Search

Linker histone H1 plays an important role in chromatin folding in vitro and in order to study the role of H1 in vivo, mouse embryonic stem cells null for three H1 genes were derived and were found to have 50% of the normal level of H1. Results indicate that linker histones can participate in epigenetic regulation of gene expression by contributing to the maintenance or establishment of specific DNA methylation patterns.

Summary: Condensin is a central regulator of mitotic genome structure with mutants showing poorly condensed chromosomes and profound segregation defects. Here, we identify NCT, a complex comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02), casein kinase II (CKII), and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions but only briefly colocalize during the periods of chromosome condensation and decondensation. This pattern of NCT binding at the core centromere, the region of maximal condensin enrichment, tracks the abundance of acetylated histone H4, as regulated by the Hat1-Mis16 acetyltransferase complex and recognized by the first Nrc1 bromodomain. Strikingly, mutants in NCT or Hat1-Mis16 restore the formation of segregation-competent chromosomes in cells containing defective condensin. These results are consistent with a model where NCT targets CKII to chromatin in a cell-cycle-directed manner in order to modulate the activity of condensin during chromosome condensation and decondensation. [Copyright &y& Elsevier]

Summary Efficiency of reprogramming of human cells into induced pluripotent stem cells (iPSCs) has remained low. We report that individual adult human CD49f+ long-term hematopoietic stem cells (LT-HSCs) can be reprogrammed into iPSCs at close to 50% efficiency using Sendai virus transduction. This exquisite sensitivity to reprogramming is specific to LT-HSCs, since it progressively decreases in committed progenitors. LT-HSC reprogramming can follow multiple paths and is most efficient when transduction is performed after the cells have exited G 0. Sequencing of 75 paired skin fibroblasts/LT-HSC samples collected from nine individuals revealed that LT-HSCs contain a lower load of somatic single-nucleotide variants (SNVs) and indels than skin fibroblasts and accumulate about 12 SNVs/year. Mutation analysis revealed that LT-HSCs and fibroblasts have very different somatic mutation signatures and that somatic mutations in iPSCs generally exist prior to reprogramming. LT-HSCs may become the preferred cell source for the production of clinical-grade iPSCs. Graphical Abstract Highlights • Single adult human LT-HSCs can be reprogrammed into iPSCs at close to 50% efficiency. • LT-HSCs contain less somatic variants than skin fibroblasts. • LT-HSCs may become the preferred source for the production of clinical-grade iPSCs. Wang et al. show that single adult human long-term hematopoietic stem cells can be reprogrammed into induced pluripotent stem cells at close to 50% efficiency and contain fewer somatic single-nucleotide variants and indels than skin fibroblasts. They may become the preferred source for the production of clinical-grade iPSCs. [ABSTRACT FROM AUTHOR]

Objective: β–thalassemia results from β–globin gene mutations that lead to ineffective erythropoiesis, shortened red cell survival, and anemia. Patients with β–thalassemia develop iron overload, despite which, hepcidin levels are low. This suggests that hepcidin regulation in β–thalassemia is more sensitive to factors unrelated to iron state. Our preliminary data demonstrates that Hbbth1/th1 mice, a model of β–thalassemia intermedia, have lower bone marrow iron levels while levels in the liver and spleen are increased; the later account for the increased systemic iron burden in β–thalassemia intermedia. We hypothesized that exogenous iron would improve anemia in β–thalassemia intermedia despite systemic iron overload and further suppress hepcidin secondary to progressive expansion of erythroid precursors. Materials and Methods: We investigate parameters involved in red cell production, precursor apoptosis, parenchymal iron distribution, and hepcidin expression in iron treated Hbbth1/th1 mice. Results: Exogenous iron results in an expansion of erythroid precursors in the liver and spleen, leading to an increase in the number of red cells, reticulocytes, and hemoglobin production. A decrease in hepcidin expression is also observed. Conclusions: These findings demonstrate for the first time that iron results in expansion of extramedullary erythropoiesis, which improves anemia and suggests that expansion of extramedullary erythropoiesis itself results in hepcidin suppression in β–thalassemia intermedia. [Copyright &y& Elsevier]

Objective: To develop a method to produce in culture large number of erythroid cells from human embryonic stem cells. Materials and Methods: Human H1 embryonic stem cells were differentiated into hematopoietic cells by coculture with a human fetal liver cell line, and the resulting CD34-positive cells were expanded in vitro in liquid culture using a three-step method. The erythroid cells produced were then analyzed by light microscopy and flow cytometry. Globin expression was characterized by quantitative reverse-transcriptase polymerase chain reaction and by high-performance liquid chromatography. Results: CD34-positive cells produced from human embryonic stem cells could be efficiently differentiated into erythroid cells in liquid culture leading to a more than 5000-fold increase in cell number. The erythroid cells produced are similar to primitive erythroid cells present in the yolk sac of early human embryos and did not enucleate. They are fully hemoglobinized and express a mixture of embryonic and fetal globins but no β-globin. Conclusions: We have developed an experimental protocol to produce large numbers of primitive erythroid cells starting from undifferentiated human embryonic stem cells. As the earliest human erythroid cells, the nucleated primitive erythroblasts, are not very well characterized because experimental material at this stage of development is very difficult to obtain, this system should prove useful to answer a number of experimental questions regarding the biology of these cells. In addition, production of mature red blood cells from human embryonic stem cells is of great potential practical importance because it could eventually become an alternate source of cell for transfusion. [Copyright &y& Elsevier]

ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motifs), a circulating multidomain zinc metalloprotease of the reprolysin subfamily, is critical for preventing yon Willebrand factor-platelet interaction under high shear stress conditions. A deficiency of the protease, due to mutations in the ADAMTS13 gene or the presence of antibodies that inhibit the activity of the protease, causes thrombotic thrombocytopenic purpura (TTP). Plasma therapy, the conventional therapy for TTP, may cause serious adverse reactions and is ineffective in some patients. In order to develop new strategies for improving the diagnosis and treatment of TTP, we produced a series of truncated ADAMTS13 proteins in mammalian cells and analyzed their binding with and suppression by the IgG derived from the TTP patients. The results revealed that truncation of the ADAMTS13 protein at its cysteine-rich region eliminated its recognition by the antibodies without abolishing its von Willebrand factor-cleaving activity. This raises the possibility that resistant ADAMTS13 variants may be exploited to circumvent inhibitory antibodies that cause TTP. [ABSTRACT FROM AUTHOR]

Objective: To find a human cell line that could support differentiation of human embryonic stem cells (hESCs) into hematopoietic cells. To determine in detail the expression profiles of the β-like globin genes in hESC-derived erythroid cells. Materials and Methods: FH-B-hTERT, a human fetal liver-derived cell line, and S17, a mouse bone marrow stromal cell line, were used as stromas to induce the differentiation of hESC into hematopoietic cells. The number of hematopoietic progenitors and surface antigen expression were monitored during time-course experiments using colony assays and flow cytometry. Globin expression patterns in individual erythroid colonies were determined by real-time quantitative reverse transcriptase polymerase chain reaction. Results: Comparison of coculture of hESCs with FH-B-hTERT or S17 cells revealed that the fraction of CD34+ cells and the number of clonogenic progenitors per 250,000 cells plated were higher with FH-B-hTERT than with S17. Analysis of β-like globin expression in individual burst-forming unit erythroid and colony-forming unit erythroid colonies revealed that erythroid cells derived from hESC cocultured for 8 to 21 days on either FH-B-hTERT or S17 produced ɛ- and γ-globin mRNAs in similar amounts. With increasing time in coculture, the mean ratio of γ/ɛ increased by more than 10-fold on both S17 and FH-B-hTERT stroma. Importantly, β-globin expression was barely detectable at all time point examined. Conclusions: FH-B-hTERT can induce hESCs differentiation into hematopoietic cells more efficiently than S17. In vitro differentiation of hESCs recapitulates the ɛ-globin to γ-globin switch but not the γ-globin to β-globin switch that occurs around birth. This experimental system will be useful for studying the regulation of globin gene expression during early human hematopoiesis. [Copyright &y& Elsevier]

Abstract: Somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion of immunoglobulin genes require activation-induced cytidine deaminase (AID). AID initiates these events by deaminating cytidines within antibody variable and switch regions. The mechanism that restricts mutation to antibody genes is not known. Although genes other than antibody genes have been found to mutate, not all highly transcribed genes mutate. Thus, somatic hypermutation does not target all genes and suggests a mechanism that either recruits AID to genes for mutation, and/or one that protects genes from promiscuous AID activity. Recent evidence suggests that AID deaminates methyl cytidines inefficiently. Methylation of cytidines could thus represent a means to protect the genome from potentially harmful AID activity that occurs outside of the immunoglobulin loci. To test this premise, we examined whether AID could deaminate methylated-CpG motifs in different sequence contexts. In agreement with a report that suggests that AID has processive-like properties in vitro, we found that AID could completely deaminate single-stranded DNA tracks in plasmid substrates that were greater than 300 nucleotides in length. In addition, methylated-CpG motifs, but not their unmethylated counterparts, were protected from AID-mediated deamination. However, methylation did not protect cytidines that neighbored CpG motifs indicating that methylation per se does not provide a more global safeguard against AID-mediated activity. These data also suggest that AID, and possibly other related cytidine deaminases, might represent a more rapid alternative to bisulfite sequencing for identifying methylated-CpG motifs. [Copyright &y& Elsevier]

• Lineage tracing of somatically acquired DNA transversions enables a sensitive method for tracking hematopoietic clones. • This approach has revealed multilineage clones persisting in two normal individuals up to 53 years of age. Characterization of human cells that sustain blood cell production lifelong has historically been inferred from phenotypically defined subsets of cells assayed in vitro , in transplanted immunodeficient mice, or in patients transplanted with genetically marked cells. These approaches have led to the concept of a persistent complex hierarchical process of differentiation divisions originating from a rare population of CD34+CD38−CD45RA−CD90+CD49f+ cells with an average self-renewal potential of >0.5 and an ability to produce some or all blood cell types for >1 year. However, the role of these "49f" cells in the unperturbed adult has remained poorly understood. To address this gap, somatic single-nucleotide polymorphisms (SNVs) have recently been exploited as lineage tracing markers to enumerate and characterize active hematopoietic clones in normal adults using a capture and recapture approach. We show here that the use of somatic transversions to identify somatically acquired variant alleles enabled their detection in bulk populations at frequencies of approximately 1 in 80,000 cells. We then applied this method to blood cells isolated from two normal adults (aged 31 and 53 years) over a 1- to 3-year period. The results revealed in both donors a continued clonal output of both T- and B-lymphoid cells as well as myeloid cells identified by the same unique transversions found to distinguish single 49f cells isolated from the same donors' initial blood samples. These findings provide the first evidence of a continuing hematopoietic stem cell–derived source of all mature blood cell types in normal (unperturbed) adult humans. [ABSTRACT FROM AUTHOR]

• Albumin-free, low-transferrin, chemically defined protocols were used to produce cultured red blood cells. • FeIII-EDTA, an iron chelator, allows transferrin recycling to take place in cell culture. • The long-PSC-RED protocol yields iPSC-derived erythroid cells that enucleate at rates as high as 50%. • Low costs, high rates of enucleation, and high yields improve the economic viability of cultured RBCs. Many methods have been developed to produce cultured red blood cells (cRBCs) in vitro but translational applications have been hampered by high costs of production and by low rates of enucleation. We have developed R6 and IMIT, two chemically defined culture media and combined them into robust erythroid differentiation (RED) protocols to differentiate induced-pluripotent stem cells (iPSCs) and peripheral blood mononuclear cells (MNCs) into enucleated erythroid cells. The RED protocols do not require any albumin or animal components and require ten- to twentyfold less transferrin (Tf) than previously, because iron is provided to the differentiating erythroblasts by small amounts of recombinant Tf supplemented with FeIII-EDTA, an iron chelator that allows Tf recycling to take place in cell culture. Importantly, cRBCs produced by iPSC differentiation using the long PSC-RED protocol enucleate at much higher rates than with previous protocols, eliminating one of the impediments to the use of these cells to produce clinically useful cRBCs. The absence of albumin, the reduced amounts of Tf, the improved reproducibility associated with the elimination of all animal components, and the high yield on the RED protocols decrease the cost of production of cultured red blood cells. RED protocols should therefore help to make translational applications of cultured RBCs more economically realistic. [ABSTRACT FROM AUTHOR]

Objective: DNA methylation has long been implicated in developmental β-globin gene regulation. However, the mechanism underlying this regulation is unclear, especially because these genes do not contain CpG islands. This has led us to propose and test the hypothesis that, just as for histone modifications, developmentally specific changes in human β-like globin gene expression are associated with long-range changes in DNA methylation. Materials and Methods: Bisulfite sequencing was used to determine the methylation state of individual CpG dinucleotides across the β-globin locus in uncultured primary human erythroblasts from fetal liver and bone marrow, and in primitive-like erythroid cells derived from human embryonic stem cells. Results: β-globin locus CpGs are generally highly methylated, but domains of DNA hypomethylation spanning thousands of base pairs are established around the most highly expressed genes during each developmental stage. These large domains of DNA hypomethylation are found within domains of histone modifications associated with gene expression. We also find hypomethylation of a small proportion of γ-globin promoters in adult erythroid cells, suggesting a mechanism by which adult erythroid cells produce fetal hemoglobin. Conclusion: This is one of the first reports to show that changes in DNA methylation patterns across large domains around non-CpG island genes correspond with changes in developmentally regulated histone modifications and gene expression. These data support a new model in which extended domains of DNA hypomethylation and active histone marks are coordinately established to achieve developmentally specific gene expression of non-CpG island genes. [Copyright &y& Elsevier]

Objective: The regulation of the β-globin switch remains undetermined, and understanding this mechanism has important benefits for clinical and basic science. Histone modifications regulate gene expression and this study determines the presence of three important histone modifications across the β-globin locus in erythroblasts with different β-like globin-expression profiles. Understanding the chromatin associated with weak γ gene expression in bone marrow cells is an important objective, with the goal of ultimately inducing postnatal expression of weak γ-globin to cure β-hemoglobinopathies. Materials and Methods: These studies use uncultured primary fetal and bone marrow erythroblasts and human embryonic stem cell−derived primitive-like erythroblasts. Chromatin immunoprecipitation with antibodies against modified histones reveals DNA associated with such histones. Precipitated DNA is quantitated by real-time polymerase chain reaction for 40 sites across the locus. Results: Distribution of histone modifications differs at each developmental stage. The most highly expressed genes at each stage are embedded within large domains of modifications associated with expression (acetylated histone H3 [H3ac] and dimethyl lysine 4 of histone H3 [H3K4me2]). Moderately expressed genes have H3ac and H3K4me2 in the immediate area around the gene. Dimethyl lysine 9 of histone H3 (H3K9me2), a mark associated with gene suppression, is present at the ε and γ genes in bone marrow cells, suggesting active suppression of these genes. Conclusion: This study reveals complex patterns of histone modifications associated with highly expressed, moderately expressed, and unexpressed genes. Activation of γ postnatally will likely require extensive modification of the histones in a large domain around the γ genes. [Copyright &y& Elsevier]