GC-rich sequences in the 5-lipoxygenase gene promoter are required for expression in Mono Mac 6 cells, characterization of a novel Sp1 binding site.
5-lipoxygenase (5LO) catalyzes formation of leukotrienes, mediators with roles in several inflammatory disorders, including atherosclerosis. The human 5LO gene promoter contain multiple GC-boxes. The relevance of these for expression of 5LO in the human monocytic cell line Mono Mac 6 (MM6) was studied. A downregulating effect of the GC-box binding compound mithramycin indicated that GC-rich sequences in the 5LO gene promoter are important for expression of native 5LO. In DNase I footprinting, mithramycin and Sp1 protected known GC-boxes, but also a novel Sp1 binding site was found, comprising 20 bp upstream of the major transcription initiation site, beside an Initiator-like sequence. Mutation of this site reduced Sp1 binding and expression of reporter genes in MM6 cells, compatible with a function as basal promoter element for the TATA-less 5LO gene. When differentiation was induced by TGFbeta and vitamin D(3), 5LO expression became prominent, but expression levels of Sp1/3 and Egr-1 were the same as for control cells. Also, 5LO reporter gene activity in transiently transfected MM6 cells was insensitive to differentiation. Thus, the GC-rich part of the 5LO gene promoter, including a novel Sp1 site, appear important for basal (rather than upregulated) transcription of 5LO in MM6 cells.
Dishart
David
D
Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II Karolinska Institutet, S-171 77 Stockholm, Sweden.
Schnur
Nicole
N
Klan
Niko
N
Werz
Oliver
O
Steinhilber
Dieter
D
Samuelsson
Bengt
B
Rådmark
Olof
O
D001094:Arachidonate 5-Lipoxygenase / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D051766:Early Growth Response Protein 1 / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D020862:GC Rich Sequence; D005786:Gene Expression Regulation; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D008926:Plicamycin / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D051705:Sp3 Transcription Factor / Q000235:genetics / Q000378:metabolism; D024363:Transcription Initiation Site
D051766:Early Growth Response Protein 1; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor; D001094:Arachidonate 5-Lipoxygenase; D008926:Plicamycin