Lin28 is induced in primed embryonic stem cells and regulates let-7-independent events.

Lin28 RNA-binding proteins play important roles in pluripotent stem cells, but the regulation of their expression and the mechanisms underlying their functions are still not definitively understood. Here we address the above-mentioned issues in the first steps of mouse embryonic stem cell (ESC) differentiation. We observed that the expression of Lin28 genes is transiently induced soon after the exit of ESCs from the naive ground state and that this induction is due to the Hmga2-dependent engagement of Otx2 with enhancers present at both Lin28 gene loci. These mechanisms are crucial for Lin28 regulation, as demonstrated by the abolishment of the Lin28 accumulation in Otx2- or Hmga2-knockout cells compared to the control cells. We have also found that Lin28 controls Hmga2 expression levels during ESC differentiation through a let-7-independent mechanism. Indeed, we found that Lin28 proteins bind a highly conserved element in the 3' UTR of Hmga2 mRNA, and this provokes a down-regulation of its translation. This mechanism prevents the inappropriate accumulation of Hmga2 that would modify the proliferation and physiological apoptosis of differentiating ESCs. In summary, we demonstrated that during ESC differentiation, Lin28 transient induction is dependent on Otx2 and Hmga2 and prevents an inappropriate excessive rise of Hmga2 levels.-Parisi, S., Passaro, F., Russo, L., Musto, A., Navarra, A., Romano, S., Petrosino, G., Russo, T. Lin28 is induced in primed embryonic stem cells and regulates let-7-independent events.

Hmga2 is necessary for Otx2-dependent exit of embryonic stem cells from the pluripotent ground state.

BACKGROUND: A crucial event in the differentiation of mouse embryonic stem cells (ESCs) is the exit from the pluripotent ground state that leads to the acquisition of the 'primed' pluripotent phenotype, characteristic of the epiblast-like stem cells (EpiLCs). The transcription factors Oct4 and Otx2 play a key role in this phenomenon. In particular, Otx2 pioneers and activates new enhancers, which are silent in ESCs and which control the transcription of genes responsible for the acquisition of the EpiLC phenotype. An important point that remains to be addressed is the mechanism through which Otx2 engages the new enhancers and stably associates with them. Hmga2 is a member of the high-mobility group family of proteins, non-histone components of chromatin whose expression is high during embryogenesis and becomes low or undetectable in adults. Its high expression during embryogenesis suggests that Hmga2 fulfills important roles in development. RESULTS: Here, we demonstrate that Hmga2 accumulates soon after the induction of ESC differentiation. Its suppression hampers the exit of ESCs from the pluripotent ground state and their differentiation into EpiLCs. Mechanistically, Hmga2 controls the differentiation process by cooperating with Otx2 in the pioneering of new enhancers. In Hmga2 null induced pluripotent stem cells we observe that Otx2 fails to regulate its target genes upon the induction of differentiation. Hmga2 associates to Otx2-bound loci in EpiLCs, and in Hmga2 KO cells Otx2 is unable to engage and activate the new enhancers, thus indicating that Hmga2 is required for the binding of Otx2 to its cis-elements. We find that this mechanism also operates on the Hmga2 gene, which is one of the targets of Otx2, thus indicating the existence of a positive feedback loop. CONCLUSIONS: Our findings reveal a novel mechanism necessary for the exit of ESCs from the pluripotent ground state. Upon the induction of ESC differentiation, Otx2 alone or in combination with Oct4 engages new enhancers, which are silent in undifferentiated ESCs. The Hmga2 gene is activated by Otx2 and Hmga2 protein binds to the enhancers targeted by Otx2, thus facilitating the engagement and/or the stable association of Otx2. Therefore, our results demonstrate that Hmga2 is a key element of the regulatory network that governs the exit of ESCs from the pluripotent ground state.

A regulatory loop involving Dies1 and miR-125a controls BMP4 signaling in mouse embryonic stem cells.

Bone morphogenetic protein 4 (BMP4) plays an important role in maintaining embryonic stem cells (ESCs) in the undifferentiated state and in the regulation of lineage commitment. We recently identified a transmembrane protein, named Dies1, the suppression of which by RNA interference blocks mouse ESC differentiation by interfering with the BMP4 signaling. We asked whether modulation of Dies1 levels could be a physiological mechanism to regulate ESC pluripotency and/or differentiation. We demonstrated that miR-125a targets Dies1 and regulates its expression in ESCs. The overexpression of miR-125a impairs differentiation, and this effect is specifically mediated by Dies1 down-regulation and accompanied by a decrease of BMP4 signaling. We also found that Dies1 is associated with BMP4 receptor complex and that BMP4 activates the transcription of miR-125a gene. Therefore, a feedback loop exists that sets ESC sensitivity to BMP4. The analysis of this regulatory mechanism revealed that miR-125a overexpression and the consequent inhibition of the BMP4 signaling arrest the cells in the epiblast stem cell (epiSC) status, due to the concomitant activation of the Nodal/Activin pathway.

miR-125b regulates the early steps of ESC differentiation through dies1 in a TGF-independent manner.

Over the past few years, it has become evident that the distinctive pattern of miRNA expression seen in embryonic stem cells (ESCs) contributes to important signals in the choice of the cell fate. Thus, the identification of miRNAs and their targets, whose expression is linked to a specific step of differentiation, as well as the modulation of these miRNAs, may prove useful in the learning of how ESC potential is regulated. In this context, we have studied the expression profile of miRNAs during neural differentiation of ESCs. We have found that miR-125b is upregulated in the first steps of neural differentiation of ESCs. This miRNA targets the BMP4 co-receptor, Dies1, and, in turn, regulates the balance between BMP4 and Nodal/Activin signaling. The ectopic expression of miR-125b blocks ESC differentiation at the epiblast stage, and this arrest is rescued by restoring the expression of Dies1. Finally, opposite to miR-125a, whose expression is under the control of the BMP4, miR-125b is not directly regulated by Transforming Growth Factor beta (TGFß) signals. These results highlight a new important role of miR-125b in the regulation of the transition from ESCs to the epiblast stage and add a new level of control on TGFß signaling in ESCs.

Author Correction: Characterization of the most frequent ATP7B mutation causing Wilson disease in hepatocytes from patient induced pluripotent stem cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characterization of the most frequent ATP7B mutation causing Wilson disease in hepatocytes from patient induced pluripotent stem cells.

H1069Q substitution represents the most frequent mutation of the copper transporter ATP7B causing Wilson disease in Caucasian population. ATP7B localizes to the Golgi complex in hepatocytes but moves in response to copper overload to the endo-lysosomal compartment to support copper excretion via bile canaliculi. In heterologous or hepatoma-derived cell lines, overexpressed ATP7B-H1069Q is strongly retained in the ER and fails to move to the post-Golgi sites, resulting in toxic copper accumulation. However, this pathogenic mechanism has never been tested in patients' hepatocytes, while animal models recapitulating this form of WD are still lacking. To reach this goal, we have reprogrammed skin fibroblasts of homozygous ATP7B-H1069Q patients into induced pluripotent stem cells and differentiated them into hepatocyte-like cells. Surprisingly, in HLCs we found one third of ATP7B-H1069Q localized in the Golgi complex and able to move to the endo-lysosomal compartment upon copper stimulation. However, despite normal mRNA levels, the expression of the mutant protein was only 20% compared to the control because of endoplasmic reticulum-associated degradation. These results pinpoint rapid degradation as the major cause for loss of ATP7B function in H1069Q patients, and thus as the primary target for designing therapeutic strategies to rescue ATP7B-H1069Q function.

Supplementing π-systems: eumelanin and graphene-like integration towards highly conductive materials for the mammalian cell culture bio-interface.

Organic (bio)electronics appears to be the first target for competitive exploitation in the materials science of eumelanins, black insoluble photoprotective human biopolymers. Nonetheless, the low conductivity of these pigments is limiting the implementation of eumelanin-based devices. Here we present a novel organic/organic hybrid material (EUGL) by integration of conductive graphene-like (GL) layers within the EUmelanin pigment (EU). GL layers were obtained by a two-step oxidation/reduction of carbon black. The stability of GL layers over a wide pH range and the self-assembling tendency place this material in a leading position for the fabrication of hybrid materials in aqueous media. EUGL was obtained by inducing the polymerization of eumelanin precursors (5,6-dihydroxyindole, DHI and 5,6-dihydroxyindole-2 carboxylic acid, DHICA) in aqueous media containing GL layers. The new material featured promising biocompatibility and an increased conductivity with respect to eumelanin by four orders of magnitude.