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, 2014). It is therefore likely that the other factors we have investigated, including nuclear receptors, can also bind directly to some hotspots and be involved in facilitating recruitment of additional factors through alternative mechanisms [http://www.selleckchem.com/products/tariquidar.html Tariquidar] as described here for C/EBPs, AP1, and KLFs, even though we do not identify a footprint for these factors in this study. The basis for the varying efficiency of footprint formation by different factors is unclear but may relate to the molecular details of how factors bind to DNA or the dynamics of the protein-DNA interactions. In favor of the latter is the finding that nuclear receptors have a very short residence time on DNA (McNally et?al., 2000?and?Voss et?al., 2011) and a poor ability to make footprints (M.-H.S., M. Guertin, S.B., and G.L.H., unpublished data; this study; He et?al., 2014). Surprisingly, we find that alternative binding events for C/EBP�� appear to be functionally as important as direct binding to its predicted sites for the formation of hotspots, recruitment of coactivators, and gene activation, even though alternative binding events are associated with significantly [http://en.wikipedia.org/wiki/PRDX5 PRDX5] lower ChIP-seq signal than footprints at predicted sites. Intriguingly, the finding that C/EBP�� footprints and alternative binding of C/EBP�� are associated with distinct gene programs suggests that it may be possible to design drugs to target specific gene programs by specifically targeting one, but not the other, of these binding mechanisms of a particular transcription factor. In conclusion, we present molecular insight into how transcription factors are organized and function at shared target regions, thereby providing a framework for understanding transcription factor cooperativity on chromatin. 3T3-L1 cells were grown and induced to differentiate as described in Siersb?k et?al. (2014). Knockdown of C/EBP�� was done using the pSicoR PGK puro (Addgene; 12084) system as described in Siersb?k et?al. (2014). ChIP was performed as described (Siersb?k et?al., 2011) using antibodies against C/EBP�� (C-19, sc-150; Santa Cruz Biotechnology), VDR (C-20, sc-1008; Santa Cruz), and STAT5A (L-20, sc-1081; Santa Cruz). Purified DNA was [http://www.selleckchem.com/autophagy.html Autophagy pathway inhibitor] analyzed by quantitative PCR (qPCR). Cells were harvested in Isol-RNA lysis reagent (5 PRIME) and purified according to the manufacturer. Purified RNA was then prepared for sequencing (Illumina) according to the instructions of the manufacturer. Sequence reads were mapped to the mouse genome (mm9) as described in Siersb?k et?al. (2014). Mapped reads in exons were counted using HOMER (Heinz et?al., 2010), and differentially expressed genes (adjusted p?