Globin Expression Patterns Research Articles

Hemoglobin switching in mice carrying the Klf1Nan variant.

Haploinsufficiency for transcription factor KLF1 causes a variety of human erythroid phenotypes, such as the In(Lu) blood type, increased HbA2 levels, and hereditary persistence of fetal hemoglobin. Severe dominant congenital dyserythropoietic anemia IV (CDA-IV) (OMIM 613673) is associated with the KLF1 p.E325K variant. CDA-IV patients display ineffective erythropoiesis and hemolysis resulting in anemia, accompanied by persistently high levels of embryonic and fetal hemoglobin. The mouse Nan strain carries a variant in the orthologous residue, KLF1 p.E339D. Klf1Nan causes dominant hemolytic anemia with many similarities to CDA-IV. Here we investigated the impact of Klf1Nan on the developmental expression patterns of the endogenous α-like and β-like globins, and the human β-like globins carried on a HBB locus transgene. We observe that the switch from primitive, yolk sac-derived, erythropoiesis to definitive, fetal liver-derived, erythropoiesis is delayed in Klf1wt/Nan embryos. This is reflected in globin expression patterns measured between embryonic day 12.5 (E12.5) and E14.5. Cultured Klf1wt/Nan E12.5 fetal liver cells display growth- and differentiation defects. These defects likely contribute to the delayed appearance of definitive erythrocytes in the circulation of Klf1wt/Nan embryos. After E14.5, expression of the embryonic/fetal globin genes is silenced rapidly. In adult Klf1wt/Nan animals, silencing of the embryonic/fetal globin genes is impeded, but only minute amounts are expressed. Thus, in contrast to human KLF1 p.E325K, mouse KLF1 p.E339D does not lead to persistent high levels of embryonic/fetal globins. Our results support the notion that KLF1 affects gene expression in a variant-specific manner, highlighting the necessity to characterize KLF1 variant-specific phenotypes of patients in detail.

IGF2BP3 Moderately Increases Fetal Hemoglobin Levels in Human Adult Erythroblasts

Recent studies demonstrated that IGF2BP1 over-expression (IGF2BP1-OE) in adult erythroblasts has robust effects on fetal hemoglobin (HbF; >65% of the total globin levels), accompanied by reversal of the beta-like globin expression patterns to a fetal-like phenotype. Here we investigated if another member of the insulin-like growth factor 2 mRNA-binding protein family, IGF2BP3, also has potential for HbF regulation that may be useful for therapeutic application among patients with beta-hemoglobin disorders.The developmental pattern and expression levels for IGF2BP3 were initially determined in cord blood versus adult blood CD34(+) samples cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. RNA samples were collected at culture day 14 and expression levels were measured by qRT-PCR. IGF2BP3 showed a developmentally regulated expression pattern similar to IGF2BP1 (IGF2BP1: cord blood: 1.3.E+03 ± 4.3.E+02 and adult blood: below detection limits; IGF2BP3: cord blood: 5.8.E+02 ± 2.4.E+02 and adult blood: below detection limits). These results were confirmed in vivo by comparing human fetal liver to adult bone marrow samples (IGF2BP1: fetal liver: 3.5.E+02 ± 5.7.E+01, adult bone marrow: below detection limits and IGF2BP3: fetal liver: 2.0.E+01 ± 2.7.E+00, adult bone marrow: below detection limits). To investigate the effects of IGF2BP3 upon erythropoiesis and globin expression, a lentiviral construct was designed for expression of IGF2BP3 driven by the erythroid-specific gene promoter region of the human SPTA1 gene (IGF2BP3-OE), with a matched empty vector control. Transductions were performed in CD34(+) cells from four adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Over-expression of IGF2BP3 was confirmedby qRT-PCR and Western blot analyses at culture day 14. IGF2BP3-OE cells maintained their ability to differentiate and enucleate ex vivo compared to donor-matched controls. The expression levels of globin genes were evaluated at culture day 14 by qRT-PCR and showed that IGF2BP3-OE caused significantly increased gamma-globin expression levels compared to control transductions (control: 7.7.E+05 ± 1.7.E+05; IGF2BP3-OE: 8.4.E+06 ± 3.2.E+06; p=0.018). Consistent with increased gamma-globin, HbF rose to moderately high levels upon IGF2BP3-OE (control: 4.0 ± 2.1%; IGF2BP3-OE: 18.6 ± 1.0%; p=0.0021). In addition, the expression pattern of the erythroid transcription factor BCL11A was investigated by qRT-PCR at culture day 14 and no significant changes were observed (control: 5.6.E+02 ± 2.7.E+02; IGF2BP3-OE: 6.7.E+02 ± 3.5.E+02; p=0.694). However, minor decreases in BCL11A protein levels were detected by Western analysis.These results demonstrate that IGF2BP3 is developmentally regulated in human erythroid tissues with silencing during the fetal-to-adult transition. However, the effects of IGF2BP3-OE on HbF levels were less robust when compared to IGF2BP1-OE in cultured adult erythroblasts. DisclosuresNo relevant conflicts of interest to declare.

Defining the minimal factors required for erythropoiesis through direct lineage conversion

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of well characterized genes. However, the minimal set of factors necessary for instructing red blood cell (RBC) development remains undefined. We employed a screen for transcription factors allowing direct lineage reprograming from fibroblasts to induced erythroid progenitors/precursors (iEPs). We show that Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs. The transcriptional signature of murine iEPs resembled mainly that of primitive erythroid progenitors in the yolk sac, whereas addition of Klf1 or Myb to the GTLM cocktail resulted in iEPs with a more adult-type globin expression pattern. Our results demonstrate that direct lineage conversion is a suitable platform for defining and studying the core factors inducing the different waves of erythroid development.

Defining the Minimal Factors Required for Erythropoiesis through Direct Lineage Conversion.

SummaryErythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of well characterized genes. However, the minimal set of factors necessary for instructing red blood cell (RBC) development remains undefined. We employed a screen for transcription factors allowing direct lineage reprograming from fibroblasts to induced erythroid progenitors/precursors (iEPs). We show that Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs. The transcriptional signature of murine iEPs resembled mainly that of primitive erythroid progenitors in the yolk sac, whereas addition of Klf1 or Myb to the GTLM cocktail resulted in iEPs with a more adult-type globin expression pattern. Our results demonstrate that direct lineage conversion is a suitable platform for defining and studying the core factors inducing the different waves of erythroid development.

Transcriptional environment and chromatin architecture interplay dictates globin expression patterns of heterospecific hybrids derived from undifferentiated human embryonic stem cells or from their erythroid progeny

To explore the response of β globin locus with established chromatin domains upon their exposure to new transcriptional environments, we transferred the chromatin-packaged β globin locus of undifferentiated human embryonic stem cells (hESCs) or hESC-derived erythroblasts into an adult transcriptional environment. Distinct globin expression patterns were observed. In hESC-derived erythroblasts where both ε and γ globin were active and marked by similar chromatin modifications, ε globin was immediately silenced upon transfer, whereas γ globin continued to be expressed for months, implying that different transcriptional environments were required for their continuing expression. Whereas β globin was silent both in hESCs and in hESC-derived erythroblasts, β globin was only activated upon transfer from hESCs, but not in the presence of dominant γ globin transferred from hESC-derived erythroblasts, confirming the competing nature of γ versus β globin expression. With time, however, silencing of γ globin occurred in the adult transcriptional environment with concurrent activation of β-globin, accompanied by a drastic change in the epigenetic landscape of γ and β globin gene regions without apparent changes in the transcriptional environment. This switching process could be manipulated by overexpression or downregulation of certain transcription factors. Our studies provide important insights into the interplay between the transcription environment and existing chromatin domains, and we offer an experimental system to study the time-dependent human globin switching.

A Screen for Regulators of Globin Switching in the Zebrafish Embryo

Abstract 826The switching of the globin genes involves critical transcriptional regulators such as BCL11A, EKLF and SOX6, and the induction of fetal globin has been shown to ameliorate the symptoms of diseases such as sickle cell anemia. Recently, there has been interest in driving iPS cells to produce mature red cells that express adult globin genes in an attempt to make these cells therapeutically useful. Here, to understand hemoglobin switching and the molecular pathways that allow the establishment of an adult fate in embryonic tissues, we utilized a screening approach in the zebrafish model. The concept of the screen is to find transcription factors that are expressed in a stage-specific manner, and manipulate the expression of these genes to alter the cell fate of embryonic erythroid cells. In order to generate a candidate list of genes, microarray analysis was performed on murine yolk sac, fetal liver and adult derived red blood cells and red blood cell precursors, which express unique sets of globin genes. Pair-wise comparison of these populations yielded 879 unique differentially regulated genes. GO term analysis was used to narrow the list to 49 transcription factors. We focused on the transcription factors that might increase adult globin expression in the embryo based on their differential expression in the microarrays. Morpholinos were used to knock down these 24 genes by individually injecting each into one-cell stage embryos, allowing the embryos to reach 24 hpf and performing in situ hybridization for the adult globin gene αa1. The number of adult globin positive cells present in each embryo was counted for a clutch control group, which on average has 2–4 positive cells per embryo, and three doses of morpholino. We identified 4 genes, Tcf7l2, Ncoa1, Hif1al and E2F5, the knock down of which results in a significant increase in the number of adult globin positive cells in at least one dose of morpholino (control [n=53, mean=6.34], 6ng [n=56, mean=15.07], p=<0.0001; control [n=35, mean=1.543], 4ng [n=56, mean=2.75], p=<0.01; control [n=19, mean=1.368], 12ng [n=16, mean=6.188], p=<0.0001; control [n=44, mean=1.091], 4ng [n=30, mean=2.7], p=<0.05, respectively). Pair-wise knock down of these genes were also tested, and the combinations of Ncoa1 and E2F5, Tcf7l2 and E2F5 and Tcf7l2 and Ncoa1 were found to synergistically increase the number of adult globin expressing cells (control [n=49, mean=0.5306], knock down [n=38, mean=9.895], p=<0.0001; control [n=49, mean=7.633], knock down [n=54, mean=17.41], p=<0.0001; control [n=20, mean=2.95], knock down [n=28, mean=too numerous to count], p=<0.0001, respectively). The combined knock down of Tcf7l2 and Ncoa1 was both the strongest inducer of adult globin expression and had the lowest toxicity of the pair-wise combinations. Further characterization of this phenotype shows that, while many globin genes are up regulated, both of the adult globin genes, αa1 and βa1, are upregulated to a higher degree than other globin genes. In order to determine if the Wnt pathway is responsible for phenotype observed with the Tcf7l2 morpholino, we tested the Wnt pathway inhibitors IWR1 and XAV939. Both drugs phenocopied the Tcf7l2 knockdown response. In addition, XAV939 synergies with the Ncoa1 morpholino to enhance the increase in adult globin observed in a similar manner to Tcf7l2 knockdown. These results indicate that modulation of Wnt signaling, rather than a Wnt-independent function of Tcf7l2, is responsible for the phenotype and regulation of globin gene expression. Chip-Seq analysis of Ncoa1 occupancy in the erythroid cell line K562 was performed to examine potential mechanisms of action. Significant binding was observed at the enhancers of the α- and β-globin loci, indicating that the nuclear hormone receptor pathway may be acting directly on the globin loci to modulate globin expression patterns. These results indicate that Wnt signaling in combination with alterations of other pathways regulated by Ncoa1 are responsible for stage-specific globin expression. Our studies have impact on the understanding of globin switching in vertebrates, and could establish new methods to activate specific globins clinically, and to make iPS cells form adult-type tissues. Disclosures:Zon:Fate Therapeutics: Founder Other.

Epigenetic Modifications and Chromosome Conformations of the Beta Globin Locus throughout Development

Human embryonic stem cells provide an alternative to using human embryos for studying developmentally regulated gene expression. The co-expression of high levels of embryonic ε and fetal γ globin by the hESC-derived erythroblasts allows the interrogation of ε globin regulation at the transcriptional and epigenetic level which could only be attained previously by studying cell lines or transgenic mice. In this study, we compared the histone modifications across the β globin locus of the undifferentiated hESCs and hESC-, FL-, and mobilized PB CD34(+) cells-derived erythroblasts, which have distinct globin expression patterns corresponding to their developmental stages. We demonstrated that the histone codes employed by the β globin locus are conserved throughout development. Furthermore, in spite of the close proximity of the ε globin promoter, as compared to the β or γ globin promoter, with the LCR, a chromatin loop was also formed between the LCR and the active ε globin promoter, similar to the loop that forms between the β or γ globin promoters and the LCR, in contrary to the previously proposed tracking mechanism.

Production of Embryonic and Fetal-Like Red Blood Cells from Human Induced Pluripotent Stem Cells

We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.

A transient definitive erythroid lineage with unique regulation of the β-globin locus in the mammalian embryo

AbstractA transient erythromyeloid wave of definitive hematopoietic progenitors (erythroid/myeloid progenitors [EMPs]) emerges in the yolk sac beginning at embryonic day 8.25 (E8.25) and colonizes the liver by E10.5, before adult-repopulating hematopoietic stem cells. At E11.5, we observe all maturational stages of erythroid precursors in the liver and the first definitive erythrocytes in the circulation. These early fetal liver erythroblasts express predominantly adult β-globins and the definitive erythroid-specific transcriptional modifiers c-myb, Sox6, and Bcl11A. Surprisingly, they also express low levels of “embryonic” βH1-, but not εy-, globin transcripts. Consistent with these results, RNA polymerase and highly modified histones are found associated with βH1- and adult globin, but not εy-globin, genes. E11.5 definitive proerythroblasts from mice transgenic for the human β-globin locus, like human fetal erythroblasts, express predominately human γ-, low β-, and no ε-globin transcripts. Significantly, E9.5 yolk sac–derived EMPs cultured in vitro have similar murine and human transgenic globin expression patterns. Later liver proerythroblasts express low levels of γ-globin, while adult marrow proerythroblasts express only β-globin transcripts. We conclude that yolk sac–derived EMPs, the first of 2 origins of definitive erythropoiesis, express a unique pattern of globin genes as they generate the first definitive erythrocytes in the liver of the mammalian embryo.

“Definitive” Erythropoiesis Has Distinct Developmental Origins and Globin Expression Patterns in the Mammalian Embryo.

Abstract 2539Poster Board II-516A transient wave of primitive erythropoiesis begins at embryonic day 7.5 (E7.5) in the mouse as yolk sac-derived primitive erythroid progenitors (EryP-CFC) generate precursors that mature in the circulation and expand in numbers until E12.5. A second wave of erythroid progenitors (BFU-E) originates in the yolk sac beginning at E8.25 that generate definitive erythroid cells in vitro. These BFU-E colonize the newly forming liver beginning at E10.5, prior to the initial appearance there of adult-repopulating hematopoietic stem cells (HSCs) between E11.5-12.5. This wave of definitive erythroid yolk sac progenitors is proposed to be the source of new blood cells required by the growing embryo after the expansion of primitive erythroid cells has ceased and before HSC-derived hematopoiesis can fulfill the erythropoietic needs of the embryo. We utilized multispectral imaging flow cytometry both to distinguish erythroid lineages and to define specific stages of erythroid precursor maturation in the mouse embryo. Consistent with this model, we found that small numbers of definitive erythrocytes first enter the embryonic circulation beginning at E11.5. All maturational stages of erythroid precursors were observed in the E11.5 liver, consistent with these first definitive erythrocytes having rapidly completed their maturation in the liver. The expression of βH1 and εy-beta globin genes is thought to be limited to primitive erythroid cells. Surprisingly, examination of globin gene expression by in situ hybridization revealed high levels of βH1-, but not εy-globin, transcripts in the parenchyma of E11.5-12.5 livers. RT-PCR analysis of globin mRNAs confirmed the expression of βH1- and adult β1-, but not εy-globin, in E11.5 liver-derived definitive (ckit+, Ter119lo) proerythroblasts sorted by flow cytometry to remove contaminating primitive (ckit-, Ter119+) erythroid cells. A similar pattern of globin gene expression was found in individual definitive erythroid colonies derived from E9.5 yolk sac and from early fetal liver. In vitro differentiation of definitive erythroid progenitors from E9.5 yolk sac revealed a maturational “switch” from βH1- and β1-globins to predominantly β1-globin. βH1-globin transcripts were not observed in proerythroblasts from bone marrow or E16.5 liver or in erythroid colonies from later fetal liver. ChIP analysis revealed that hyperacetylated domains encompass all beta globin genes in primitive erythroid cells but only the adult β1- and β2-globin genes in E16.5 liver proerythroblasts. Consistent with their unique gene expression, E11.5 liver proerythroblasts have hyperacetylated domains encompassing the βh1-, β1- and β2-, but not εy-globin genes. We also examined human globin transgene expression in mice carrying a single copy of the human beta globin locus. Because of the overlapping presence and changing proportion of primitive and definitive erythroid cells during development, we analyzed sorted cell populations whose identities were confirmed by murine globin gene expression. We confirmed that primitive erythroid cells express higher levels of γ- than ε-globin and little β-globin. E11.5 proerythroblasts and cultured E9.5 progenitors express γ- and β-, but not ε-globin. E16.5 liver proerythroblasts express β- and low levels of γ-globin, while adult marrow proerythroblasts express only β-globin transcripts. In summary, two forms of definitive erythropoiesis emerge in the murine embryo, each with distinct globin expression patterns and chromatin modifications of the β-globin locus. While both lineages predominantly express adult globins, the first, yolk sac-derived lineage uniquely expresses low levels of the embryonic βH1-globin gene as well as the human γ-globin transgene. The second definitive erythroid lineage, found in the later fetal liver and postnatal marrow, expresses only adult murine globins as well as low levels of the human γ-globin transgene only in the fetus. Our studies reveal a surprising complexity to the ontogeny of erythropoiesis. Disclosures:No relevant conflicts of interest to declare.

Extensively Self-Renewing Erythroid Precursors Emerge from the Embryo but Not from the Adult

The only cell in the hematopoietic hierarchy thought to be capable of long-term selfrenewal is the stem cell. An erythroid progenitor derived from mammalian hematopoietic tissue, fetal or adult, is capable of limited proliferation (103–106 fold expansion; Bauer, 1999; Panzenböck, 1998; von Lindern, 1999). Here we report that an erythroid precursor derived from the mouse embryo is capable not only of limited, but also of extensive proliferation (~1030 fold expansion). These cells resemble proerythroblasts and basophilic erythroblasts based on their morphology, their globin gene expression profile, and their immunophenotype. While aneuploidy is not necessary for extensive proliferation, it sporadically begins to accumulate after prolonged culture. These cells are capable of massive (>100 days) daily proliferation in vitro in the presence of Epo, SCF, IGF-1, and dexamethasone. Examination of cultures lacking each of these factors support that glucocorticoids play an important role in this expansion by uncoupling erythroid precursor proliferation from maturation. Despite prolonged in vitro culture, these cells preserve their potential to fully differentiate into enucleated red blood cells with the removal of dexamethasone. Differentiation occurs over 2–3 days and is characterized by the accumulation of adult (α, β1, and β2), but not embryonic (ζ, εy, and βH1), globins. The retention of full differentiation potential despite >1030 fold expansion indicates that this proliferation represents self-renewal. To determine the developmental origin of these extensively self-renewing erythroblasts (ESREs), we initiated in vitro cultures from staged mouse embryos as well as adult tissues. E7.5 embryos, that contain primitive but not definitive erythroid progenitors, failed to generate ESREs. In contrast, ESREs can be derived from E8.5–E10.5 yolk sac and E11.5–E14.5 fetal liver. These findings along with the globin expression pattern indicate that erythroblast self-renewal is associated with definitive, but not primitive, erythropoiesis. Surprisingly, marrow from adult steady-state hematopoiesis failed to yield ESREs. Furthermore, despite the characteristics shared by stress erythropoiesis (adult spleen) and fetal erythropoiesis (liver), stress erythropoiesis only yielded erythroblasts with limited, and not extensive, self-renewal capacity. This result suggests that extensive self-renewal potential is linked either to the transient yolk sac-derived definitive erythroid lineage or to the fetal hematopoietic microenvironment. We are currently investigating the mechanisms responsible for the extensive self-renewal capacity of such lineage-restricted and mature hematopoietic precursors. Our findings raise the possibility that the expansive cellular output of the erythron within the midgestation mammalian embryo may be regulated, in part, at the level of late stage erythroid precursors.

Human erythroid progenitors from adult bone marrow and cord blood in optimized liquid culture systems respectively maintained adult and neonatal characteristics of globin gene expression

In vitro suspension culture procedures for erythroid progenitor cells make it possible for us to obtain large cultures of erythrocyte populations for the investigation of globin gene switching. In this study we aimed to establish optimized culture systems for neonatal and adult erythroblasts and to explore the globin expression patterns in these culture systems. To culture CD34+ cells purified from human umbilical cord blood (CB) and adult bone marrow (BM), we respectively replaced the fetal bovine serum (FBS) with human cord serum and human adult serum. These CD34+ cells were then induced to erythroid differentiation. All the globin mRNA (including alpha-, zeta-, beta-, gamma-and epsilon-globin), the hemoglobin (Hb)-producing erythroid cells and the cellular distribution of fetal hemoglobin (Hb F) were identified during the culture process. The results showed that the globin expression pattern during erythroid differentiation in our culture systems closely recapitulated neonatal and adult patterns of globin expression in vivo, suggesting that our specially optimized culture systems not only overcame the higher Hb F levels in the BM-derived CD34+ culture in FBS-containing medium but also eliminated the disadvantages of low cell proliferation rate and low globin mRNA levels in serum-free medium.

Definitive-like erythroid cells derived from human embryonic stem cells coexpress high levels of embryonic and fetal globins with little or no adult globin

Human embryonic stem cells are a promising tool to study events associated with the earliest ontogenetic stages of hematopoiesis. We describe the generation of erythroid cells from hES (H1) by subsequent processing of cells present at early and late stages of embryoid body (EB) differentiation. Kinetics of hematopoietic marker emergence suggest that CD45+ hematopoiesis peaks at late D14EB differentiation stages, although low-level CD45- erythroid differentiation can be seen before that stage. By morphologic criteria, hES-derived erythroid cells were of definitive type, but these cells both at mRNA and protein levels coexpressed high levels of embryonic (epsilon) and fetal (gamma) globins, with little or no adult globin (beta). This globin expression pattern was not altered by the presence or absence of fetal bovine serum, vascular endothelial growth factor, Flt3-L, or coculture with OP-9 during erythroid differentiation and was not culture time dependent. The coexpression of both embryonic and fetal globins by definitive-type erythroid cells does not faithfully mimic either yolk sac embryonic or their fetal liver counterparts. Nevertheless, the high frequency of erythroid cells coexpressing embryonic and fetal globin generated from embryonic stem cells can serve as an invaluable tool to further explore molecular mechanisms.

“Maturational” globin switching in primary primitive erythroid cells

Mammals have 2 distinct erythroid lineages. The primitive erythroid lineage originates in the yolk sac and generates a cohort of large erythroblasts that terminally differentiate in the bloodstream. The definitive erythroid lineage generates smaller enucleated erythrocytes that become the predominant cell in fetal and postnatal circulation. These lineages also have distinct globin expression patterns. Our studies in primary murine primitive erythroid cells indicate that betaH1 is the predominant beta-globin transcript in the early yolk sac. Thus, unlike the human, murine beta-globin genes are not up-regulated in the order of their chromosomal arrangement. As primitive erythroblasts mature from proerythroblasts to reticulocytes, they undergo a betaH1- to epsilony-globin switch, up-regulate adult beta1- and beta2-globins, and down-regulate zeta-globin. These changes in transcript levels correlate with changes in RNA polymerase II density at their promoters and transcribed regions. Furthermore, the epsilony- and betaH1-globin genes in primitive erythroblasts reside within a single large hyperacetylated domain. These data suggest that this "maturational" betaH1- to epsilony-globin switch is dynamically regulated at the transcriptional level. Globin switching during ontogeny is due not only to the sequential appearance of primitive and definitive lineages but also to changes in globin expression as primitive erythroblasts mature in the bloodstream.

Differentiation of human embryonic stem cells into hematopoietic cells by coculture with human fetal liver cells recapitulates the globin switch that occurs early in development

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 beta-like globin genes in hESC-derived erythroid cells. 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. 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 beta-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 epsilon- and gamma-globin mRNAs in similar amounts. With increasing time in coculture, the mean ratio of gamma/epsilon increased by more than 10-fold on both S17 and FH-B-hTERT stroma. Importantly, beta-globin expression was barely detectable at all time point examined. FH-B-hTERT can induce hESCs differentiation into hematopoietic cells more efficiently than S17. In vitro differentiation of hESCs recapitulates the epsilon-globin to gamma-globin switch but not the gamma-globin to beta-globin switch that occurs around birth. This experimental system will be useful for studying the regulation of globin gene expression during early human hematopoiesis.

In vitro maturation of erythroid progenitors from human umbilical cord blood and patterns of globin gene expression: Serum from different developmental stage plays important roles in liquid culture

To investigate the maturation of neonatal erythrocytes and kinetics of globin expression, we induced CD34 + cells purified from human umbilical cord blood to erythroid differentiation in different suspension culture systems containing human cord serum (CHS) and adult serum (AHS), respectively. The ζ- to α-globin switching and the ε- to γ-globin switching were observed in CHS + cultures but not in AHS + cultures. A reduced proportion of F cell and two day postponed cell enucleation in AHS + cultures compared with in CHS + cultures was also found. However, predominant γ-globin expression compared with ε- and β-globin was always observed from days 5 in either CHS + or AHS + cultures. Our data showed that neonatal erythrocytes in CHS + cultures maintained their fetal characteristics which differed from their counterpart from adult. Serum from different developmental stage significantly affected the maturation and globin expression pattern of cultured neonatal erythrocytes. Obviously, some uncertain serum components play important roles in cultures, but they are not enough to fundamentally change the developmentally specific globin gene transcription program, suggesting that this program is mainly controlled by determinants within the erythroid progenitors.

Two Cellular Mechanisms for Gamma-Globin Gene and Protein Silencing in Humans.

Although the genetic processes responsible for gamma-globin gene and protein silencing are not known, the prevailing model is that gamma-globin silencing results from a gradual change within a single hematopoietic cell lineage that is governed by intrinsic properties of the cells. In order to provide a more complete characterization of the silencing phenomenon, we studied globin expression patterns directly from clinical samples using single-cell, quantitative PCR, and globin protein phenotyping. We collected blood samples from untransfused donors: umbilical cords (n=3), infants (n=11; ages 1 day to 35 months), and adults (n=3). All samples were maintained at 4°C and analyzed within 72 hours. Flow cytometry (30,000 cells per donor) and HPLC analyses were used for globin protein phenotyping. For globin gene expression, we identified reticulocytes using a strategy that required no membrane permeabilization, and sorted them as single cells directly into lysis buffer. Oligo-dT reverse transcription of mRNA was followed by real-time PCR quantitation. Globin cDNA copy numbers were calculated using standard curves from serial dilutions of a plasmid DNA. We analyzed approximately 1000 single-cell quantitative PCR amplifications for gamma- and beta-globin gene expression. In cord blood, we detected both gamma- and beta-globin gene expression in 97.4% (112/115) of the reticulocytes. The average gamma-globin cDNA copy number was 1870±1390 copies, compared with an average beta-globin cDNA copy number of 2181±2138 copies per reticulocyte. HbF and HbA were also detected in >95% of the cord blood erythrocytes. In the adult samples, HbF was detected in <5% of the circulating erythrocytes and gamma-globin gene expression in only 1.5% (3/206) of the reticulocytes. The average gamma-globin cDNA copy number in the minor population of gamma(+) adult reticulocytes was 468±198 copies, and the average beta-globin cDNA copy number in the beta(+) adult reticulocytes was 3869±3733 copies. Compared with the relatively monotonous patterns of gamma-globin gene and protein expression in cord and adult blood, we clearly detected an age-based fluctuation between those patterns in the infant blood samples. During the first three years of life, a gradual loss in the level of gamma-globin gene and protein expression was identified among the gamma(+)beta(+) reticulocytes and the HbF(+)HbA(+) erythrocytes. In addition, discrete populations of gamma(−)beta(+)reticulocytes and HbF(−)HbA(+) erythrocytes were detected. Rapid expansion of those gamma-silenced populations became apparent soon after birth. Within four months, the proportion of gamma-silenced cells eclipsed that of the gamma(+)beta(+) cells to become the predominant population. By three years after birth, the two cell populations essentially merged to become a single, gamma-silenced population similar to that found in adults. These data suggest two cellular mechanisms for gamma-globin silencing in humans: 1) a gradual loss in gamma-globin expression in the gamma(+)beta(+) cells beginning prior to delivery and continuing during infancy, and 2) replacement of the gamma(+)beta(+) cells with a population of gamma-silenced cells that rapidly accumulate after birth, possibly in response to the dramatic increase in oxygenation or other environmental changes.

The Genomic Record of Humankind's Evolutionary Roots

Taxa above the genus level that are referred to in the text are indicated here in boldface. Order Primates (63 Ma) Semiorder Strepsirhini (50 Ma) Suborder Lemuriformes (45 Ma) Infraorder Chiromyiformes Family Daubentoniidae, Daubentonia: aye-ayes Infraorder Eulemurides Superfamily Lemuroidea (28 Ma) Family Cheirogaleidae (22 Ma) Subfamily Microcebinae, Microcebus: mouse lemurs Subfamily Cheirogaleinae, Cheirogaleus: dwarf lemurs Family Indridae, Propithecus: sifakas Family Lemuridae, Eulemur: brown lemurs Suborder Loriformes Family Loridae (23 Ma) Subfamily Galagoninae, Otolemur: bush babies Subfamily Perodicticinae, Perodicticus: pottos Subfamily Lorinae, Nycticebus: slow lorises Semiorder Haplorhini (58 Ma) Suborder Tarsiiformes Family Tarsiidae, Tarsius: tarsiers Suborder Anthropoidea (40 Ma) Infraorder Platyrrhini Superfamily Ceboidea (26 Ma) Family Cebidae (22 Ma) Subfamily Cebinae (20 Ma) Tribe Cebini, Cebus: capuchin monkeys Tribe Saimiriini, Saimiri: squirrel monkeys Subfamily Aotinae, Aotus: night monkeys Subfamily Callitrichinae Tribe Callitrichini (13 Ma) Subtribe Saguinina, Saguinus: tamarins Subtribe Leontopithecina, Leontopithecus: golden lion tamarins Subtribe Callimiconina, Callimico: goeldi's monkeys Subtribe Callitrichina Callithrix (Callithrix): marmosets (jacchus group) Callithrix (Cebuella): pygmy marmosets Callithrix (Mico): marmosets (argentata group) Family Pitheciidae Subfamily Pitheciinae (19 Ma) Tribe Callicebini, Callicebus: titi monkeys Tribe Pitheciini Subtribe Pitheciina (11 Ma) Pithecia: saki monkeys Chiropotes (Chiropotes): bearded saki monkeys Chiropotes (Cacajao): uacari monkeys Family Atelidae Subfamily Atelinae (17 Ma) Tribe Alouattini, Alouatta: howler monkeys Tribe Atelini (12 Ma) Subtribe Atelina, Ateles: spider monkeys Subtribe Brachytelina (10 Ma) Lagothrix: woolly monkeys Brachyteles: woolly spider monkeys Infraorder Catarrhini Superfamily Cercopithecoidea (25 Ma) Family Cercopithecidae Subfamily Cercopithecinae (15 Ma) Tribe Colobini (10 Ma) Subtribe Colobina, Colobus: colobus monkeys Subtribe Presbytina (7 Ma) Trachypithecus: langurs Nasalis: proboscis monkeys Tribe Cercopithecini (10 Ma) Subtribe Cercopithecina (9 Ma) Cercopithecus: guenons Erythrocebus: patas monkeys Chlorocebus: green monkeys Subtribe Papionina (9 Ma) Macaca: macaques Cercocebus (Cercocebus): terrestrial mangabeys Cercocebus (Mandrillus): mandrills, drills Papio (Papio): baboons (hamadryas group) Papio (Theropithecus): gelada baboons Papio (Lophocebus): arboreal mangabeys Family Hominidae Subfamily Homininae (18 Ma) Tribe Hylobatini Subtribe Hylobatina (8 Ma) Symphalangus: siamangs Hylobates: gibbons Tribe Hominini (14 Ma) Subtribe Pongina, Pongo: orangutans Subtribe Hominina (7 Ma) Gorilla: gorillas Homo (Homo): humans Homo (Pan): chimpanzees, bonobosAdditional relevant references are included in a Supplemental Reading List, which appears, in the electronic version of this article, immediately after the References.

Additional evidence for the Precolumbian migration of Black African peoples to the American continent

Evolutionary approaches to the identification of DNA sequences required for transcription of the genes of the β-globin cluster are reviewed. Sequence alignments of non-coding regions from widely divergent species revealed many conserved motifs (phylogenetic footprints) that were putative transcription factor binding sites and candidate binding proteins were identified. The differential timing of the prosimian and simian γ-globin genes was analyzed by identifying base changes in the vicinity of the phylogenetic footprints. These differential phylogenetic footprints were shown to bind different nuclear factors, and the behavior of constructs with human or galago γ-promoters in transgenic mice indicated that DNA motifs near the γ-globin genes are sufficient to determine the developmental stage of expression. Locus control region alignments have identified many conserved sequence differences outside of the hypersensitive sites. Globin protein and mRNA expression profiles during embryological development in a series of catarrhine (Old World monkeys and apes) and platyrrhine (New World monkeys) primates have been determined. While all catarrhines examined to date have globin expression patterns that are highly similar to the well-established human switching behavior, platyrrhines have inactivated their γ1 genes by a variety of mechanisms, and have an earlier γ–β switch.