Xiao-bo Zhong

Noncanonical Wnt signaling maintains hematopoietic stem cells in the niche

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 Ca(2+)-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.

Cloning, characterization, and chromosomal mapping of a human electroneutral Na(+)-driven Cl-HCO3 exchanger.

The electroneutral Na+-driven Cl-HCO3 exchanger is a key mechanism for regulating intracellular pH (pH i ) in neurons, glia, and other cells. Here we report the cloning, tissue distribution, chromosomal location, and functional characterization of the cDNA of such a transporter (NDCBE1) from human brain (GenBankTM accession number AF069512). NDCBE1, which encodes 1044 amino acids, is 34% identical to the mammalian anion exchanger (AE2); ∼50% to the electrogenic Na/HCO3 cotransporter (NBCe1) from salamander, rat, and humans; ∼73% to mammalian electroneutral Na/HCO3 cotransporters (NBCn1); 71% to mouse NCBE; and 47% to a Na+-driven anion exchanger (NDAE1) fromDrosophila. Northern blot analysis of NDCBE1 shows a robust ∼12-kilobase signal in all major regions of human brain and in testis, and weaker signals in kidney and ovary. This human gene (SLC4A8) maps to chromosome 12q13. When expressed inXenopus oocytes and running in the forward direction, NDCBE1 is electroneutral and mediates increases in both pH i and [Na+] i (monitored with microelectrodes) that require HCO 3 − and are blocked by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). The pH i increase also requires extracellular Na+. The Na+:HCO 3 − stoichiometry is 1:2. Forward-running NDCBE1 mediates a36Cl efflux that requires extracellular Na+ and HCO 3 − and is blocked by DIDS. Running in reverse, NDCBE1 requires extracellular Cl−. Thus, NDCBE1 encodes a human, electroneutral Na+-driven Cl-HCO3 exchanger.

A comparison of whole genome gene expression profiles of HepaRG cells and HepG2 cells to primary human hepatocytes and human liver tissues

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.

Maternal imprinting at the H19–Igf2 locus maintains adult haematopoietic stem cell quiescence

The epigenetic regulation of imprinted genes by monoallelic DNA methylation of either maternal or paternal alleles is critical for embryonic growth and development. Imprinted genes were recently shown to be expressed in mammalian adult stem cells to support self-renewal of neural and lung stem cells; however, a role for imprinting per se in adult stem cells remains elusive. Here we show upregulation of growth-restricting imprinted genes, including in the H19–Igf2 locus, in long-term haematopoietic stem cells and their downregulation upon haematopoietic stem cell activation and proliferation. A differentially methylated region 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 allele. In addition, H19 serves as a source of miR-675, which restricts Igf1r expression. We demonstrate that conditional deletion of the maternal but not the paternal H19-DMR reduces adult haematopoietic stem cell quiescence, a state required for long-term maintenance of haematopoietic stem cells, and compromises haematopoietic stem cell function. Maternal-specific H19-DMR deletion results in activation of the Igf2–Igfr1 pathway, as shown 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 haematopoietic stem cells. Mechanistically, maternal-specific H19-DMR deletion leads to Igf2 upregulation and increased translation of Igf1r, which is normally suppressed by H19-derived miR-675. Similarly, genetic inactivation of Igf1r partly rescues the H19-DMR deletion phenotype. Our work establishes a new role for this unique form of epigenetic control at the H19–Igf2 locus in maintaining adult stem cells.

Genome-Wide Tissue-Specific Farnesoid X Receptor Binding in Mouse Liver and Intestine

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. (HEPATOLOGY 2010.)

Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibers using rolling circle DNA amplification.

Rolling circle amplification (RCA) is a surface-anchored DNA replication reaction that can be exploited to visualize single molecular recognition events. Here we report the use of RCA to visualize target DNA sequences as small as 50 nts in peripheral blood lymphocytes or in stretched DNA fibers. Three unique target sequences within the cystic fibrosis transmembrane conductance regulator gene could be detected simultaneously in interphase nuclei, and could be ordered in a linear map in stretched DNA. Allele-discriminating oligonucleotide probes in conjunction with RCA also were used to discriminate wild-type and mutant alleles in the cystic fibrosis transmembrane conductance regulator, p53, BRCA-1, and Gorlin syndrome genes in the nuclei of cultured cells or in DNA fibers. These observations demonstrate that signal amplification by RCA can be coupled to nucleic acid hybridization and multicolor fluorescence imaging to detect single nucleotide changes in DNA within a cytological context or in single DNA molecules. This provides a means for direct physical haplotyping and the analysis of somatic mutations on a cell-by-cell basis.

Simultaneous detection of microsatellite repeats and SNPs in the macrophage migration inhibitory factor (MIF) gene by thin-film biosensor chips and application to rural field studies

Microsatellite repeat and single nucleotide polymorphisms (SNPs) are abundant sources of genetic variation, but existing methodologies cannot simultaneously detect these variants in a facile or inexpensive way. We describe herein a thin-film biosensor chip based on an allele-discriminating oligonucleotide array that enables genotyping for both microsatellite repeats and SNPs in a single analysis. We validated this methodology for the functionally polymorphic −794 CATT5–8 repeat and −173 G/C SNP present in the promoter of the human gene for macrophage migration inhibitory factor (MIF). In a comparison of 30 samples collected at a rural hospital in Zambia, we observed a 100% concordance for both the CATT repeat and G/C SNP between the biosensor methodology and the conventional capillary electrophoresis. The biosensor chips are low in cost and once printed, they are robust and require no instrumentation for analysis. When combined with multiple displacement amplification, this methodology can be utilized in primitive settings for the genotyping of nanogram quantities of DNA present in blood, dried and stored on filter paper samples. We applied this methodology to a field study of MIF genotype in children with malaria, and provide first evidence for a potential association between MIF alleles and malaria infection. We also present data supporting significant population stratification of the low- versus high-expression forms of MIF that may bear on the role of this gene in infectious diseases.

Single-nucleotide polymorphism genotyping on optical thin-film biosensor chips

Single-nucleotide polymorphisms (SNPs) constitute the bulk of human genetic variation and provide excellent markers to identify genetic factors contributing to complex disease susceptibility. A rapid, sensitive, and inexpensive assay is important for large-scale SNP scoring. Here we report the development of a multiplex SNP detection system using silicon chips coated to create a thin-film optical biosensor. Allele-discriminating, aldehyde-labeled oligonucleotides are arrayed and covalently attached to a hydrazinederivatized chip surface. Target sequences (e.g., PCR amplicons) then are hybridized in the presence of a mixture of biotinylated detector probes, one for each SNP, and a thermostable DNA ligase. After a stringent wash (0.01 M NaOH), ligation of biotinylated detector probes to perfectly matched capture oligomers is visualized as a color change on the chip surface (gold to blue/purple) after brief incubations with an anti-biotin IgG-horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate. Testing of PCR fragments is completed in 30–40 min. Up to several hundred SNPs can be assayed on a 36-mm2 chip, and SNP scoring can be done by eye or with a simple digital-camera system. This assay is extremely robust, exhibits high sensitivity and specificity, and is format-flexible and economical. In studies of mutations associated with risk for venous thrombosis and genotyping/haplotyping of African-American samples, we document high-fidelity analysis with 0 misassignments in 500 assays performed in duplicate.

Genetic polymorphisms in cytochrome P450 oxidoreductase influence microsomal P450-catalyzed drug metabolism.

Objectives Cytochrome P450 oxidoreductase (POR) is the only flavoprotein that donates electrons to all microsomal P450 enzymes, which catalyze the biosynthesis of steroids, fatty acids, and bile acids, as well as metabolism of more than 80% of prescription drugs. Although mutations in POR have been identified in several disease states with disordered steroidogenesis, effects of polymorphisms on drug metabolism in the general population are unclear. In this report, we performed a comprehensive study to correlate POR polymorphisms with POR gene expression, POR activity, and P450-catalyzed drug metabolism. Methods A set of human liver samples (n=99) were used in this study. POR polymorphisms were identified by sequencing the exons and surrounding introns of the POR gene and mRNA levels were quantified by branched DNA technology. POR activity was quantified by measuring cytochrome c reduction in liver microsomes and activities of 10 drug-metabolizing P450 enzymes were quantified by high performance liquid chromatography methods with drugs known to be specific for each enzyme. Results Of the 34 polymorphisms identified in this cohort, four polymorphisms changed an amino acid: K49N, L420M, A503V, and L577P. L577P likely resulted in an &agr; helix change, possible disruption of the nicotinamide adenine dinucleotide phosphate interaction, and decreased POR activity (P=0.003) and several drug-metabolizing P450 activities. We also found an intronic polymorphisms rs41301427, which was associated with altered POR, but not P450 activities. Conclusion Polymorphisms in the POR gene can affect POR and P450-catalyzed drug oxidation. These results suggest that POR has the potential to serve as a predictive biomarker for pharmacogenomic testing.

Three Patterns of Cytochrome P450 Gene Expression during Liver Maturation in Mice

The neonatal period of liver development is an often overlooked phase of development. For instance, ontogeny of xenobiotic-metabolizing enzymes can markedly affect biotransformation as the liver matures. To systematically examine the ontogenic gene expression patterns of cytochrome P450 genes (P450) in mice, the gene expression profiles of 19 xenobiotic-metabolizing P450 in Cyp1 to 4 families were determined. The mRNA levels in C57BL/6 mouse livers were quantified using branched DNA technology at the following ages: gestational day 17 (2 days before birth) and postnatal days 0, 1, 3, 5, 10, 15, 20, 30, and 45. Among the 13 P450 genes expressed in mouse livers, three distinct ontogenic expression patterns were identified by cluster analysis. Genes in group 1 (Cyp3a16 as well as 3a41b in male) were expressed in the perinatal period, but they were essentially nondetectable by 30 days of age. Genes in group 2 (Cyp2e1, 3a11, and 4a10 as well as 3a41b in female) quickly increased after birth and reached maximal expression levels by day 5. Genes in group 3 (Cyp1a2, 2a4, 2b10, 2c29, 2d22, 2f2, 3a13, and 3a25) were expressed at low levels until days 10 to 15, but they markedly increased at day 20 to a high and stable level. In conclusion, the developmental expression of P450 in mouse liver can be divided into three patterns, suggesting that different mechanisms are responsible for the expression of P450 during liver maturation.