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,§
,¶
*
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia;
Intramural Research Support Program, Science Applications International Corp.-Frederick and Laboratory of Molecular Immunoregulation, Division of Basic Sciences, and
Molecular Basis of Carcinogenesis Laboratory, Advanced BioScience Laboratories-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702;
§
Institute of Molecular Medicine and Department of Paediatrics, John Radcliffe Hospital, Oxford University, Oxford, United Kingdom; and
¶
Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia
Transcription of the TNF gene is rapidly and transiently induced by
LPS in cells of monocyte/macrophage lineage. Previous data suggested
that multiple NF-
B/Rel binding sites play a role in the
transcriptional response to LPS of the murine gene. However, the
relevance of homologous sites in the human TNF gene remained a matter
of controversy, partly because the high affinity NF-B/Rel site
located at -510 in the murine promoter is not conserved in humans.
Here we used two sets of similarly designed human and mouse TNF
promoter deletion constructs and overexpression of IB in the murine
macrophage cell line ANA-1 to show remarkable similarity in the pattern
of the transcriptional response to LPS, further demonstrating the
functional role of the distal promoter region located between -600 and
-650. This region was characterized by mutagenesis of protein binding
sites, including two relatively low affinity NF-B/Rel sites, #2 and
2a. Mutation in each of the NF-B sites resulted in 2- to 3-fold
lower transcriptional activity in response to LPS. In contrast to LPS
activation, the response to PMA was substantially lower in magnitude
and required only the proximal promoter region. In summary, the
functional topography of human and murine promoters when assayed in the
same system has some marked similarities. Our observations support the
notion that full LPS response of TNF gene requires both NF-B and
non-NF-B nuclear proteins. Our data also suggest that the functional
activity of a given B site depends on the entire DNA sequence
context in the promoter region.
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