Comprehensive genome scans involving many thousands of SNP assays will require significant amounts of genomic DNA from each sample. We report two successful methods for amplifying whole-genomic DNA prior to SNP analysis, multiple displacement amplification, and OmniPlex technology. We determined the coverage of amplification by analyzing a SNP linkage marker set that contained 2320 SNP markers spread across the genome at an average distance of 2.5 cM. We observed a concordance of >99.8% in genotyping results from genomic DNA and amplified DNA, strongly indicating the ability of both methods used to amplify genomic DNA in a highly representative manner. Furthermore, we were able to achieve a SNP call rate of >98% in both genomic and amplified DNA. The combination of whole-genome amplification and comprehensive SNP linkage analysis offers new opportunities for genetic analysis in clinical trials, disease association studies, and archiving of DNA samples.

Host APCs are required for initiating T cell-dependent acute graft-vs-host disease (GVHD), but the role of APCs in the effector phase of acute GVHD is not known. To measure the effect of tissue-resident APCs on the local development of acute GVHD, we selectively depleted host macrophages and DCs from the livers and spleens, but not from the skin, peripheral lymph nodes (PLN), or mesenteric lymph nodes (MLN), of C57BL/6 (B6) mice by i.v. administration of liposomal clodronate before allogeneic bone marrow transplantation. Depletion of host hepatic and splenic macrophages and DCs significantly inhibited the proliferation of donor C3H.SW CD8+ T cells in the spleen, but not in the PLN or MLN, of B6 mice. Such organ-selective depletion of host tissue APCs also markedly reduced the trafficking of allogeneic CD8+ T cells into the livers and spleens, but not PLN and MLN, of B6 recipients compared with that of the control mice. Acute hepatic, but not cutaneous, GVHD was inhibited as well, resulting in improved survival of liposomal clodronate-treated B6 recipients. When C3H.SW CD8+ T cells were activated in normal B6 recipients, recovered, and adoptively transferred into secondary B6 recipients, activated donor CD8+ T cells rapidly migrated into the livers and spleens of control B6 recipients but were markedly decreased in B6 mice that were depleted of hepatic and splenic macrophages and DCs. Thus, tissue-resident APCs control the local recruitment of allo-reactive donor T cells and the subsequent development of acute GVHD.

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Interleukin 3 (IL-3) is a hematopoietic stem-cell growth and differentiation factor that is expressed solely in activated T and NK cells. Studies to date have identified elements 5' to the IL-3 coding sequences that regulate its transcription, but the sequences that confer T cell-specific expression remain to be clearly defined. We have now identified DNA sequences that are required for T cell-restricted IL- 3 gene transcription. A series of transient transfections performed with human IL-3-chloramphenicol acetyltransferase (CAT) reporter plasmids in T and non-T cells revealed that a plasmid containing 319 bp of 5' flanking sequences was active exclusively in T cells. Deletion analysis revealed that T cell specificity was conferred by a 49-bp fragment (bp -319 to -270) that included a potential binding site for AP-1 transcription factors 6 bp upstream of a binding site for Elf-1, a member of the Ets family of transcription factors. DNaseI footprint and electrophoretic mobility shift assay analyses performed with MLA-144 T cell nuclear extracts demonstrated that this 49-bp region contains a nuclear protein binding region that includes consensus AP-1 and Elf-1 binding sites. In addition, extracts prepared from purified human T cells contained proteins that bound to synthetic oligonucleotides corresponding to the AP-1 and Elf-1 binding sites. In vitro-transcribed and -translated Elf-1 protein bound specifically to the Elf-1 site, and Elf-1 antisera competed and super shifted nuclear protein complexes present in MLA-144 nuclear extracts. Moreover, addition of anti-Jun family antiserum in electrophoretic mobility shift assay reactions completely blocked formation of the AP-1-related complexes. Transient transfection studies in MLA-144 T cells revealed that constructs containing mutations in the AP-1 site almost completely abolished CAT activity while mutation of the Elf-1 site or the NF-IL-3 site, a previously described nuclear protein binding site (bp. -155 to -148) in the IL-3 promoter, reduced CAT activity to < 25% of the activity given by wild-type constructs. We conclude that expression of the human IL-3 gene requires the AP-1 and Elf-1 binding sites; however, unlike other previously characterized cytokine genes such as IL-2, the AP-1 and Elf- 1 factors can bind independently in the IL-3 gene.(ABSTRACT TRUNCATED AT 400 WORDS)

The homeobox genes encode a family of transcription factors that regulate development and postnatal tissue homeostasis. Since HOXB4 plays a key role in regulating the balance between hematopoietic stem cell renewal and differentiation, we studied the molecular regulation of HOXB4 expression in human hematopoietic stem cells. HOXB4 expression in K562 cells is regulated at the level of transcription, and transient transfection defines primary HOXB4 regulatory sequences within a 99-bp 5′ promoter. Culture of highly purified human CD34+ bone marrow cells in thrombopoietin/Flt-3 ligand/stem cell factor induced HOXB4 3–10-fold, whereas culture in granulocyte/macrophage colony-stimulating factor, only increased HOXB4/luciferase expression 20–50%. Mutations within the HOXB4 promoter identified a potential E box binding site (HOX response element [HXRE]-2) as the most critical regulatory sequence, and yeast one hybrid assays evaluating bone marrow and K562 libraries for HXRE-2 interaction identified upstream stimulating factor (USF)-2 and micropthalmia transcription factor (MITF). Electrophoretic mobility shift assay with K562 extracts confirmed that these proteins, along with USF-1, bind to the HOXB4 promoter in vitro. Cotransfection assays in both K562 and CD34+ cells showed that USF-1 and USF-2, but not MITF, induce the HOXB4 promoter in response to signals stimulating stem cell self-renewal, through activation of the mitogen-activated protein kinase pathway. Thus hematopoietic expression of the human HOXB4 gene is regulated by the binding of USF-1 and USF-2, and this process may be favored by cytokines promoting stem cell self-renewal versus differentiation.