Kinetics of a Gamma Interferon Response: Expression and Assembly of CIITA Promoter IV and Inhibition by Methylation

Abstract

Chromatin immunoprecipitation assays were employed to assess the kinetics of transcription factor assembly and histone modifications that occur during gamma interferon (IFN-γ) induction of CIITA gene expression. CIITA is the master regulator of major histocompatibility complex class II transcription. Promoter IV (PIV), the major IFN-γ responsive promoter for CIITA expression, requires both STAT1 and IFN regulatory factor 1 (IRF-1) for induction by IFN-γ. STAT1 binding to PIV was detected first and was accompanied by a modest acetylation of histones H3 and H4 that were associated with the region. Despite these changes, which occurred within 30 min of IFN-γ treatment, CIITA mRNA was not detected until IRF-1 protein was synthesized and bound to its site, a process that required >120 min. In contrast to these events, fetal trophoblast-like cell lines, which are refractory to CIITA induction by IFN-γ, failed to assemble the above factors or modify their chromatin, suggesting that accessibility to the promoter is blocked. Bisulfite sequencing of PIV showed strong hypermethylation of PIV, providing a link between methylation, chromatin structure, and factor binding. Together, this analysis provides a kinetic view of the activation of the CIITA gene in response to IFN-γ and shows that regulatory factor assembly, chromatin modification, and gene expression proceed in discrete steps.

We acknowledge U. Nagarajan for help with flow cytometry, as well as members of the laboratory and M. Brown, P. Wade, and D. Reines for discussions about the results.

This work was supported by NIH grants AI34000 and HD34440. M. Mooney is supported by NIH fellowship GM20675.

A.C.M. and G.W.B. contributed equally to this study.