Differential Regulation of NF-κB by Elongation Factors Is Determined by Core Promoter Type

Abstract

NF-B transcription factors activate genes important for immune response, inflammation, and cell survival. P-TEFb and DSIF, which are positive and negative transcription elongation factors, respectively, both regulate NF-B-induced transcription, but the mechanism underlying their recruitment to NF-B target genes is unknown. We show here that upon induction of NF-B, a subset of target genes is regulated differentially by either P-TEFb or DSIF. The regulation of these genes and their occupancy by these elongation factors are dependent on the NF-B enhancer and the core promoter type. Converting a TATA-less promoter to a TATA promoter switches the regulation of NF-B from DSIF to P-TEFb. Accumulation or displacement of DSIF and P-TEFb is dictated by the formation of distinct initiation complexes (TFIID dependent or independent) on the two types of core promoter. The underlying mechanism for the dissociation of DSIF from TATA promoters upon NF-B activation involves the phosphorylation of RNA polymerase II by P-TEFb. The results highlight a regulatory link between the initiation and the elongation phases of the transcription reaction and broaden our comprehension of the NF-B pathway.Transcription of protein-coding genes by RNA polymerase II (Pol II) is a multistep process, each step being a target for regulation and critical for the production of mature mRNA (27,29). A number of factors that control RNA Pol II elongation have been characterized in recent years. Among these are the positive elongation factor P-TEFb, which induces Pol II processivity by facilitating the transition from the early to the late elongation phase (24), and two negative elongation factors, DSIF (DRB sensitivity inducing factor) (31) and NELF (negative elongation factor) (37). In vitro, P-TEFb alleviates transcription inhibition by DSIF (25,32).NF-B is a transcription factor central to the cellular response to a broad range of extracellular signals, including inflammatory cytokines, tumor promoters, and chemotherapeutic agents. In response to these agents, NF-B induces the expression of cell cycle regulators, pro-and antiapoptotic factors, inflammatory cytokines, chemokines, adhesion molecules, and many other factors (22). In unstimulated cells, NF-B is retained in the cytoplasm by IB proteins. NF-B-activating signals trigger degradation of IB and nuclear translocation of NF-B, which result in activation of responsive genes (14). A subset of early response genes that includes IB␣ and A20 are themselves negative regulators of the NF-B pathway and so form a negative feedback loop. Transcriptional control of these genes is likely to influence the strength and the duration of the inflammatory signal.Induction of NF-B target genes is remarkably fast, and the mechanism underlying their rapid transcriptional activation was investigated previously. It was found that the promoters of NF-B-regulated genes are bound by the general transcription machinery prior to NF-B activation, and subsequent activation by NF-B increases the rate of the transcription cycl...

TAF4/4b·TAF12 Displays a Unique Mode of DNA Binding and Is Required for Core Promoter Function of a Subset of Genes

“…For each of these elements these frequencies are significantly higher (p ϭ 0.007 and 4.6EϪ10, respectively) than those in promoters in general (also with up to two mismatches) (28,29). Because genes driven by the Initiator are TAF-dependent (14,30,31), we can deduce that the core promoter features of the down-regulated genes are TAFdependent. These characteristics are specific to the down-regulated set, because enrichment of TATA and Initiator motifs was not found in the up-regulated genes.…”

Disparity between microRNA levels and promoter strength is associated with initiation rate and Pol II pausing

“…To investigate further the influence of transcription initiation rate directed by the A20 promoter on productive miRNA synthesis, we utilized a previously characterized TATA-box version of this promoter (A20-TATA) 19 . The A20-TATA is more active than the native A20 and the miR-22 promoters (Fig.…”

“…The promoters of miR-22, A20 WT, A20 TATA, miR-375 WT and miR-375 TATA mutant were previously described 19,24,48 . For generation of miR-22-TATA promoter mutant, a PCR fragment bearing the TATA-box Cotranscriptional miRNA processing pauses PolII, leading to a 'traffic-jam', which causes some of the following PolII molecules to abort.…”

“…THP-1 cells were maintained in RPMI-1640 and 10% fetal calf serum. Transfection into HEK293T cells was done using the CaPO 4 method 19 . For reporter gene assay, cells were harvested 24 h after transfection.…”

“…It is present in a fraction of PolII genes: between 20 and 46% in yeast (depending on the definition of the TATA-box sequence) 9,10 , B30% of Drosophila genes 11 and up to B20% of human genes [12][13][14][15] . The presence or absence of a TATA box in core promoters has been linked in yeast 9,10,[16][17][18] and humans 19 to two pathways of PIC assembly, one being TFIID-dependent (weak TATA or TATA-less) and the other TFIID-independent and SAGA dependent, suggesting that the mechanism of transcription initiation in TATA-less promoters is quite different from TATA promoters 20 .…”