An Intracellular Allosteric Modulator Binding Pocket in SK2 Ion Channels Is Shared by Multiple Chemotypes.

Small conductance potassium (SK) ion channels define neuronal firing rates by conducting the after-hyperpolarization current. They are key targets in developing therapies where neuronal firing rates are dysfunctional, such as in epilepsy, Parkinson's, and amyotrophic lateral sclerosis (ALS). Here, we characterize a binding pocket situated at the intracellular interface of SK2 and calmodulin, which we show to be shared by multiple small-molecule chemotypes. Crystallization of this complex revealed that riluzole (approved for ALS) and an analog of the anti-ataxic agent (4-chloro-phenyl)-[2-(3,5-dimethyl-pyrazol-1-yl)-pyrimidin-4-yl]-amine (CyPPA) bind to and allosterically modulate via this site. Solution-state nuclear magnetic resonance demonstrates that riluzole, NS309, and CyPPA analogs bind at this bipartite pocket. We demonstrate, by patch-clamp electrophysiology, that both classes of ligand interact with overlapping but distinct residues within this pocket. These data define a clinically important site, laying the foundations for further studies of the mechanism of action of riluzole and related molecules.

Gata6 potently initiates reprograming of pluripotent and differentiated cells to extraembryonic endoderm stem cells.

Transcription factor-mediated reprograming is a powerful method to study cell fate changes. In this study, we demonstrate that the transcription factor Gata6 can initiate reprograming of multiple cell types to induced extraembryonic endoderm stem (iXEN) cells. Intriguingly, Gata6 is sufficient to drive iXEN cells from mouse pluripotent cells and differentiated neural cells. Furthermore, GATA6 induction in human embryonic stem (hES) cells also down-regulates pluripotency gene expression and up-regulates extraembryonic endoderm (ExEn) genes, revealing a conserved function in mediating this cell fate switch. Profiling transcriptional changes following Gata6 induction in mES cells reveals step-wise pluripotency factor disengagement, with initial repression of Nanog and Esrrb, then Sox2, and finally Oct4, alongside step-wise activation of ExEn genes. Chromatin immunoprecipitation and subsequent high-throughput sequencing analysis shows Gata6 enrichment near pluripotency and endoderm genes, suggesting that Gata6 functions as both a direct repressor and activator. Together, this demonstrates that Gata6 is a versatile and potent reprograming factor that can act alone to drive a cell fate switch from diverse cell types.

Dynamic Proteomic Profiling of Extra-Embryonic Endoderm Differentiation in Mouse Embryonic Stem Cells.

During mammalian preimplantation development, the cells of the blastocyst's inner cell mass differentiate into the epiblast and primitive endoderm lineages, which give rise to the fetus and extra-embryonic tissues, respectively. Extra-embryonic endoderm (XEN) differentiation can be modeled in vitro by induced expression of GATA transcription factors in mouse embryonic stem cells. Here, we use this GATA-inducible system to quantitatively monitor the dynamics of global proteomic changes during the early stages of this differentiation event and also investigate the fully differentiated phenotype, as represented by embryo-derived XEN cells. Using mass spectrometry-based quantitative proteomic profiling with multivariate data analysis tools, we reproducibly quantified 2,336 proteins across three biological replicates and have identified clusters of proteins characterized by distinct, dynamic temporal abundance profiles. We first used this approach to highlight novel marker candidates of the pluripotent state and XEN differentiation. Through functional annotation enrichment analysis, we have shown that the downregulation of chromatin-modifying enzymes, the reorganization of membrane trafficking machinery, and the breakdown of cell-cell adhesion are successive steps of the extra-embryonic differentiation process. Thus, applying a range of sophisticated clustering approaches to a time-resolved proteomic dataset has allowed the elucidation of complex biological processes which characterize stem cell differentiation and could establish a general paradigm for the investigation of these processes.

Derivation of extraembryonic endoderm stem (XEN) cells from mouse embryos and embryonic stem cells.

At the time of implantation in the maternal uterus, the mouse blastocyst possesses an inner cell mass comprising two lineages: epiblast (Epi) and primitive endoderm (PrE). Representative stem cells derived from these two cell lineages can be expanded and maintained indefinitely in vitro as either embryonic stem (ES) or XEN cells, respectively. Here we describe protocols that can be used to establish XEN cell lines. These include the establishment of XEN cells from blastocyst-stage embryos in either standard embryonic or trophoblast stem (TS) cell culture conditions. We also describe protocols for establishing XEN cells directly from ES cells by either retinoic acid and activin-based conversion or by overexpression of the GATA transcription factor Gata6. XEN cells are a useful model of PrE cells, with which they share gene expression, differentiation potential and lineage restriction. The robust protocols for deriving XEN cells described here can be completed within 2-3 weeks.

Conversion from mouse embryonic to extra-embryonic endoderm stem cells reveals distinct differentiation capacities of pluripotent stem cell states.

The inner cell mass of the mouse pre-implantation blastocyst comprises epiblast progenitor and primitive endoderm cells of which cognate embryonic (mESCs) or extra-embryonic (XEN) stem cell lines can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of their in vivo tissue of origin. Recently, we demonstrated that XEN-like cells arise within mESC cultures. This raises the possibility that mESCs can generate self-renewing XEN cells without the requirement for gene manipulation. We have developed a novel approach to convert mESCs to XEN cells (cXEN) using growth factors. We confirm that the downregulation of the pluripotency transcription factor Nanog and the expression of primitive endoderm-associated genes Gata6, Gata4, Sox17 and Pdgfra are necessary for cXEN cell derivation. This approach highlights an important function for Fgf4 in cXEN cell derivation. Paracrine FGF signalling compensates for the loss of endogenous Fgf4, which is necessary to exit mESC self-renewal, but not for XEN cell maintenance. Our cXEN protocol also reveals that distinct pluripotent stem cells respond uniquely to differentiation promoting signals. cXEN cells can be derived from mESCs cultured with Erk and Gsk3 inhibitors (2i), and LIF, similar to conventional mESCs. However, we find that epiblast stem cells (EpiSCs) derived from the post-implantation embryo are refractory to cXEN cell establishment, consistent with the hypothesis that EpiSCs represent a pluripotent state distinct from mESCs. In all, these findings suggest that the potential of mESCs includes the capacity to give rise to both extra-embryonic and embryonic lineages.