Oct-1 cooperates with the TATA binding initiation complex to control rapid transcription of human iNOS

Abstract

Expression of the human inducible nitric oxide synthase (hiNOS) is generally undetectable in resting cells, but stimulation by a variety of signals including cytokines induces transcription in most cell types. The tight transcriptional regulation of the enzyme is a complex mechanism many aspects of which remain unknown. Here, we describe an octamer (Oct) element in hiNOS proximal promoter, located close to the TATA box. This site constitutively binds Oct-1 and its deletion abrogates cytokine-induced transcription, showing that it is indispensable though not sufficient for transcription. Increasing the distance between Oct and the TATA box by inserting inert DNA sequence inhibits transcription, and footprinting of this region shows no other protein binding in resting cells, suggesting an interaction between the two complexes. Chromatin immunoprecipitation assays detect the presence of Oct-1, RNA polymerase II and trimethyl K4 histone H3 on the proximal promoter in resting cells, confirming that the gene is primed for transcription before stimulation. RT-PCR of various fragments along the hiNOS gene shows that transcription is initiated in resting cells and this is inhibited by interference with Oct-1 binding to the proximal site of the promoter. We propose that, through interaction with the initiation complex, Oct-1 regulates hiNOS transcription by priming the gene for the rapid response required in an immune response.

Leishmania (Leishmania) amazonensis induces macrophage miR-294 and miR-721 expression and modulates infection by targeting NOS2 and L-arginine metabolism

“…The miR-29 was upregulated mediated by NOD2-stimulation, that downregulates IL-12p40, but not alter IL-6, TGF-β or IL-10 levels51. However, NOD2-stimulation amplifying the release of IL-1β, IL-6, and IL-234950 and induces NOS2 expression and NO production4142. Among the dysregulated miRNAs, miR-294-3p and miR-721 drew our attention, as these miRNAs were less regulated in La - arg − -infected macrophages and were predicted as candidates for regulating the expression of proteins involved in the L-arginine pathway.…”

“…Nos2 expression can be regulated at the transcriptional level by factors such as NF- k B48, Oct-149 and STAT50. Indeed, L. major and L. amazonensis infections increase the nuclear translocation of the NF-kB p50/p50 dimer, which correlate with the decrease of Nos2 and Cat2B transcription levels12.…”

The Oct1 transcription factor and epithelial malignancies: Old protein learns new tricks

“…In breast cancer cell lines, Oct1 regulates rapid induction of Nos2 ( iNOS ) through a binding site that must be positioned close to the TATA box in the proximal promoter. Prior to induction, this gene has an engaged Pol II, indicating that Oct1 likely regulates this gene at the level of transcript elongation [62]. Oct1 activation of Nos2 can be inhibited by expression of, and association with, Oct2 [28].…”

Tailoring the Models of Transcription

“…The primers contain the flanking sequence on either side of the binding element, so that it is excluded during amplification (Figure 2b), or a few nucleotide changes that will prevent the transcription factor from binding (Figure 2c). Mutagenesis led to the identification of crucial transcription factors for the activation of iNOS transcription in response to a variety of stimuli, including NFκB [23], AP-1 [28], STAT-1 [24] and Oct-1 [29,30]. The responsiveness of specific transcription sites to signalling pathways can also be examined by genetic manipulation.…”

“…We confirmed these observations in the context of iNOS transcription, by inserting small inert DNA sequences into a luciferase reporter of the iNOS promoter, to increase the separation between the TATA box and the Oct site by half a turn of DNA (5 bp) at a time (Figure 2d). Any increase disrupted transcriptional activity, indicating that the proximity between TBP and Oct-1 is important for PIC formation [30]. We went on to suggest that this mechanism allows recruitment of Pol II and initiation of transcription, so that the rate limiting-step is elongation of the transcripts rather than recruitment of Pol II [3].…”