Summary
The signaling module that specifies nuclear factor-κB (NF-κB) activation is a three-component system: NF-κB, inhibitor of NFκB (IκB), and IκB kinase complex (IKK). IKK receives upstream signals from the surface or inside the cell and converts itself into a catalytically active form leading to the destruction of IκB in the inhibited IκB: NF-κB complex, leaving active NF-κB free to regulate target genes. Hidden within this simple module are family members that all can undergo various modifications resulting in expansion of functional spectrum. Three-dimensional structures representing all three components are now available. These structures have allowed us to interpret cellular observations in molecular terms and at the same time helped us to bring forward new concepts focused towards understanding the specificity in the NF-κB activation pathway.
The transcription cycle of RNA polymerase II (Pol II) is regulated at discrete transition points by cyclin-dependent kinases (CDKs). Positive transcription elongation factor b (P-TEFb), a complex of Cdk9 and cyclin T1, promotes release of paused Pol II into elongation, but the precise mechanisms and targets of Cdk9 action remain largely unknown. Here, by a chemical genetic strategy, we identified ∼100 putative substrates of human P-TEFb, which were enriched for proteins implicated in transcription and RNA catabolism. Among the RNA processing factors phosphorylated by Cdk9 was the 5 ′ -to-3 ′ "torpedo" exoribonuclease Xrn2, required in transcription termination by Pol II, which we validated as a bona fide P-TEFb substrate in vivo and in vitro. Phosphorylation by Cdk9 or phosphomimetic substitution of its target residue, Thr439, enhanced enzymatic activity of Xrn2 on synthetic substrates in vitro. Conversely, inhibition or depletion of Cdk9 or mutation of Xrn2-Thr439 to a nonphosphorylatable Ala residue caused phenotypes consistent with inefficient termination in human cells: impaired Xrn2 chromatin localization and increased readthrough transcription of endogenous genes. Therefore, in addition to its role in elongation, P-TEFb regulates termination by promoting chromatin recruitment and activation of a cotranscriptional RNA processing enzyme, Xrn2.
SUMMARY
Nuclear factor κB (NF-κB) regulates gene expression by binding to specific DNA elements, known collectively as κB sites, that are contained within the promoters/enhancers of target genes. We found that the identity of the central base pair (bp) of κB sites profoundly affects the transcriptional activity of NF-κB dimers. RelA dimers prefer an A/T bp at this position for optimal transcriptional activation (A/T-centric) and discriminate against G/C-centric κB sites. The p52 homodimer, in contrast, activates transcription from G/C-centric κB sites in complex with Bcl3 but represses transcription from the A/T-centric sites. The p52:Bcl3 complex binds to these two classes of κB sites in distinct modes, permitting the recruitment of coactivator, corepressor, or both coactivator and corepressor complexes in promoters that contain G/C-, A/T-, or both G/C- and A/T-centric sites. Therefore, through sensing of bp differences within κB sites, NF-κB dimers modulate biological programs by activating, repressing, and altering the expression of effector genes.