Cytokine-mediated increases in fetal hemoglobin are associated with globin gene histone modification and transcription factor reprogramming.

Therapeutic regulation of globin genes is a primary goal of translational research aimed toward hemoglobinopathies. Signal transduction was used to identify chromatin modifications and transcription factor expression patterns that are associated with globin gene regulation. Histone modification and transcriptome profiling were performed using adult primary CD34(+) cells cultured with cytokine combinations that produced low versus high levels of gamma-globin mRNA and fetal hemoglobin (HbF). Embryonic, fetal, and adult globin transcript and protein expression patterns were determined for comparison. Chromatin immunoprecipitation assays revealed RNA polymerase II occupancy and histone tail modifications consistent with transcriptional activation only in the high-HbF culture condition. Transcriptome profiling studies demonstrated reproducible changes in expression of nuclear transcription factors associated with high HbF. Among the 13 genes that demonstrated differential transcript levels, 8 demonstrated nuclear protein expression levels that were significantly changed by cytokine signal transduction. Five of the 8 genes are recognized regulators of erythropoiesis or globin genes (MAFF, ID2, HHEX, SOX6, and EGR1). Thus, cytokine-mediated signal transduction in adult erythroid cells causes significant changes in the pattern of globin gene and protein expression that are associated with distinct histone modifications as well as nuclear reprogramming of erythroid transcription factors.

Inhibition of erythroblast growth and fetal hemoglobin production by ribofuranose-substituted adenosine derivatives.

In vivo, inhibition of fetal hemoglobin (HbF) expression in humans around the time of birth causes the clinical manifestation of sickle cell and beta-thalassemia syndromes. Inhibition of HbF among cultured cells was recently described by the adenosine derivative molecule named SQ22536. Here, a primary cell culture model was utilized to further explore the inhibition of HbF by adenosine derivative molecules. SQ22536 demonstrated down-regulation of growth and HbF expression among erythroblasts cultured from fetal and adult human blood. The effects upon HbF were noted in a majority of cells, and quantitative PCR analysis demonstrated a transcriptional mechanism. Screening assays demonstrated that two additional molecules named 5'-deoxy adenosine and 2',3'-dideoxy adenosine had effects on HbF comparable to SQ22536. Other adenosine derivative molecules, adenosine receptor binding ligands, and cAMP-signaling regulators failed to inhibit HbF in matched cultures. These results suggest that structurally related ribofuranose-substituted adenosine analogues act through an unknown mechanism to inhibit HbF expression in fetal and adult human erythroblasts.

Cl-IB-MECA inhibits human erythropoiesis.

Candidate drugs are being sought for the suppression of human erythropoiesis. Cl-IB-MECA [2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide] is a derivative of adenosine that inhibits the growth of leukaemic cell lines. To determine the effects of Cl-IB-MECA upon erythropoiesis, studies were performed by using an ex vivo culture system of primary human CD34+ cells. Cl-IB-MECA suppressed erythroblast growth and maturation at doses >/=50 mumol/l through a mechanism of cell cycle inhibition and accumulation of cells in the G1/G0 phase. These findings demonstrate that Cl-IB-MECA inhibits human erythropoiesis, and suggest that further consideration of this drug is warranted for patients with erythrocytosis or polycythemia syndromes.

Functional effects of monoclonal antibodies to the purified amino-terminal extracellular domain of the human Ca(2+) receptor.

UNLABELLED: We generated three functionally unique monoclonal antibodies to the purified human CaR extracellular domain. Flow cytometry studies of chimeric receptors localized their epitopes to lobe 2 of the VFT domain. These results lead us to propose a mechanism for the functional effects of these antibodies. INTRODUCTION: The human Ca(2+) receptor (CaR), which plays a central role in the regulation of [Ca(2+)](0) homeostasis, has a distinctively large extracellular domain that consists of a bilobed Venus flytrap (VFT) domain, involved in agonist binding, and a cysteine-rich domain. Functional antibodies that specifically bind to this domain would have therapeutic potential and could be used as a tool to gain insights into receptor activation as well. MATERIALS AND METHODS: We generated three monoclonal antibodies (mAbs), 7F8, 5C8, and 1A8, to the purified human CaR extracellular domain. Functional characterization of these antibodies included Ca(2+) stimulation of phosphoinositide hydrolysis to examine effects of intact or protease digested antibodies on sensitivity of the receptor to extracellular Ca(2+) and flow cytometry assay of binding of the antibodies to HEK-293 cells expressing chimeric receptors to map antibody epitopes. RESULTS: We found these mAbs specifically recognize native but not denatured human CaR or homologous native Fugu CaR. Sensitivity of the human CaR to extracellular calcium was increased by binding of 5C8 but decreased by binding of 1A8. A chimeric receptor FCFCF, with lobe 2 region of the human CaR VFT domain in the Fugu CaR backbone, bound all three mAbs, and the sensitivity of this chimeric CaR to extracellular Ca(2+) was also increased by binding of 5C8 and decreased by binding of 1A8. CONCLUSIONS: The epitopes of these mAbs reside in the lobe 2 region of the human CaR VFT domain. 5C8 might activate the receptor by facilitating closure and/or rotation of the VFT domains on agonist binding, whereas 1A8 might inhibit the receptor by impeding such agonist-induced conformational changes. Recombinant antibodies with antigen binding domains of 5C8 and 1A8 could be useful in the treatment of hyperparathyroidism and osteoporosis, respectively.

Fetal hemoglobin silencing in humans.

Interruption of the normal fetal-to-adult transition of hemoglobin expression should largely ameliorate sickle cell and beta-thalassemia syndromes. Achievement of this clinical goal requires a robust understanding of gamma-globin gene and protein silencing during human development. For this purpose, age-related changes in globin phenotypes of circulating human erythroid cells were examined from 5 umbilical cords, 99 infants, and 5 adult donors. Unexpectedly, an average of 95% of the cord blood erythrocytes and reticulocytes expressed HbA and the adult beta-globin gene, as well as HbF and the gamma-globin genes. The distribution of hemoglobin and globin gene expression then changed abruptly due to the expansion of cells lacking HbF or gamma-globin mRNA (silenced cells). In adult reticulocytes, less than 5% expressed gamma-globin mRNA. These data are consistent with a "switching" model in humans that initially results largely from gamma- and beta-globin gene coexpression and competition during fetal development. In contrast, early postnatal life is marked by the rapid accumulation of cells that possess undetectable gamma-globin mRNA and HbF. The silencing phenomenon is mediated by a mechanism of cellular replacement. This novel silencing pattern may be important for the development of HbF-enhancing therapies.

A sustained and pancellular reversal of gamma-globin gene silencing in adult human erythroid precursor cells.

We systematically compared cytokine-mediated increases or decreases in proliferation with globin gene and protein expression in adult human erythroblasts. Despite their opposite effects on growth, stem cell factor (SCF) and transforming growth factor beta (TGF-B) had synergistic effects with respect to fetal hemoglobin (HbF): average HbF/HbF + adult hemoglobin (HbA) ratio in erythropoietin (EPO) = 1.4 +/- 1.0%; EPO + TGF-B = 10.8 +/- 1.9%; EPO + SCF = 19.1 +/- 6.2%; and EPO + SCF + TGF-B (EST) = 39.3 +/- 6.3%. Polymerase chain reaction (PCR) revealed significant increases in gamma-globin transcripts that were balanced by reduced beta-globin transcripts. Single-cell quantitative PCR demonstrated a complete reversal of gamma-globin gene silencing with detectable gamma-globin mRNA in more than 95% of the cells. Immunostaining with HbF antibodies also showed a pancellular distribution in EST (96.2 +/- 0.01% HbF positive) compared with a heterocellular distribution in EPO (42.9 +/- 0.01% HbF positive). As shown here for the first time, a robust and pancellular reversal of gamma-globin gene silencing among hemoglobinized erythroblasts from adult humans may be achieved in the absence of hereditary mutation or direct genomic manipulation.