Architecture of Sp1 and κB Regulatory Elements within HIV-1 LTR.

<p>(A) Schematic representing the U3, R, and U5 regions of the HIV LTR. Several important transcriptional elements within the U3 region are shown, including the TATA box (−27/−23) and binding elements Sp1 (−55/−46, −66/−57, and −77/−68), κB (−90/−81 and −104/−95), LBP-1 (−16/+27), LEF-1 (−37/−51), NFAT-1 (−254/−216), and AP-1 (−247/−222). (B) Inactivating point mutations in the Sp1 and κB sites were engineered into the <i>LGIT</i> lentiviral plasmid. Mutation sites for κB <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000260#ppat.1000260-Chen1" target="_blank">[50]</a> and Sp1 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000260#ppat.1000260-Berkhout2" target="_blank">[48]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000260#ppat.1000260-Jones1" target="_blank">[49]</a> were previously described, and primer sequences are supplied in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000260#ppat.1000260.s007" target="_blank">Table S1</a>. Infections of <i>LGIT</i> and mutant lentivirus are detailed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000260#s4" target="_blank">Materials and Methods</a>. (C) A sample bifurcating clonal population of <i>LGIT</i>-infected Jurkats. Gene expression of GFP and Tat is amplified by Tat-transactivation, and the two modes of fluorescence (Off and Bright) correspond to the two states in this genetic circuit (Off and On). We hypothesize that transcriptional bimodality is regulated by repressing and activating complexes, which stabilize Off and Bright modes, respectively. These factors may include repressing histone deacetylase (HDACs, including HDAC1) complexes−recruited by p50-p50 homodimer (at κB sites) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000260#ppat.1000260-Williams1" target="_blank">[3]</a> and Sp1 protein (at Sp1 sites)−and activating histone acetyltransferases (HATs, including p300)−recruited in conjunction with p50-RelA heterodimer (κB sites) and Sp1 protein (at Sp1 sites). The largely unstable Mid region, which may result from stochastic fluctuations in Tat and switching between Off and Bright states, is regulated by dynamic interplay between repressing and activating complexes. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000260#ppat.1000260.s006" target="_blank">Figure S6</a> for further detail.</p>