REQUIREMENT OF HISTONE DEACETYLASE ACTIVITY FOR SIGNALING BY STAT1 Running title: HDAC and signaling by IFNγ

STAT1 is a transcription factor which plays a crucial role in signaling by IFNs. In this study we demonstrated that inhibitors of HDAC activity, butyrate, TSA and SAHA, prevented IFNγ-induced JAK1 activation, STAT1 phosphorylation, its nuclear translocation, and STAT1-dependent gene activation. Furthermore, we showed that silencing of HDAC1, HDAC2 and HDAC3 through RNA interference markedly decreased IFNγ-driven gene activation, and that overexpression of HDAC1, HDAC2 and HDAC3 enhanced STAT1 dependent transcriptional activity. Our data therefore established the essential role of deacetylase activity in STAT1 signaling. Induction of IRF-1 by IFNγ requires functional STAT-1 signaling and was abrogated by butyrate, TSA, SAHA, and by STAT1 siRNA. In contrast, silencing of STAT1 did not interfere with IFNγ-induced expression of STAT2 and caspase-7, and HDAC inhibitors did not preclude IFNγ− induced expression of STAT1, STAT2 and caspase-7, suggesting that HDAC inhibitors impede the expression of IFNγ target genes whose expression depends on STAT1, but do not interfere with STAT1 independent signaling by IFNγ. Finally, we showed that inhibitors of deacetylase activity sensitized colon cancer cells to IFNγ-induced apoptosis through cooperative negative regulation of Bcl-x expression, demonstrating that interruption of the balance between STAT1 dependent and STAT1 independent signaling significantly alters the biological activity of IFNγ.