, 59). ICI 164,384, on the other hand, is a complete antagonist which inhibits transcriptional activation by both AF-1 and AF-2 (57, 60). No known antiestrogens prevent DNA binding by hER␣, although ICI 164,384 treatment reduces the half-life of ER␣ (24,29,71) and results in the progressive loss of ER␣ from the nucleus (23). Furthermore, in vivo or in vitro treatment with ICI 164,384 results in a loss in the ability of hER␣ to bind DNA in vitro at elevated temperatures through a mechanism which is at present unclear (60).Phosphorylation is a common covalent modification of proteins which provides an important mecha...
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