The long term goal of this project is to define the molecular mechanisms that control the switch from fetal (gamma) to adult (beta) globin synthesis; this switch is controlled at the level of globin gene transcription. Individuals with Hereditary Persistence of Fetal Hemoglobin (HPFH) continue to express gamma-globin genes in adult RBCs, and frequently have mutations just upstream from the gamma-globin genes that probably cause the HPFH phenotype. We intend to use these mutations to define the cis-acting DNA sequences and trans-regulatory factors that control gamma- globin transcription in erythroid cells, as follows: 1) We will define the cis-acting DNA sequences in the human beta-globin gene cluster that silence the gamma-globin gene in mouse erythroleukemia (MEL) cells. gamma-globin genes are minimally expressed in """"""""adult"""""""" erythroid MEL cells when introduced on whole chromosomes or cosmids. We will define the sequences that normally silence gamma-globin transcription in MEL cells using stable transfection techniques, and will determine whether these elements interact with HPFH mutations. 2) We will further define an S1 nuclease hypersensitive site (S1-HSS) in the -210 region of the gamma-globin promoter, and define how this site is altered by HPFH mutations. The region from -216 to -208 contains an S1-HSS in supercoiled plasmids; this site is destabilized by some HPFH mutations. We will define the sequences that contribute to the formation of the S1-HSS with site-directed mutations, and will examine the relevance of the site for the binding of potential regulatory factors. 3) We will characterize trans-acting factors that discriminate between wild-type and HPFH-associated gamma-globin alleles. We have identified a MEL cell protein(s) that binds with increased affinity to a gamma-globin allele with the -202 c -> G HPFH substitution. This protein will be characterized, purified, and cloned. A newly region will be further characterized. Functional analysis of this cloned protein will examine its relevance for globin gene switching. 4) We will further characterize the structure and function of the murine beta-globin locus control region (LCR). The region upstream from murine beta-globin LCR 5' HS-2 will be sequenced and examined with a variety of functional assays. Structure/function relationships with the human beta- globin LCR will be established.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
Project #
Application #
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Washington University
Internal Medicine/Medicine
Schools of Medicine
Saint Louis
United States
Zip Code
Young, Margaret A; Larson, David E; Sun, Chiao-Wang et al. (2012) Background mutations in parental cells account for most of the genetic heterogeneity of induced pluripotent stem cells. Cell Stem Cell 10:570-82
Lu, Zhi Hong; Books, Jason T; Ley, Timothy J (2006) Cold shock domain family members YB-1 and MSY4 share essential functions during murine embryogenesis. Mol Cell Biol 26:8410-7
Lu, Zhi Hong; Books, Jason T; Ley, Timothy J (2005) YB-1 is important for late-stage embryonic development, optimal cellular stress responses, and the prevention of premature senescence. Mol Cell Biol 25:4625-37
Lu, Zhi Hong; Books, Jason T; Kaufman, Richard M et al. (2003) Long targeting arms do not increase the efficiency of homologous recombination in the beta-globin locus of murine embryonic stem cells. Blood 102:1531-3
Kaufman, R M; Lu, Z H; Behl, R et al. (2001) Lack of neighborhood effects from a transcriptionally active phosphoglycerate kinase-neo cassette located between the murine beta-major and beta-minor globin genes. Blood 98:65-73
Kaufman, R M; Pham, C T; Ley, T J (1999) Transgenic analysis of a 100-kb human beta-globin cluster-containing DNA fragment propagated as a bacterial artificial chromosome. Blood 94:3178-84
Graubert, T A; Hug, B A; Wesselschmidt, R et al. (1998) Stochastic, stage-specific mechanisms account for the variegation of a human globin transgene. Nucleic Acids Res 26:2849-58
Ley, T J; Hug, B; Fiering, S et al. (1998) Reduced beta-globin gene expression in adult mice containing deletions of locus control region 5' HS-2 or 5' HS-3. Ann N Y Acad Sci 850:45-53
Graubert, T A; Russell, J H; Ley, T J (1996) The role of granzyme B in murine models of acute graft-versus-host disease and graft rejection. Blood 87:1232-7
Graubert, T A; Ley, T J (1996) How do lymphocytes kill tumor cells? Clin Cancer Res 2:785-9

Showing the most recent 10 out of 38 publications