The treatment of a number of diseases including a broad range of anemias, hemoglobinopathies and malarial diseases will require a fundamental understanding of both cellular and molecular aspects of human erythropoiesis. We have taken a direct approach toward the prospective study of the early and late transcriptional events that encompass human erythropoiesis by examining cells that proliferate in culture specifically in response to the hormone erythropoietin. Our goal is to fully characterize these cell populations using current molecular genetic methods in order to understand and manipulate their transcriptional patterns. Several accomplishments have been achieved to date: #1. Generation of a transcription-based description of human erythropoiesis. Using pilot sequence analyses from several erythroblast libraries, larger scale sequencing efforts were pursued. The sequencing efforts have consisted of a single pass for each harvested clone to generate an Expressed Sequence Tag (EST) dataset (http://hembase.niddk.nih.gov/). In silico comparisons of our EST with other publicly available databases, including those from NCBI and the internet-linked human genome map browser, were used in lieu of further sequencing to define gene homologies, genomic loci, and ?finished? sequences. These efforts have revealed the existence of several hundred novel genes or splice variants expressed in erythroid cells. Full-length descriptions of novel erythroid-specific splice variants of the heme enzyme PBDG and arginase genes, as well as several novel transcripts from intergenic regions of the beta globin locus have already been reported or deposited in Genbank. We have also sequenced and begun the informatic and biological characterizations of other genes prioritized on the basis of their novelty or perceived clinical relevance. #2. A redefinition of the hemoglobin biosynthesis patterns in adult humans. Current hypotheses for reactivation of the fetal hemoglobin genes are based on a model conceived over a decade ago of hemoglobin accumulation during adult erythropoiesis. This model includes a switch from fetal-type to adult-type hemoglobin during the proerythroblast-basophilic normoblast stage of differentiation. Accordingly, we expected to identify a majority of fetal globin transcripts within our database derived from the isolated populations of less mature cells. Instead, we determined that the globin ESTs encoded over 85% adult-type transcripts, a result not consistent with the previous model for erythropoiesis. We hypothesized that fetal hemoglobin may instead accumulate in adult human cells with a pattern similar to that of adult hemoglobin. To test our hypothesis, the accumulation of hemoglobin was directly measured among differentiating cells harvested from the peripheral blood and bone marrow of volunteers. Using the peripheral blood cells, we were able to monitor several cell biology parameters in addition to hemoglobin accumulation, including morphology, cell cycle phase, and surface phenotype. Direct quantitation of hemoglobin species was performed using high-pressure liquid chromatography. Contrary to the standard model, we found that fetal hemoglobin does not precede adult hemoglobin accumulation during the differentiation process. Our analysis was consistent with our hypothesis and revealed that fetal and adult hemoglobin share a coordinated pattern of accumulation. Examination of fresh bone marrow as well as quantitative PCR analyses revealed the same coordinated patterns. Hence, these results directly challenge the prevailing model of hemoglobin accumulation in adults. As a result, we have proposed a model of globin ?modulation? rather than ?switching? to explain the control of hemoglobin biosynthesis in adults. Importantly, we also found that the pattern of hemoglobin accumulation during adult erythropoiesis is directly related to the proliferation of the cells. This new data has led us to actively explore the novel hypothesis that regulation of signal transduction cascades and growth among fully committed erythroid cells may be used to increase levels of fetal hemoglobin. #3. Identification of the Dombrock blood group carrier molecule. Blood transfusion is one of the top three procedures performed in U.S. hospitals with at least 1.2 million units transfused annually in the inpatient setting alone. Patients who receive numerous units of blood often develop immune-based reactions to the transfused cells. Those immune reactions have provided the basis for blood crossmatching and the identification of 25 genetically distinct molecules that carry the responsible antigens. One such blood group antigen carrier molecule termed ?Dombrock? was identified serologically in 1965. Dombrock antigenicity had been linked genetically to the short arm of chromosome 12, and the molecule itself was thought to be GPI-anchored. However, further efforts to identify and define the Dombrock molecule or its genetic basis had been unsuccessful. The clinical significance of solving this problem has recently been highlighted by the description of Dombrock-based hemolysis in sickle cell patients receiving multiple blood transfusions. We hypothesized that our genomic-based analysis of erythropoiesis could be used to identify the Dombrock blood group carrier molecule. This hypothesis was based upon the fact that approximately half of the known blood group carrier molecules were identifiable in our database by homology analyses. By applying previously reported studies regarding the Dombrock encoding gene, we examined our database for EST mapped to the short arm of chromosome 12 that also carry the genetic signature of GPI-anchored proteins. One candidate EST was identified that met these criteria which we designated DOK1. Our sequence analysis of the entire clonal insert, as well as expression studies among differentiating erythroid cells were consistent with our hypothesis. As a result, I pursued a collaboration with Dr. Marion Reid (Lindsley F. Kimball Research Institute, New York Blood Center). Using flow cytometry, we demonstrated the binding of anti-Dombrock serum antibodies from patients to DOK1 transfected cells. We further defined the Dombrock encoded gene as sharing at least two exons with a member of the ADP ribosyltransferase gene family. Furthermore, we defined the molecular basis of the DO(a) and DO(b) blood types as being due to a SNP that results in an amino-acid substitution within an RGD motif of the molecule. Together, these findings confirmed our discovery that DOK1 encodes the Dombrock blood group carrier molecule. We are now engaged in further collaborative efforts to develop clinical assays to identify units of blood according to their Dombrock blood type, and define other Dombrock polymorphisms at the molecular level. The identification of the Dombrock gene and its polymorphisms has already resulted in molecular screens in blood banks for the purpose of preventing hemolytic reactions. #4. Analysis of myelodysplastic bone marrow using erythroid-focused cDNA arrays. Our working hypothesis is that array technologies may be useful for defining abnormal transcriptional patterns in complex diseases involving erythroid cells. For this purpose, we examined gene transcription in bone marrow samples from patients with myelodysplasia with erythroid-focused arrays. We compared bone marrow gene expression patterns sampled from 10 normal donors and 5 patients with myelodysplasia. By comparing the patterns of gene expression in normal bone marrow, we identified a steady state of erythroid-relevant gene activity among mixed populations of bone marrow cell types. We validated our hypothesis and demonstrated distinct patterns of abnormal gene expression consistent with morphology-based predictions of imbalances between proliferation and differentiation.
| de Vasconcellos, Jaira F; Lee, Y Terry; Byrnes, Colleen et al. (2016) HMGA2 Moderately Increases Fetal Hemoglobin Expression in Human Adult Erythroblasts. PLoS One 11:e0166928 |
| Oneal, Patricia A; Gantt, Nicole M; Schwartz, Joseph D et al. (2006) Fetal hemoglobin silencing in humans. Blood 108:2081-6 |
| Miller, Jeffery Lynn (2006) Patchwork HBA1 and HBA2 genes. Haematologica 91:289A |
| Bhanu, Natarajan V; Trice, Tiffany A; Lee, Y Terry et al. (2005) A sustained and pancellular reversal of gamma-globin gene silencing in adult human erythroid precursor cells. Blood 105:387-93 |
| Miller, Jeffery L (2005) Signaled expression of fetal hemoglobin during development. Transfusion 45:1229-32 |
| Goh, Sung-Ho; Lee, Y Terry; Bhanu, Natarajan V et al. (2005) A newly discovered human alpha-globin gene. Blood 106:1466-72 |
| Aerbajinai, Wulin; Lee, Y Terry; Wojda, Urszula et al. (2004) Cloning and characterization of a gene expressed during terminal differentiation that encodes a novel inhibitor of growth. J Biol Chem 279:1916-21 |
| Pal, Saumen; Nemeth, Michael J; Bodine, David et al. (2004) Neurokinin-B transcription in erythroid cells: direct activation by the hematopoietic transcription factor GATA-1. J Biol Chem 279:31348-56 |
| Bhanu, Natarajan V; Trice, Tiffany A; Lee, Y Terry et al. (2004) A signaling mechanism for growth-related expression of fetal hemoglobin. Blood 103:1929-33 |
| Goh, Sung-Ho; Jackson, Kaedrea A; Terry Lee, Y et al. (2004) Identification of an alternate delta-globin mRNA in adult human erythroid cells. Genomics 84:431-4 |
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