Developmentally regulated alterations in Polycomb repressive complex 1 proteins on the inactive X chromosome

Polycomb group (PcG) proteins belonging to the polycomb (Pc) repressive complexes 1 and 2 (PRC1 and PRC2) maintain homeotic gene silencing. In Drosophila, PRC2 methylates histone H3 on lysine 27, and this epigenetic mark facilitates recruitment of PRC1. Mouse PRC2 (mPRC2) has been implicated in X inactivation, as mPRC2 proteins transiently accumulate on the inactive X chromosome (Xi) at the onset of X inactivation to methylate histone H3 lysine 27 (H3-K27). In this study, we demonstrate that mPRC1 proteins localize to the Xi, and that different mPRC1 proteins accumulate on the Xi during initiation and maintenance of X inactivation in embryonic cells. The Xi accumulation of mPRC1 proteins requires Xist RNA and is not solely regulated by the presence of H3-K27 methylation, as not all cells that exhibit this epigenetic mark on the Xi show Xi enrichment of mPRC1 proteins. Our results implicate mPRC1 in X inactivation and suggest that the regulated assembly of PcG protein complexes on the Xi contributes to this multistep process

Long-term culture of genome-stable bipotent stem cells from adult human liver.

Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.This work was supported by grants to MH (EU/236954) and to HC (The United European Gastroenterology Federation (UEGF) Research Prize 2010, EU/232814-StemCellMark and NWO/116002008). MH is supported by The Wellcome Trust Sir Henry Dale fellowship. The Rspo cell line was kindly provided by Dr. Calvin Kuo.This is the final published version. It first appeared at http://www.cell.com/abstract/S0092-8674%2814%2901566-9

The Use of a Stringent Selection System Allows the Identification of DNA Elements that Augment Gene Expression

The use of high stringency selection systems often results in the induction of very few recombinant mammalian cell lines, which limits the ability to isolate a cell line with favorable characteristics. The employment of for instance STAR elements in DNA constructs elevates the induced number of colonies and also the protein expression levels in these colonies. Here, we describe a method to systematically identify genomic DNA elements that are able to induce many stably transfected mammalian cell lines. We isolated genomic DNA fragments upstream from the human Rb1 and p73 gene loci and cloned them around an expression cassette that contains a very stringent selection marker. Due to the stringency of the selection marker, hardly any colony survives without flanking DNA elements. We tested fourteen ~3500 bp DNA stretches from the Rb1 and p73 loci. Only two ~3500 bp long DNA fragments, called Rb1E and Rb1F, induced many colonies in the context of the stringent selection system and these colonies displayed high protein expression levels. Functional analysis showed that the Rb1 DNA fragments contained no enhancer, promoter, or STAR activity. Our data show the potential of a methodology to identify novel gene expression augmenting DNA elements in an unbiased manner

Long-term culture of genome-stable bipotent stem cells from adult human liver

Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy

The Polycomb Group Protein EED Interacts with YY1, and Both Proteins Induce Neural Tissue in Xenopus Embryos

Polycomb group (PcG) proteins form multimeric protein complexes which are involved in the heritable stable repression of genes. Previously, we identified two distinct human PcG protein complexes. The EED-EZH protein complex contains the EED and EZH2 PcG proteins, and the HPC-HPH PcG complex contains the HPC, HPH, BMI1, and RING1 PcG proteins. Here we show that YY1, a homolog of the Drosophila PcG protein pleiohomeotic (Pho), interacts specificially with the human PcG protein EED but not with proteins of the HPC-HPH PcG complex. Since YY1 and Pho are DNA-binding proteins, the interaction between YY1 and EED provides a direct link between the chromatin-associated EED-EZH PcG complex and the DNA of target genes. To study the functional significance of the interaction, we expressed the Xenopus homologs of EED and YY1 in Xenopus embryos. Both Xeed and XYY1 induce an ectopic neural axis but do not induce mesodermal tissues. In contrast, members of the HPC-HPH PcG complex do not induce neural tissue. The exclusive, direct neuralizing activity of both the Xeed and XYY1 proteins underlines the significance of the interaction between the two proteins. Our data also indicate a role for chromatin-associated proteins, such as PcG proteins, in Xenopus neural induction

Identification of Enteroendocrine Regulators by Real-Time Single-Cell Differentiation Mapping

Homeostatic regulation of the intestinal enteroendocrine lineage hierarchy is a poorly understood process. We resolved transcriptional changes during enteroendocrine differentiation in real time at single-cell level using a novel knockin allele of Neurog3, the master regulator gene briefly expressed at the onset of enteroendocrine specification. A bi-fluorescent reporter, Neurog3Chrono, measures time from the onset of enteroendocrine differentiation and enables precise positioning of single-cell transcriptomes along an absolute time axis. This approach yielded a definitive description of the enteroendocrine hierarchy and its sub-lineages, uncovered differential kinetics between sub-lineages, and revealed time-dependent hormonal plasticity in enterochromaffin and L cells. The time-resolved map of transcriptional changes predicted multiple novel molecular regulators. Nine of these were validated by conditional knockout in mice or CRISPR modification in intestinal organoids. Six novel candidate regulators (Sox4, Rfx6, Tox3, Myt1, Runx1t1, and Zcchc12) yielded specific enteroendocrine phenotypes. Our time-resolved single-cell transcriptional map presents a rich resource to unravel enteroendocrine differentiation

Distinct BMI-1 and EZH2 expression patterns in thymocytes and mature T cells suggest a role for polycomb genes in human T cell differentiation

BMI-1 and EZH2 Polycomb-group (PcG) proteins belong to two distinct protein complexes involved in the regulation of hematopoiesis. Using unique PcG-specific antisera and triple immunofluorescence, we found that mature resting peripheral T cells expressed BMI-1, whereas dividing blasts were EZH2+. By contrast, subcapsular immature double-negative (DN) (CD4-/CD8-) T cells in the thymus coexpressed BMI-1 and EZH2 or were BMI-1 single positive. Their descendants, double-positive (DP; CD4+/CD8+) cortical thymocytes, expressed EZH2 without BMI-1. Most EZH2+ DN and DP thymocytes were dividing, while DN BMI-1+/EZH2- thymocytes were resting and proliferation was occasionally noted in DN BMI-1+/EZH2+ cells. Maturation of DP cortical thymocytes to single-positive (CD4+/CD8- or CD8+/CD4-) medullar thymocytes correlated with decreased detectability of EZH2 and continued relative absence of BMI-1. Our data show that BMI-1 and EZH2 expression in mature peripheral T cells is mutually exclusive and linked to proliferation status, and that this pattern is not yet established in thymocytes of the cortex and medulla. T cell stage-specific PcG expression profiles suggest that PcG genes contribute to regulation of T cell differentiation. They probably reflect stabilization of cell type-specific gene expression and irreversibility of lineage choice. The difference in PcG expression between medullar thymocytes and mature interfollicular T cells indicates that additional maturation processes occur after thymocyte transportation from the thymus.</p