Higher order chromatin structure in eukaryotes can lead to differential gene 
expression in response to the same transcription factor; however, how 
transcription factor inputs integrate with quantitative features of the 
chromatin environment to regulate gene expression is not clear. In vitro models 
of HIV gene regulation, in which repressive mechanisms acting locally at an 
integration site keep proviruses transcriptionally silent until appropriately 
stimulated, provide a powerful system to study gene expression regulation in 
different chromatin environments. Here we quantified HIV expression as a 
function of activating transcription factor nuclear factor-κB RelA/p65 (RelA) 
levels and chromatin features at a panel of viral integration sites. Variable 
RelA overexpression demonstrated that the viral genomic location sets a 
threshold RelA level necessary to induce gene expression. However, once the 
induction threshold is reached, gene expression increases similarly for all 
integration sites. Furthermore, we found that higher induction thresholds are 
associated with repressive histone marks and a decreased sensitivity to nuclease 
digestion at the LTR promoter. Increasing chromatin accessibility via inhibition 
of histone deacetylation or DNA methylation lowered the induction threshold, 
demonstrating that chromatin accessibility sets the level of RelA required to 
activate gene expression. Finally, a functional relationship between gene 
expression, RelA level, and chromatin accessibility accurately predicted 
synergistic HIV activation in response to combinatorial pharmacological 
perturbations. Different genomic environments thus set a threshold for 
transcription factor activation of a key viral promoter, which may point toward 
biological principles that underlie selective gene expression and inform 
strategies for combinatorial therapies to combat latent HIV.