We have used a reconstituted cell-free transcription system to investigate the molecular basis of mitotic repression of RNA polymerase I (pol I) transcription. We demonstrate that SL1, the TBP-containing promoter-binding factor, is inactivated by cdc2/cyclin B-directed phosphorylation, and reactivated by dephosphorylation. Transcriptional inactivation in vitro is accompanied by phosphorylation of two subunits, e.g. TBP and hTAF I 110. To distinguish whether transcriptional repression is due to phosphorylation of TBP, hTAF I 110 or both, SL1 was purified from two HeLa cell lines that express either full-length or the core domain of TBP only. Both TBP-TAF I complexes exhibit similar activity and both are repressed at mitosis, indicating that the variable N-terminal domain which contains multiple target sites for cdc2/cyclin B phosphorylation is dispensable for mitotic repression. Protein-protein interaction studies reveal that mitotic phosphorylation impairs the interaction of SL1 with UBF. The results suggest that phosphorylation of SL1 is used as a molecular switch to prevent pre-initiation complex formation and to shut down rDNA transcription at mitosis.

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Hypoxia-inducible factor-1K K (HIF-1K K) is a master regulator to sense decreased oxygen partial pressure. HIF-1K K stability regulation initiates a complex biological response that allows cells to act appropriately to meet patho-physiological situations of decreased oxygen availability. Recently, nitric oxide emerged as a messenger with the ability to stabilize HIF-1K K and to transactivate HIF-1 under normoxia. Considering that reactive nitrogen species are recognized for post-translation protein modi¢cations, among others S-nitrosation, we asked whether HIF-1K K is a target for S-nitrosation. In vitro NO+ donating NO donors such as GSNO and SNAP provoked massive S-nitrosation of puri¢ed HIF-1K K. All 15 free thiol groups found in human HIF-1K K are subjected to S-nitrosation. Thiol modi¢cation is not shared by spermine-NONOate, a NO radical donating compound. However, spermine-NONOate in the presence of O 3 3 2 , generated by xanthine/xanthine oxidase, regained S-nitrosation, most likely via formation of a N 2 O 3 -like species. In vitro, S-nitrosation of HIF-1K K was attenuated by the addition of GSH or ascorbate. In RCC4 and HEK293 cells GSNO or SNAP reproduced S-nitrosation of HIF-1K K, however with a signi¢cantly reduced potency that amounted to modi¢ca-tion of three to four thiols, only. Importantly, endogenous formation of NO in RCC4 cells via inducible NO synthase elicited S-nitrosation of HIF-1K K that was sensitive to inhibition of inducible NO synthase activity with N-monomethyl-L-arginine. NO-stabilized HIF-1K K was susceptible to the addition of N-acetyl-cysteine that destabilized HIF-1K K in close correlation to the disappearance of S-nitrosated HIF-1K K. In conclusion, HIF-1K K is a target for S-nitrosation by exogenously and endogenously produced NO. ß