The MHC class II transactivator Ciita has a highly restricted transcriptional footprint in murine classical dendritic cells

Ciita has been suggested to control the expression of a number of genes based on ChIP-Seq or reporter anaysis but in vivo regulation beyong MHC class II has largely not been confirmed. We crossed Ciita knock out mice to Zbtb46 GFP knock-in knock out mice to identify classical dendritic cells in vivo in a Ciita deficient background. Expression microarray analysis of CD24+ and CD172a+ DCs from homeostatic spleen or activated BM Flt3l cultures show a highly resitrcited transcriptional footprint of Ciita. These findings underscore the fact that canonical binding sites active in transient expression assays may not always be functional in vivo. In addition, ChIP-Seq alone is not sufficient to discriminate binding from control of expression of a nearby gene. CD24+ and CD172a+ DCs were sorted from the spleens Ciita -/- Zbtb46 GFP/+ and Ciita +/+ Zbtb46 GFP/+ mice. Alternatively, CD24+ and CD172a+ DCs were sorted from the BM cultures of Ciita -/- Zbtb46 GFP/+ and Ciita +/+ Zbtb46 GFP/+ mice after 9 days in Flt3l followed by activation with gamma interferon for 16 hours and LPS for 4 hours. Total RNA was extracted from cells and used for hybridization on Affymetrix Mouse Gene 1.0 ST microarrays.