SUMMARY Wnt signaling is involved in self-renewal and maintenance of hematopoietic stem cells (HSCs); however, the particular role of noncanonical Wnt signaling in regulating HSCs in vivo is largely unknown. Here, we show Flamingo (Fmi) and Frizzled (Fz) 8, members of noncanonical Wnt signaling, both express in and functionally maintain quiescent long-term HSCs. Fmi regulates Fz8 distribution at the interface between HSCs and N-cadherin+ osteoblasts (N-cad+OBs that enrich osteoprogenitors) in the niche. We further found that N-cad+OBs predominantly express noncanonical Wnt ligands and inhibitors of canonical Wnt signaling under homeostasis. Under stress, noncanonical Wnt signaling is attenuated and canonical Wnt signaling is enhanced in activation of HSCs. Mechanistically, noncanonical Wnt signaling mediated by Fz8 suppresses the Ca2+-NFAT- IFNγ pathway, directly or indirectly through the CDC42-CK1α complex and also antagonizes canonical Wnt signaling in HSCs. Taken together, our findings demonstrate that noncanonical Wnt signaling maintains quiescent long-term HSCs through Fmi and Fz8 interaction in the niche.

The epigenetic regulation of imprinted genes via monoallelic DNA methylation of either maternal or paternal alleles is critical for embryonic growth and development1. Imprinted genes were recently shown to be expressed in mammalian adult stem cells to support self-renewal of neural and lung stem cells2, 3,4; however, a role for imprinting per se in adult stem cells remains elusive. Here we show up-regulation of growth-restricting imprinted genes, including within the H19-Igf2 locus5, in long-term hematopoietic stem cells (LT-HSCs) and their down-regulation upon HSC activation and proliferation. A differentially methylated region (DMR) upstream of H19 (H19-DMR), serving as the imprinting control region, determines the reciprocal expression of H19 from the maternal allele and Igf2 from the paternal allele1. In addition, H19 also serves as a source of miR-675, which restricts Igf1r expression6. We demonstrated that conditional deletion of the maternal but not the paternal H19-DMR reduced adult HSC quiescence, a state required for long-term maintenance of HSCs, and compromised HSC function. Maternal-specific H19-DMR deletion resulted in activation of the Igf2-Igfr1 pathway as revealed by the translocation of phosphorylated Foxo3 (an inactive form) from nucleus to cytoplasm and the release of Foxo3-mediated cell-cycle arrest, thus leading to increased activation, proliferation, and eventual exhaustion of HSCs. Mechanistically, maternal-specific H19-DMR deletion led to Igf2 up-regulation and increased translation of Igf1r, which is normally suppressed by H19-derived miR-675. Similarly, genetic inactivation of Igf1r partially rescued the H19-DMR deletion phenotype. Our work establishes a novel role for this unique form of epigenetic control at the H19-Igf2 locus in maintaining adult stem cells.

Farnesoid X receptor (FXR) is a bile acid-activated transcription factor belonging to the nuclear receptor superfamily. FXR is highly expressed in liver and intestine and crosstalk mediated by FXR in these two organs is critical in maintaining bile acid homeostasis. FXR deficiency has been implicated in many liver and intestine diseases. However, regulation of transcription by FXR at the genomic level is not known. This study analyzed genome-wide FXR binding in liver and intestine of mice treated with a synthetic FXR ligand (GW4064) by chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-seq). The results showed a large degree of tissue-specific FXR binding, with only 11% of total sites shared between liver and intestine. The sites were widely distributed between intergenic, upstream, intragenic, and downstream of genes, with novel sites identified within even known FXR target genes. Motif analysis revealed a half nuclear receptor binding site, normally bound by a few orphan nuclear receptors, adjacent to the FXR response elements, indicating possible involvement of some orphan nuclear receptors in modulating FXR function. Furthermore, pathway analysis indicated that FXR may be extensively involved in multiple cellular metabolic pathways. Conclusion This study reports genome-wide FXR binding in vivo and the results clearly demonstrate tissue-specific FXR/gene interaction. In addition, FXR may be involved in regulating broader biological pathways in maintaining hepatic and intestinal homeostasis.

ABSTRACT:HepaRG cells, derived from a female hepatocarcinoma patient, are capable of differentiating into biliary epithelial cells and hepatocytes. More importantly, differentiated HepaRG cells are able to maintain activities of many xenobiotic-metabolizing enzymes, and expression of the metabolizing enzyme genes can be induced by xenobiotics. The ability of these cells to express and induce xenobiotic-metabolizing enzymes is in stark contrast to the frequently used HepG2 cells. The previous studies have mainly focused on a set of selected genes; therefore, it is of significant interest to know the extent of similarity of gene expression at whole genome levels in HepaRG cells and HepG2 cells compared with primary human hepatocytes and human liver tissues. To accomplish this objective, we used Affymetrix (Santa Clara, CA) U133 Plus 2.0 arrays to characterize the whole genome gene expression profiles in triplicate biological samples from HepG2 cells, HepaRG cells (undifferentiated and differentiated cells), freshly isolated primary human hepatocytes, and frozen liver tissues. After using similarity matrix, principal components, and hierarchical clustering methods, we found that HepaRG cells globally transcribe genes at levels more similar to human primary hepatocytes and human liver tissues than HepG2 cells. In particular, many genes encoding drug-processing proteins are transcribed at a more similar level in HepaRG cells than in HepG2 cells compared with primary human hepatocytes and liver samples. The transcriptomic similarity of HepaRG with primary human hepatocytes is encouraging for use of HepaRG cells in the study of xenobiotic metabolism, hepatotoxicology, and hepatocyte differentiation.

BackgroundRecently, many studies utilizing next generation sequencing have investigated plant evolution and domestication in annual crops. Peach, Prunus persica, is a typical perennial fruit crop that has ornamental and edible varieties. Unlike other fruit crops, cultivated peach includes a large number of phenotypes but few polymorphisms. In this study, we explore the genetic basis of domestication in peach and the influence of humans on its evolution.ResultsWe perform large-scale resequencing of 10 wild and 74 cultivated peach varieties, including 9 ornamental, 23 breeding, and 42 landrace lines. We identify 4.6 million SNPs, a large number of which could explain the phenotypic variation in cultivated peach. Population analysis shows a single domestication event, the speciation of P. persica from wild peach. Ornamental and edible peach both belong to P. persica, along with another geographically separated subgroup, Prunus ferganensis.We identify 147 and 262 genes under edible and ornamental selection, respectively. Some of these genes are associated with important biological features. We perform a population heterozygosity analysis in different plants that indicates that free recombination effects could affect domestication history. By applying artificial selection during the domestication of the peach and facilitating its asexual propagation, humans have caused a sharp decline of the heterozygote ratio of SNPs.ConclusionsOur analyses enhance our knowledge of the domestication history of perennial fruit crops, and the dataset we generated could be useful for future research on comparative population genomics.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0415-1) contains supplementary material, which is available to authorized users.