Renate Voit

Acetylation of TAFI68, a subunit of TIF-IB/SL1, activates RNA polymerase I transcription

Mammalian rRNA genes are preceded by a terminator element that is recognized by the transcription termination factor TTF‐I. In exploring the functional significance of the promoter‐proximal terminator, we found that TTF‐I associates with the p300/CBP‐associated factor PCAF, suggesting that TTF‐I may target histone acetyltransferase to the rDNA promoter. We demonstrate that PCAF acetylates TAFI68, the second largest subunit of the TATA box‐binding protein (TBP)‐containing factor TIF‐IB/SL1, and acetylation enhances binding of TAFI68 to the rDNA promoter. Moreover, PCAF stimulates RNA polymerase I (Pol I) transcription in a reconstituted in vitro system. Consistent with acetylation of TIF‐IB/SL1 being required for rDNA transcription, the NAD+‐dependent histone deacetylase mSir2a deacetyl ates TAFI68 and represses Pol I transcription. The results demonstrate that acetylation of the basal Pol I transcription machinery has functional consequences and suggest that reversible acetylation of TIF‐IB/SL1 may be an effective means to regulate rDNA transcription in response to external signals.

Phosphorylation by G1‐specific cdk–cyclin complexes activates the nucleolar transcription factor UBF

Transcription of rRNA genes by RNA polymerase I increases following serum stimulation of quiescent NIH 3T3 fibroblasts. To elucidate the mechanism underlying transcriptional activation during progression through the G1 phase of the cell cycle, we have analyzed the activity and phosphorylation pattern of the nucleolar transcription factor upstream binding factor (UBF). Using a combination of tryptic phosphopeptide mapping and site‐directed mutagenesis, we have identified Ser484 as a direct target for cyclin‐dependent kinase 4 (cdk4)–cyclin D1‐ and cdk2–cyclin E‐directed phosphorylation. Mutation of Ser484 impairs rDNA transcription in vivo and in vitro. The data demonstrate that UBF is regulated in a cell cycle‐dependent manner and suggest a link between G1 cdks–cyclins, UBF phosphorylation and rDNA transcription activation.

The nucleolar transcription factor mUBF is phosphorylated by casein kinase II in the C-terminal hyperacidic tail which is essential for transactivation.

UBF is a DNA binding protein which interacts with both the promoter and the enhancer of various vertebrate ribosomal RNA genes and functions as a transcription initiation factor for RNA polymerase I (pol I). We have purified murine UBF to apparent molecular homogeneity and demonstrate that its transactivating potential, but not its DNA binding activity, is modulated in response to cell growth. In vivo labelling experiments demonstrate that UBF is a phosphoprotein and that the phosphorylation state is different in growing and quiescent cells. We show that UBF is phosphorylated in vitro by a cellular protein kinase which by several criteria closely resembles casein kinase II (CKII). A major modification involves serine phosphoesterifications in the carboxy terminal hyperacidic tail of UBF. Deletions of this C‐terminal domain severely decreases the UBF directed activation of transcription. The data suggest that phosphorylation of UBF by CKII may play an important role in growth dependent control of rRNA synthesis.

Mechanism of Repression of RNA Polymerase I Transcription by the Retinoblastoma Protein

The retinoblastoma susceptibility gene product pRb restricts cellular proliferation by affecting gene expression by all three classes of nuclear RNA polymerases. To elucidate the molecular mechanisms underlying pRb-mediated repression of ribosomal DNA (rDNA) transcription by RNA polymerase I, we have analyzed the effect of pRb in a reconstituted transcription system. We demonstrate that pRb, but not the related protein p107, acts as a transcriptional repressor by interfering with the assembly of transcription initiation complexes. The HMG box-containing transcription factor UBF is the main target for pRb-induced transcriptional repression. UBF and pRb form in vitro complexes involving the C-terminal part of pRb and HMG boxes 1 and 2 of UBF. We show that the interactions between UBF and TIF-IB and between UBF and RNA polymerase I, respectively, are not perturbed by pRb. However, the DNA binding activity of UBF to both synthetic cruciform DNA and the rDNA promoter is severely impaired in the presence of pRb. These studies reveal another mechanism by which pRb suppresses cell proliferation, namely, by direct inhibition of cellular rRNA synthesis.

Mitotic silencing of human rRNA synthesis: inactivation of the promoter selectivity factor SL1 by cdc2/cyclin B-mediated phosphorylation

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 hTAFI110. To distinguish whether transcriptional repression is due to phosphorylation of TBP, hTAFI110 or both, SL1 was purified from two HeLa cell lines that express either full‐length or the core domain of TBP only. Both TBP‐TAFI 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.

The chromatin remodelling complex WSTF–SNF2h interacts with nuclear myosin 1 and has a role in RNA polymerase I transcription

Nuclear actin and myosin 1 (NM1) are key regulators of gene transcription. Here, we show by biochemical fractionation of nuclear extracts, protein–protein interaction studies and chromatin immunoprecipitation assays that NM1 is part of a multiprotein complex that contains WICH, a chromatin remodelling complex containing WSTF (Williams syndrome transcription factor) and SNF2h. NM1, WSTF and SNF2h were found to be associated with RNA polymerase I (Pol I) and ribosomal RNA genes (rDNA). RNA interference‐mediated knockdown of NM1 and WSTF reduced pre‐rRNA synthesis in vivo, and antibodies to WSTF inhibited Pol I transcription on pre‐assembled chromatin templates but not on naked DNA. The results indicate that NM1 cooperates with WICH to facilitate transcription on chromatin.

AMP-activated protein kinase adapts rRNA synthesis to cellular energy supply

AMP-activated protein kinase (AMPK) senses changes in the intracellular AMP/ATP ratio, switching off energy-consuming processes and switching on catabolic pathways in response to energy depletion. Here, we show that AMPK down-regulates rRNA synthesis under glucose restriction by phosphorylating the RNA polymerase I (Pol I)-associated transcription factor TIF-IA at a single serine residue (Ser-635). Phosphorylation by AMPK impairs the interaction of TIF-IA with the TBP-containing promoter selectivity factor SL1, thereby precluding the assembly of functional transcription initiation complexes. Mutation of Ser-635 compromises down-regulation of Pol I transcription in response to low energy supply, supporting that activation of AMPK adapts rRNA synthesis to nutrient availability and the cellular energy status.

Phosphorylation of UBF at serine 388 is required for interaction with RNA polymerase I and activation of rDNA transcription

Modulation of the activity of the upstream binding factor (UBF) plays a key role in cell cycle-dependent regulation of rRNA synthesis. Activation of rDNA transcription on serum stimulation requires phosphorylation of UBF at serine 484 by G1-specific cyclin-dependent kinase (cdk)/cyclin complexes. After G1 progression UBF is phosphorylated at serine 388 by cdk2/cyclin E and cdk2/cyclin A. Conversion of serine 388 to glycine abolishes UBF activity, whereas substitution by aspartate enhances the transactivating function of UBF. Protein–protein interaction studies reveal that phosphorylation at serine 388 is required for the interaction between RNA polymerase I and UBF. The results suggest that phosphorylation of UBF represents a powerful means of modulating the assembly of the transcription initiation complex in a proliferation- and cell cycle-dependent fashion.

Identification of novel functional TBP-binding sites and general factor repertoires.

Our current knowledge of the general factor requirement in transcription by the three mammalian RNA polymerases is based on a small number of model promoters. Here, we present a comprehensive chromatin immunoprecipitation (ChIP)‐on‐chip analysis for 28 transcription factors on a large set of known and novel TATA‐binding protein (TBP)‐binding sites experimentally identified via ChIP cloning. A large fraction of identified TBP‐binding sites is located in introns or lacks a gene/mRNA annotation and is found to direct transcription. Integrated analysis of the ChIP‐on‐chip data and functional studies revealed that TAF12 hitherto regarded as RNA polymerase II (RNAP II)‐specific was found to be also involved in RNAP I transcription. Distinct profiles for general transcription factors and TAF‐containing complexes were uncovered for RNAP II promoters located in CpG and non‐CpG islands suggesting distinct transcription initiation pathways. Our study broadens the spectrum of general transcription factor function and uncovers a plethora of novel, functional TBP‐binding sites in the human genome.

The RNA polymerase I-specific transcription initiation factor UBF is associated with transcriptionally active and inactive ribosomal genes

We have characterized an anti-NOR (nucleolar organizer region) serum (P419) from a patient with rheumatoid arthritis and show that it contains antibodies directed against the RNA polymerase I-specific transcription initiation factor UBF. This serum reacts with UBF from a variety of vertebrate cells as revealed both by immunoblotting and by indirect immunofluorescence. We have used the P419 serum to study the intracellular localization of this transcription factor at the light and electron microscopic level. In interphase cells, UBF exhibits a pronounced punctate pattern and is found to be associated with necklace-like structures, which appear to reflect the transcriptionally active state of the nucleolus. Inhibition of rRNA synthetic activity caused either by nutritional starvation or by actinomycin D treatment resulted in a marked decrease in the number and in a significant increase in the size of UBF-positive granules. Under all experimental conditions applied, UBF was exclusively found within the nucleolus and was not released into the nucleoplasm or cytoplasm. During mitosis, UBF was found to be concentrated at the chromosomal NOR indicating that a significant quantity, if not all, of this factor remains bound to the ribosomal transcription units. From this we conclude that UBF is associated both with transcriptionally active and inactive rRNA genes and, therefore, changes in the intracellular localization of UBF are very likely not involved in rDNA transcription regulation.