ABSTRACT: The chromatin structure of the human beta-globin gene locus assumes a transcriptionally-active conformation in erythroid cells. One feature of this chromatin reorganization is the formation of DNase 1 hypersensitive sites in the regions of active globin gene promoters. This reorganization requires the globin locus control region and is associated with normal expression of the beta-like globin genes. To determine whether it is possible to artificially enhance the opening of the chromatin structure of a minimal beta-globin promoter, we placed a 101bp, erythroid-specific DNase 1 hypersensitive site-forming element (HSFE) immediately upstream of the beta-globin promoter and gene. This element includes binding sites for NF-E2, AP-1, GATA-1 and Sp-1. Constructs were stably transfected into murine erythroleukemia cells and promoter chromatin structure and gene expression were analyzed. The HSFE induced an area of enhanced DNase 1 hypersensitivity extending from the transcriptional start site to -300bp of the artificial promoter and significantly increased the proportion of beta-globin promoters in an open chromatin configuration. This remodeling of promoter chromatin structure resulted in 3 fold increases in beta-globin gene transcription and induction, and inhibited long-term beta-globin gene silencing. These results indicate that a relatively small cis-acting element is able to enhance remodeling of promoter chromatin structure resulting in increased beta-globin gene expression.

Reprint requests to: Christopher H. Lowrey, M.D., Department of Medicine, Hinman Box 7650, Dartmouth Medical School, Hanover, New Hampshire 03755, phone: (603) 650-1682, fax: (603) 650-1129, e-mail: c.lowrey@dartmouth.edu.

ABSTRACT: HFE is a non-typical MHC class 1-type protein that is mutated in hereditary hemochromatosis. The purpose of this study was to identify possible splice variants of HFE mRNA and investigate the regulation of these isoforms in duodenum and liver of patients with normal and altered iron stores. RT-PCR was performed using HFE specific primers and duodenal RNA obtained from patients with hemochromatosis, iron deficiency, secondary iron overload and normal controls. The reaction products were visualized by Southern blot and identified by DNA sequence analysis. Additional studies were performed on RNA isolated from liver and a range of human tissues. A truncated (soluble) form of HFE protein was identified that lacks the transmembrane domain and occurs as a result of alternative splicing. Soluble HFE was found predominantly in the duodenum, spleen, breast, skin and testicle. In hereditary hemochromatosis full length HFE was the predominant isoform present in the duodenum similar to iron deficiency. Alternate splicing produces soluble HFE that may have a unique function to regulate cellular iron transport.

Reprint requests to: Dr. P.C. Adams, Department of Medicine, London Health Sciences Center, London, Ontario N6A 5A5, CANADA, phone: 519-663-4939, fax: 519-663-3232.

ABSTRACT: Proper expression of the c-myb proto-oncogene is essential for definitive, but not primitive erythropoiesis. To examine the role of c-myb during adult erythropoiesis, we incubated purified murine colony-forming units (CFU-E) with a c-myb-specific antisense oligodeoxynucleotide (AS-oligo) in order to diminish expression levels. By western blot analysis, c-myb expression was reduced during the first seven hours of AS-oligo treatment as compared to untreated cells. We then quantitated the amount of heme synthesized in CFU-E treated with c-myb AS-oligo, a random sequence oligo or no oligo. No significant differences were seen in the amount of heme synthesized during 42 hours of erythroid culture with either high levels (1 U/mL) or physiological levels (20 mU/mL) of Epo. In contrast, CFU-E treated with an AS-oligo directed toward mRNA encoding the first enzyme of the heme biosynthetic pathway in erythroid cells (d- aminolevulinate synthase) demonstrated a 65% reduction in the amount of heme synthesized. We conclude that the major role of c-myb during hematopoiesis must be in progenitor cells antecedent to the CFU-E stage and may possibly involve the establishment of the genetic program directing the formation of red blood cells.

Keywords: CFU-E, c-myb, antisense oligodeoxynucleotides, heme, erythropoietin.

Reprint requests to: Terry Rogers Bishop, The Johns Hopkins University, 720 Rutland Avenue, Ross Research Building, Room 1125, Baltimore, Maryland 21205, phone: (410) 614-0712, fax: (410) 955-8208, e-mail: tbishop@welchlink.welch,jhu.edu.