Epigenetic silencing of the CIITA gene and posttranscriptional regulation of class II MHC genes in ocular melanoma cells.
OBJECTIVE
Primary uveal melanocytes and many ocular melanoma cells are resistant to interferon (IFN)-gamma-mediated induction of major histocompatibility complex (MHC) class II molecule expression. This suppression of class II MHC induction is considered to be one of the ways in which the eye is able to inhibit inflammatory responses. However, the mechanism(s) of this suppression is unknown. In this study, we have probed the molecular basis of this phenotype and report two distinct mechanisms underlying this phenotype.


METHODS
Primary ocular melanocytes and ocular melanoma cell lines (retaining this IFN-gamma-resistant class II MHC phenotype) were examined for the expression of class II MHC molecules on the cell surface by flow cytometry. Class II MHC gene expression was further examined using Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses.


RESULTS
The IFN-gamma signal-transduction pathway was found to be intact by electrophoretic mobility shift assay (EMSA) and transfection of reporter constructs. The lack of class II MHC gene expression appears to result from at least two mechanisms: (1) a specific inhibition of CIITA (class II transactivator) gene expression (reminiscent of trophoblasts), and (2) posttranscriptional regulation of class II MHC genes.


CONCLUSIONS
The inability of primary uveal melanocytes and ocular melanoma cells to express class II MHC molecules after treatment with IFN-gamma has been found to map to two distinct points in the class II MHC biosynthetic pathway. The predominant mechanism appears to involve the silencing of the endogenous gene encoding the class II transactivator (CIITA). Here, the blockade does not involve signal transduction from the IFN-gamma receptor, but rather involves a specific silencing of the CIITA gene. A second mechanism involves the posttranscriptional regulation of class II MHC genes.
Radosevich
Michael
M
Department of Immunology, University College London, University of London, Moorfields Eye Hospital, London, United Kingdom.
Song
Zhimin
Z
Gorga
Joan C
JC
Ksander
Bruce
B
Ono
Santa Jeremy
SJ
D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D005134:Eye Neoplasms / Q000235:genetics* / Q000378:metabolism; D020868:Gene Silencing; D005802:Genes, MHC Class II; D005822:Genetic Vectors; D006684:HLA-DR Antigens / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D000949:Histocompatibility Antigens Class II / Q000378:metabolism; D006801:Humans; D050856:Interferon Regulatory Factor-1; D007371:Interferon-gamma / Q000494:pharmacology; D008544:Melanocytes / Q000378:metabolism; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D050794:STAT1 Transcription Factor; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014602:Uvea / Q000378:metabolism
D004268:DNA-Binding Proteins; D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II; C494257:IRF1 protein, human; D050856:Interferon Regulatory Factor-1; C083728:MHC class II transactivator protein; D009687:Nuclear Proteins; D010750:Phosphoproteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D007371:Interferon-gamma