SP100B Repression of Gene Expression

Abstract

Abstract: : Upon infection, viral DNA localizes near nuclear structures termed PML bodies. These structures contain numerous proteins including PML and SP100A. SP100B, a splice variant derived from the same gene as SP100A, is a potent repressor of viral gene expression, which localizes to discrete nuclear sites that are distinct from PML bodies. It has been hypothesized that SP100B represses gene expression by inducing heterochromatin formation on DNA entering the nucleus. Purpose. To determine whether SP100B repression is consistent with heterochromatin formation on viral DNA. Methods. Chromatin immunoprecipitation (Chip) assays were used to determine whether SP100B alters the composition of histones associated with DNA introduced into cells by transfection. Yeast two-hybrid assays and immunoprecipitation assays using transient transfected cells were used to determine whether SP100B or SP100A, which does not repress gene expression, interacts with heterochromatin protein 1α (HP1α), a protein that is important in heterochromatin condensation. Colocalization of HP1 α with SP100A and SP100B was evaluated by immunofluorescence of transfected cells. Deacetylation of histones is a feature of heterochromatin. The effect of an inhibitor of histone deacetylase activity (trichostatin A) on repression of gene expression by SP100B was determined. Results. In Chip assays, transfected DNA was precipitated using anti-histone antibodies, confirming previous reports that transfected DNA is assembled into nucleosomes. SP100A and SP100B associated with HP1α ; in yeast two hybrid assays, but only SP100A associated with HP1α in transfected mammalian cells. HP1α did not specifically localize with either SP100A or SP100B. Treatment of transfected cells with trichostatin A enhanced expression of transfected genes, but did not reverse the repressive activity of SP100B. Conclusion. SP100B appears to be a potent component of the innate antiviral response, repressing viral gene expression. Although transfected DNA becomes associated with histones after entry into cells and appears to be deacetylated, SP100B does not appear to function by association with the heterochromatin protein, HP1α, nor by enhancing histone deacetylation, suggesting that assembly of DNA into condensed heterochromatin is not the mechanism of repression of gene expression by SP100B.

Keywords: herpes simplex virus • gene/expression