Abstract
The components of the antigen processing machinery, low molecular mass polypeptide (LMP) 2 and transporter associated with antigen processing (TAP) 1, are encoded by closely linked genes within the major histocompatibility complex class II subregion. Although the two genes share a bi-directional promoter, LMP2 and TAP1 have differential cellular expression. TAP1 is expressed constitutively. However, LMP2 expression requires induction by interferon-gamma in most cells. The regulatory elements within the LMP2/TAP1 promoter and the transcription factors that bind these elements have been defined. However, how these transactivators regulate differential TAP1 and LMP2 gene transcription is not known. We have addressed this question by analyzing three human melanoma cell lines with distinct phenotypes of LMP2 and TAP1 expression. Whereas binding of either interferon regulatory factor 1 or Stat1 to the overlapping interferon consensus sequence-2/GAS is sufficient for regulating transcription of the TAP1 gene, binding of both factors is required for LMP2 gene transcription. This conclusion is supported by restoration of LMP2 gene transcription following transfection of wild type Stat1alpha or interferon regulatory factor 1 cDNA into cells lacking these transcription factors. The flexibility in the regulation of the TAP1 gene may reflect its role in maintaining immune surveillance. Furthermore, lack of LMP2 gene transcription in quiescent human cells suggests that LMP2 expression reflects a state of cell activation.
Highlights
From the ‡Department of Microbiology and Immunology, New York Medical College, Valhalla, New York 10595 and the §Surgery Branch, National Cancer Institute, NIH, Bethesda, Maryland 20892
Lack of Stat1␣ or IRF1 Binding at the LMP2/TAP1 Promoter Is Associated with Lack of LMP2 Gene Transcription in 526 and 624.c Human Melanoma Cell Lines—We examined by Electrophoretic Mobility Shift Assay (EMSA) the binding activity of transcription factors to the GAS, interferon consensus sequence (ICS)-2, ICS-1, B, and SP-1 cis-regulatory regions of the TAP1/ LMP2 promoter (Fig. 3) in untreated and IFN-␥-treated 526, 624.c, and 677 melanoma cell lines
These results demonstrate that binding of both Stat1␣ and IRF1 to the overlapping ICS-2/GAS site within the LMP2/TAP1 promoter is required for LMP2 gene transcription
Summary
Vol 273, No 26, Issue of June 26, pp. 16177–16183, 1998 Printed in U.S.A. Different Requirements for Signal Transducer and Activator of Transcription 1␣ and Interferon Regulatory Factor 1 in the Regulation of Low Molecular Mass Polypeptide 2 and Transporter Associated with Antigen Processing 1 Gene Expression*. Transcription of the LMP2 gene in fibroblasts, in melanocytes, and in about 50% of melanoma cell lines that do not constitutively express LMP2 was induced following incubation with IFN-␥ This observation implies the role of IFN-␥-inducible factors in transcriptional regulation of the LMP2 gene. In the present study we have characterized the IFN-␥-inducible trans-acting factors required for LMP2 gene transcription, because this information contributes to our understanding of the molecular mechanism(s) regulating the antigen presentation machinery. To this end, we have analyzed the melanoma cell lines 526, 624.c, and 677, which constitutively express TAP1 but exhibit three distinct phenotypes of LMP2 gene expression. The present study demonstrates for the first time that discordant LMP2 and TAP1 gene transcription reflects differential requirements of Stat1␣ and IRF1
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