Background: The rearrangement of nucleosomes along the DNA fiber profoundly affects gene expression, but little is known about how signalling reshapes the chromatin landscape, in three-dimensional space and over time, to allow establishment of new transcriptional programs. Results: Using micrococcal nuclease treatment and high-throughput sequencing, we map genome-wide changes in nucleosome positioning in primary human endothelial cells stimulated with tumour necrosis factor alpha (TNF alpha) - a proinflammatory cytokine that signals through nuclear factor kappa-B (NF-kappa B). Within 10 min, nucleosomes reposition at regions both proximal and distal to NF-kappa B binding sites, before the transcription factor quantitatively binds thereon. Similarly, in long TNF alpha-responsive genes, repositioning precedes transcription by pioneering elongating polymerases and appears to nucleate from intragenic enhancer clusters resembling super-enhancers. By 30 min, widespread repositioning throughout megabase pair-long chromosomal segments, with consequential effects on three-dimensional structure (detected using chromosome conformation capture), is seen. Conclusions: Whilst nucleosome repositioning is viewed as a local phenomenon, our results point to effects occurring over multiple scales. Here, we present data in support of a TNF alpha-induced priming mechanism, mostly independent of NF-kappa B binding and/or elongating RNA polymerases, leading to a plastic network of interactions that affects DNA accessibility over large domains.