Chromatin structure and developmental expression of the human alpha-globin cluster

1986 ◽  
Vol 6 (4) ◽  
pp. 1108-1116
Author(s):  
M Yagi ◽  
R Gelinas ◽  
J T Elder ◽  
M Peretz ◽  
T Papayannopoulou ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chromatin Structure ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Globin Gene ◽  
Erythroid Cells ◽  
Adult Bone Marrow ◽  
Hypersensitive Sites ◽  
Alpha Globin ◽  
Adult Bone

The human alpha-like globins undergo a switch from the embryonic zeta-chain to the alpha-chain early in human development, at approximately the same time as the beta-like globins switch from the embryonic epsilon-to the fetal gamma-chains. We investigated the chromatin structure of the human alpha-globin gene cluster in fetal and adult erythroid cells. Our results indicate that DNase I-hypersensitive sites exist at the 5' ends of the alpha 1- and alpha 2-globin genes as well as at several other sites in the cluster in all erythroid cells examined. In addition, early and late fetal liver erythroid cells and adult bone marrow cells contain hypersensitive sites at the 5' end of the zeta gene, and in a purified population of 130-day-old fetal erythroid cells, the entire zeta-to alpha-globin region is sensitive to DNase I digestion. The presence of features of active chromatin in the zeta-globin region in fetal liver and adult bone marrow cells led us to investigate the transcription of zeta in these cells. By nuclear runoff transcription studies, we showed that initiated polymerases are present on the zeta-globin gene in these normal erythroid cells. Immunofluorescence with anti-zeta-globin antibodies also showed that late fetal liver cells contain zeta-globin. These findings demonstrate that expression of the embryonic zeta-globin continues at a low level in normal cells beyond the embryonic to fetal globin switch.

scholarly journals Chromatin structure and developmental expression of the human alpha-globin cluster.

1986 ◽  
Vol 6 (4) ◽  
pp. 1108-1116 ◽  
Author(s):  
M Yagi ◽  
R Gelinas ◽  
J T Elder ◽  
M Peretz ◽  
T Papayannopoulou ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chromatin Structure ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Globin Gene ◽  
Erythroid Cells ◽  
Adult Bone Marrow ◽  
Hypersensitive Sites ◽  
Alpha Globin ◽  
Adult Bone

The human alpha-like globins undergo a switch from the embryonic zeta-chain to the alpha-chain early in human development, at approximately the same time as the beta-like globins switch from the embryonic epsilon-to the fetal gamma-chains. We investigated the chromatin structure of the human alpha-globin gene cluster in fetal and adult erythroid cells. Our results indicate that DNase I-hypersensitive sites exist at the 5' ends of the alpha 1- and alpha 2-globin genes as well as at several other sites in the cluster in all erythroid cells examined. In addition, early and late fetal liver erythroid cells and adult bone marrow cells contain hypersensitive sites at the 5' end of the zeta gene, and in a purified population of 130-day-old fetal erythroid cells, the entire zeta-to alpha-globin region is sensitive to DNase I digestion. The presence of features of active chromatin in the zeta-globin region in fetal liver and adult bone marrow cells led us to investigate the transcription of zeta in these cells. By nuclear runoff transcription studies, we showed that initiated polymerases are present on the zeta-globin gene in these normal erythroid cells. Immunofluorescence with anti-zeta-globin antibodies also showed that late fetal liver cells contain zeta-globin. These findings demonstrate that expression of the embryonic zeta-globin continues at a low level in normal cells beyond the embryonic to fetal globin switch.

scholarly journals Gamma-interferon alters globin gene expression in neonatal and adult erythroid cells

Blood ◽  
1987 ◽  
Vol 69 (6) ◽  
pp. 1674-1681 ◽  
Author(s):  
BA Miller ◽  
SP Perrine ◽  
G Antognetti ◽  
DH Perlmutter ◽  
SG Emerson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Fetal Liver ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Gamma Interferon ◽  
Erythroid Progenitors ◽  
Adult Bone Marrow ◽  
Hemoglobin Production ◽  
Adult Bone

Abstract Interferons have the ability to enhance or diminish the expression of specific genes and have been shown to affect the proliferation of certain cells. Here, the effect of gamma-interferon on fetal hemoglobin synthesis by purified cord blood, fetal liver, and adult bone marrow erythroid progenitors was studied with a radioligand assay to measure hemoglobin production by BFU-E-derived erythroblasts. Coculture with recombinant gamma-interferon resulted in a significant and dose- dependent decrease in fetal hemoglobin production by neonatal and adult, but not fetal, BFU-E-derived erythroblasts. Accumulation of fetal hemoglobin by cord blood BFU-E-derived erythroblasts decreased up to 38.1% of control cultures (erythropoietin only). Synthesis of both G gamma/A gamma globin was decreased, since the G gamma/A gamma ratio was unchanged. Picograms fetal hemoglobin per cell was decreased by gamma- interferon addition, but picograms total hemoglobin was unchanged, demonstrating that a reciprocal increase in beta-globin production occurred in cultures treated with gamma-interferon. No toxic effect of gamma-interferon on colony growth was noted. The addition of gamma- interferon to cultures resulted in a decrease in the percentage of HbF produced by adult BFU-E-derived cells to 45.6% of control. Fetal hemoglobin production by cord blood, fetal liver, and adult bone marrow erythroid progenitors, was not significantly affected by the addition of recombinant GM-CSF, recombinant interleukin 1 (IL-1), recombinant IL- 2, or recombinant alpha-interferon. Although fetal progenitor cells appear unable to alter their fetal hemoglobin program in response to any of the growth factors added here, the interaction of neonatal and adult erythroid progenitors with gamma-interferon results in an altered expression of globin genes. This supports the concept that developmental globin gene switching can be regulated by environmental factors.

scholarly journals Production of fetal antigen-bearing erythrocytes in irradiated adult mice grafted with fetal liver hematopoietic cells

Blood ◽  
1979 ◽  
Vol 54 (5) ◽  
pp. 1091-1100 ◽  
Author(s):  
JP Blanchet ◽  
J Samarut ◽  
G Mouchiroud
Keyword(s):  
Bone Marrow ◽  
Environmental Factors ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Liver Cells ◽  
Adult Bone Marrow ◽  
Adult Mice ◽  
Fetal Liver Cells ◽  
Adult Bone ◽  
Fetal Antigen

Abstract The production of erythrocytes bearing an “immature” antigen (Im+ cells) and a “fetal” antigen (Ft+ cells) has been studied in irradiated adult mice grafted either with fetal liver or adult bone marrow cells. The Im+ cells reach a peak 8–11 days after grafting. Ft+ cells are detected only after graft of fetal liver cells; the younger the liver, the greater the number. Since Ft+ cells are rapidly and briefly produced, they could be the progeny of erythroid-committed precursors, which are particularly numerous among fetal liver cells. Environmental factors directing the erythropoietic differentiation towards Ft+ erythrocytes in fetuses or Ft- erythrocytes in adults are proposed.

scholarly journals Monoclonal origin of B lymphocyte colony-forming cells in spleen colonies formed by multipotential hemopoietic stem cells

1978 ◽  
Vol 148 (6) ◽  
pp. 1468-1477 ◽  
Author(s):  
PK Lala ◽  
GR Johnson
Keyword(s):  
Bone Marrow ◽  
B Lymphocyte ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Absolute Number ◽  
Hemopoietic Stem Cell ◽  
Adult Bone Marrow ◽  
Clonal Origin ◽  
Adult Bone ◽  
Marrow Cells

Spleen colonies produced by transplanting lethally irradiated mice with either 12 day fetal liver or adult bone marrow cells were found to contain B- lymphocyte colony-forming cells (BL-CFC) . The proportion of BL-CFC positive spleen colonies did not increase substantially between 8 and 14 days after transplantation, the range being 18-45 percent. However, the absolute number of BL-CFC per spleen colony varied considerably (between 1 and 10,318), although the majority of colonies contained less than 200 BL-CFC. Irrespective of the time after transplantation, smaller spleen colonies were found to have a higher frequency of BL-CFC than larger spleen colonies. To determine the possible clonal origin of BL-CFC from spleen colony- forming unit (CFU-S), CBA mice were injected with equal numbers of CBA and CBA T(6)/T(6) fetal liver or adult bone marrow cells. Analysis of 7-15-day spleen colonies demonstrated that 90 percent were either exclusively T(6) positive or T(6) negative and approximately equal numbers ofboth colony types were observed. B-lymphocyte colonies were grown and successfully karyotyped from 19 spleen colonies. When compared with the original spleen colony karyotype the B-lymphocyte colony cells karyotype was identical in all 19 cases. In 3 of the 19 colonies analyzed a mixture of T(6) positive and T(6) negative karyotypes was present and identical proportions of the karyotypes were present in the pooled B-lymphocyte colony cells and spleen colony cells. The data indicate that the B-lymphocyte colony-forming cells detected in spleen colonies are genuine members of the hemopoietic clone derived from the initiating hemopoietic stem cell (CFU-S).

Developmental and Differentiation-Specific Patterns of γ- and β-Globin Promoter DNA Methylation in Primary Human Erythroid Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1586-1586
Author(s):  
Christine Richardson ◽  
Rodwell Mabaera ◽  
Christopher H. Lowrey
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Fetal Liver ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Globin Promoter ◽  
Methylation Patterns ◽  
Adult Bone

Abstract Despite years of investigation in a variety of experimental systems, the mechanisms underlying human β-globin locus developmental gene switching remain elusive. Several lines of evidence implicate DNA methylation in this process. As an initial step in studying the role of epigenetic modifications in the human switching process and in determining the mechanisms by which DNA methyltransferase inhibitors reverse the switch, we have characterized the DNA methylation patterns of the individual CpGs in the γ- and β-globin promoters in fetal liver (FL) and adult bone marrow (BM) primary erythroid cells and during in vitro differentiation of adult erythroid cells. Using the bisulfite conversion method we evaluated all CpGs in the ~500 bp regions centered on the γ- and β-globin promoter start sites. Fetal liver (FL) and adult bone marrow (BM) samples were obtained using IRB approved protocols and informed consent procedures. Erythroid cells were purified using anti-glycophorin A (glyA) magnetic beads. Purity was confirmed to be greater than 95%. Samples from five independent BM and FL samples were analyzed and 8–20 bisulfite converted sequences were determined for each promoter in each sample. Our results show that all 8 CpGs between −249 and +210 of the Gγ and Aγ-globin promoters are less than 20% methylated in FL and greater than 80% methylated in BM except for the −158 CpG which is only 40% methylated in BM(p<0.002). The 6 CpGs between −415 and +110 of the β-globin promoter show an inverse pattern with lower levels of DNA methylation in BM. Histone H3 acetylation of the γ-globin promoter, as determined by ChIP analysis, showed a complimentary pattern with higher levels in FL than BM. We next evaluated γ-globin promoter methylation patterns during in vitro erythroid differentiation from CD34+ BM cells. In this experiment, cells were grown with SCF, Flt3 ligand and IL-3 for 7 days and then in EPO for 14 days producing erythroid cells which express 99%HbA and 1% HbF. The initial day 0 CD34+ cells showed 90–100% methylation of all γ promoter sites. By day 3 in culture, before the initiation of erythroid differentiation, methylation at all sites upstream of the promoter had decreased to less than 60% and the CpG at −53 (the site of Stage Selector Protein complex binding) had decreased to less than 20%. The three CpG sites down-stream of the promoter (+6, +17 and +50) remained highly methylated. The pattern was unchanged at day 10, early in erythroid differentiation, when γ-globin mRNA expression was beginning. By day 14, when β-globin expression was peaking, methylation of the upstream promoter had increased back to the 70–100% level at all CpGs. These experiments provide a comprehensive picture of γ- and β-globin promoter methylation during the fetal and adult stages of erythroid development and of the γ-globin promoter during adult erythroid differentiation. The finding of transient γ-promoter hypomethylation during differentiation offers a potential mechanism to explain the transient γ-globin gene expression seen during normal adult erythropoiesis. Our results also raise the possibility that, just as domains of altered histone modification exist in β-globin gene loci, there may also be developmentally-specific domains of DNA methylation.

Decitabine Targets the Erythroid Progenitor/Precursor Subpopulations for the γ-Globin Gene Demethylation in Baboon.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 556-556
Author(s):  
Mahipal Singh ◽  
Kestas Vaitkus ◽  
Donald Lavelle ◽  
Maria Hankeywich ◽  
Nadim Mahmud ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Fetal Liver ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Cell Subpopulation ◽  
Gene Methylation ◽  
Adult Bone Marrow ◽  
Pre Treatment ◽  
Adult Bone

Abstract The DNA demethylating drug (5–Az–2′–deoxycytidine) elevates fetal hemoglobin (HbF) to therapeutic levels in patients with sickle cell disease. To further investigate the mechanism of action of this drug and the role of DNA methylation in γ–globin gene silencing, we have analyzed the level of methylation of five CpG sites in the 5′ region of the γ–globin gene in highly purified subpopulations of cells representing different stages of erythroid differentiation from baboon (P. Anubis) using bisulfite sequencing. Baboons were treated with three different doses of decitabine (0.52, 0.26, 0.17mg/kg/day) for 10 consecutive days and pre-treatment and post-treatment adult bone marrow (ABM) were analyzed. Fetal liver (FL;n=2) and ABM cells were purified by depletion of the erythroblast subpopulation using an anti-RBC antibody (Pharmingen) in combination with immunomagnetic columns (Miltenyi) and FACS purification of CD34+CD36−, CD34+CD36+ and CD34− CD36+ subpopulations. Clonal analysis of sorted subpopulations demonstrated enrichment of CFUe in the CD34−CD36+ subpopulation, BFUe in the CD34+CD36+ subpopulation and both BFUe and CFU-GM in the CD34+CD36− subpopulation, thus confirming that these sorted subpopulations were enriched for the cells representing different stages of erythroid differentiation. A progressive decrease in the level of γ-globin gene methylation, as the degree of differentiation increased, was observed in the subpopulations purified from FL (Table 1). In pre-treatment ABM the level of γ-globin gene methylation was significantly (P<0.05) reduced in erythroblasts when compared to the CD34+CD36− subpopulation. Decitabine treatment reduced the level of γ-globin gene methylation in a dose dependant manner to a similar extent in each subpopulation except the CD34+CD36− subpopulation that exhibited only minor reduction in the γ-globin gene methylation. These results demonstrate that decitabine treatment demethylates the γ-globin gene primarily in late erythroid progenitors (CD34+CD36+) and erythroid precursors (CD34−CD36+). Methylation of the γ-globin gene is not significantly reduced in the more primitive CD34+CD36- subpopulation after decitabine treatment. The greater sensitivity of the progenitor/precursor subpopulations may be due to increased cell cycle kinetics. The increased levels of DNA methytransferase in CD34+ cells may also contribute to the relative insensitivity of the most primitive subpopulation to decitabine. This analysis identifies the late progenitor/precursor subpopulation as the target subpopulation most sensitive to DNA demethylation by decitabine while the early progenitor/stem cell subpopulation is insensitive to the drug. Table 1: DNA methylation (%) of the γ-globin gene in purified cells of fetal liver and pre- and post-decitabine treated adult bone marrow samples Samples CD34+CD36− CD34+CD36+ CD34−CD36+ Erythroblasts Note: Decitabine doses for PA6973=0.52mg; PA6974=0.26mg; PA7002=0.17mg/kg/day Fetal liver (n=2) 95.4±3.96 66.25±4.17 27.3±1.41 3.7±5.23 ABM-pretreated (n=3) 96.23±0.48 87.21±5.96 79.59±13.42 74.87±8.87 BM-post treated PA6973 85.40 41.30 31.10 37.80 BM-post treated PA6974 94.83 61.90 50.79 52.46 BM-post treated PA7002 92.31 71.93 66.00 58.00

scholarly journals Expression of murine CD38 defines a population of long-term reconstituting hematopoietic stem cells

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4057-4067 ◽  
Author(s):  
TD Randall ◽  
FE Lund ◽  
MC Howard ◽  
IL Weissman
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Mouse Strains ◽  
Hematopoietic Stem ◽  
Adult Bone Marrow ◽  
Adult Bone

Using a monoclonal antibody to murine CD38, we showed that a population of adult bone marrow cells that expressed the markers Sca-1 and c-kit but lacked the lineage markers Mac-1, GR-1, B220, IgM, CD3, CD4, CD8 and CD5 could be subdivided by the expression of CD38. We showed that CD38high c-kit+ Sca-1+, linlow/-cells sorted from adult bone marrow cultured with interleukin-3 (IL-3), IL-6, and kit-L produced much larger colonies in liquid culture at a greater frequency than their CD38low/- counterparts. In addition, we found that CD36low/ - cells contained most of the day-12 colony-forming units-spleen (CFU-S) but were not long-term reconstituting cells, whereas the population that expressed higher levels of CD38 contained few, but significant, day-12 CFU-S and virtually all the long-term reconstituting stem cells. Interestingly, the CD38high Sca-1+ c-kit+ linlow/- cells isolated from day-E14.5 fetal liver were also found to be long-term reconstituting stem cells. This is in striking contrast to human hematopoietic progenitors in which the most primitive hematopoietic cells from fetal tissues lack the expression of CD38. Furthermore, because antibodies to CD38 could functionally replace antibodies to Thy-1.1 in a stem cell purification procedure, the use of anti-CD38 may be more generally applicable to the purification of hematopoietic stem cells from mouse strains that do not express the Thy-1.1 allele.

scholarly journals Production of fetal antigen-bearing erythrocytes in irradiated adult mice grafted with fetal liver hematopoietic cells

Blood ◽  
1979 ◽  
Vol 54 (5) ◽  
pp. 1091-1100
Author(s):  
JP Blanchet ◽  
J Samarut ◽  
G Mouchiroud
Keyword(s):  
Bone Marrow ◽  
Environmental Factors ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Liver Cells ◽  
Adult Bone Marrow ◽  
Adult Mice ◽  
Fetal Liver Cells ◽  
Adult Bone ◽  
Fetal Antigen

The production of erythrocytes bearing an “immature” antigen (Im+ cells) and a “fetal” antigen (Ft+ cells) has been studied in irradiated adult mice grafted either with fetal liver or adult bone marrow cells. The Im+ cells reach a peak 8–11 days after grafting. Ft+ cells are detected only after graft of fetal liver cells; the younger the liver, the greater the number. Since Ft+ cells are rapidly and briefly produced, they could be the progeny of erythroid-committed precursors, which are particularly numerous among fetal liver cells. Environmental factors directing the erythropoietic differentiation towards Ft+ erythrocytes in fetuses or Ft- erythrocytes in adults are proposed.

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