This proposal examines the molecular mechanisms that regulate tissue-specific gene expression and development in myelomonocytic cells (phagocytes). The phagocyte-specific gp9l-phox gene, which when defective causes chronic granulomatous disease, encodes a component of the respiratory burst NADPH-oxidase complex. We have previously shown that the proximal gp9l-phox promoter will direct the expression of linked reporter genes in a subset of monocyte/macrophages in, transgenic mice. In addition, macrophage tumors arise at 6-12 months of age in animals carrying a gp9l-phox promoter/SV40 T-antigen transgene. A major goal of the proposed work is to identify the cis-elements within the gp9l-phox promoter, and the DNA-binding proteins interacting with these sequences, that mediate monocyte/macrophage-specific transcription. Transgene constructs consisting of various portions of the gp9l-phox promoter linked to reporter genes will be introduced into mice, and the tissue-distribution of transgene expression determined by Northern blot analysis and immunohistochemical detection of the reporter protein. In addition, efforts will be made to develop a tissue culture system as a more convenient method to assay gp9l-phox promoter function. Nuclear extracts will be used in in vitro DNA-binding protein assays to identify DNA-binding activities that interact with the proximal gp9l-phox promoter. DNA-binding protein recognition sites will then be ablated by site-directed mutagenesis, and mutant promoter constructs will be introduced into transgenic mice (or suitable tissue culture cells) to evaluate the effect that loss of protein binding has on transgene expression. DNA-binding proteins found to be functionally relevant to the regulation of gp9l-phox expression will be molecularly cloned by ligand screening of a lambda-GT11 expression library, or by library screening with antibody or degenerate oligonucleotide probes following biochemical (DNA affinity chromatography) purification. Isolation of transcription factor cDNA clones will allow a detailed study of the regulation of their activity, with the ultimate goal of understanding how a progenitor cell becomes committed to differentiate along the myelomonocytic lineage. The time lag prior to the appearance of macrophage malignancies in gp9l-phox promoter/SV40 T-antigen transgenic mice suggests that somatic events, in addition to T-antigen expression, are required for tumorigenesis. Genetic loci that contribute to tumor progression in these cells will be identified by proviral """"""""tagging"""""""" with Moloney murine leukemia virus, followed by recovery of proviral integration sites from tumors that arise at unusually early times.
|Voo, K S; Jacobsen, B M (1998) Rapid resuspension of pelleted bacterial cells for miniprep plasmid DNA isolation. Biotechniques 24:240-3|
|Pattison, S; Skalnik, D G; Roman, A (1997) CCAAT displacement protein, a regulator of differentiation-specific gene expression, binds a negative regulatory element within the 5' end of the human papillomavirus type 6 long control region. J Virol 71:2013-22|
|Eklund, E A; Lee, S W; Skalnik, D G (1995) Cloning of a cDNA encoding a human DNA-binding protein similar to ribosomal protein S1. Gene 155:231-5|