Acetylation of UBF changes during the cell cycle and regulates the interaction of UBF with RNA polymerase I

The upstream binding factor UBF, an activator of RNA polymerase I transcription, is posttranslationally modified by phosphorylation and acetylation. We found that in NIH3T3 cells, UBF is acetylated in S-phase but not in G(1)-phase. To assess the role of acetylation in regulation of UBF activity, we have established an NIH3T3 cell line that inducibly overexpresses HDAC1. Both in vivo and in vitro, HDAC1 efficiently hypoacetylates UBF. Immunoprecipitation with antibodies against the Pol I-associated factor PAF53 co-precipitated UBF in mock cells but not in cells overexpressing HDAC1. Pull-down experiments showed that acetylation of UBF augments the interaction with Pol I. Consistent with acetylation of UBF being important for association of PAF53 and recruitment of Pol I, the level of Pol I associated with rDNA and pre-rRNA synthesis were reduced in cells overexpressing HDAC1. The results suggest that acetylation and deacetylation of UBF regulate rRNA synthesis during cell cycle progression

Acetylation of TAF(I)68, 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 TAF(I)68, the second largest subunit of the TATA box-binding protein (TBP)-containing factor TIF-IB/SL1, and acetylation enhances binding of TAF(I)68 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 TAF(I)68 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

SIRT7 and the DEAD-box helicase DDX21 cooperate to resolve genomic R loops and safeguard genome stability

R loops are three-stranded nucleic acid structures consisting of an RNA:DNA heteroduplex and a “looped-out” nontemplate strand. As aberrant formation and persistence of R loops block transcription elongation and cause DNA damage, mechanisms that resolve R loops are essential for genome stability. Here we show that the DEAD (Asp–Glu–Ala–Asp)-box RNA helicase DDX21 efficiently unwinds R loops and that depletion of DDX21 leads to accumulation of cellular R loops and DNA damage. Significantly, the activity of DDX21 is regulated by acetylation. Acetylation by CBP inhibits DDX21 activity, while deacetylation by SIRT7 augments helicase activity and overcomes R-loop-mediated stalling of RNA polymerases. Knockdown of SIRT7 leads to the same phenotype as depletion of DDX21 (i.e., increased formation of R loops and DNA double-strand breaks), indicating that SIRT7 and DDX21 cooperate to prevent R-loop accumulation, thus safeguarding genome integrity. Moreover, DDX21 resolves estrogen-induced R loops on estrogen-responsive genes in breast cancer cells, which prevents the blocking of transcription elongation on these genes.</jats:p

Cooperative Action of Cdk1/cyclin B and SIRT1 Is Required for Mitotic Repression of rRNA Synthesis

<div><p>Mitotic repression of rRNA synthesis requires inactivation of the RNA polymerase I (Pol I)-specific transcription factor SL1 by Cdk1/cyclin B-dependent phosphorylation of TAF_I110 (TBP-associated factor 110) at a single threonine residue (T852). Upon exit from mitosis, T852 is dephosphorylated by Cdc14B, which is sequestered in nucleoli during interphase and is activated upon release from nucleoli at prometaphase. Mitotic repression of Pol I transcription correlates with transient nucleolar enrichment of the NAD⁺-dependent deacetylase SIRT1, which deacetylates another subunit of SL1, TAF_I68. Hypoacetylation of TAF_I68 destabilizes SL1 binding to the rDNA promoter, thereby impairing transcription complex assembly. Inhibition of SIRT1 activity alleviates mitotic repression of Pol I transcription if phosphorylation of TAF_I110 is prevented. The results demonstrate that reversible phosphorylation of TAF_I110 and acetylation of TAF_I68 are key modifications that regulate SL1 activity and mediate fluctuations of pre-rRNA synthesis during cell cycle progression.</p></div

Human phosphatase CDC14A is recruited to the cell leading edge to regulate cell migration and adhesion

Cell adhesion and migration are highly dynamic biological processes that play important roles in organ development and cancer metastasis. Their tight regulation by small GTPases and protein phosphorylation make interrogation of these key processes of great importance. We now show that the conserved dual-specificity phosphatase human cell-division cycle 14A (hCDC14A) associates with the actin cytoskeleton of human cells. To understand hCDC14A function at this location, we manipulated native loci to ablate hCDC14A phosphatase activity (hCDC14A(PD)) in untransformed hTERT-RPE1 and colorectal cancer (HCT116) cell lines and expressed the phosphatase in HeLa FRT T-Rex cells. Ectopic expression of hCDC14A induced stress fiber formation, whereas stress fibers were diminished in hCDC14A(PD) cells. hCDC14A(PD) cells displayed faster cell migration and less adhesion than wild-type controls. hCDC14A colocalized with the hCDC14A substrate kidney- and brain-expressed protein (KIBRA) at the cell leading edge and overexpression of KIBRA was able to reverse the phenotypes of hCDC14A(PD) cells. Finally, we show that ablation of hCDC14A activity increased the aggressive nature of cells in an in vitro tumor formation assay. Consistently, hCDC14A is down-regulated in many tumor tissues and reduced hCDC14A expression is correlated with poorer survival of patients with cancer, to suggest that hCDC14A may directly contribute to the metastatic potential of tumors. Thus, we have uncovered an unanticipated role for hCDC14A in cell migration and adhesion that is clearly distinct from the mitotic and cytokinesis functions of Cdc14/Flp1 in budding and fission yeast

Reactivation of mitotic SL1 by Cdc14B.

<p>(A) Transcriptional activity of mitotic SL1 is increased by hCdc14B. SL1 was immunopurified from asynchronous (as) or nocodazole-arrested (M) HeLa cells and transcriptional activity was assayed in a reconstituted SL1-responsive transcription system in the presence or absence of GST-hCdc14B. The numbers below indicate the relative amount of run-off transcripts quantified by PhosphorImaging (rel. tx.). The Western blot below shows that comparable amounts of SL1 from asynchronous and mitotic cells were used in the transcription assays. (B) Mutation of T852 does not impair formation of SL1 complexes. Flag-hTAF_I110/WT or Flag-hTAF_I110/T852 was immunoprecipitated from Hela cells stably expressing wildtype or mutant TAF_I110 and co-precipitated hTAF_I68 and TBP were analyzed on immunoblots with anti-TAF_I68 and anti-TBP antibodies. The input lanes contain 10–30 μg of nuclear lysate. (C) Mutation of T852 does not affect SL1 binding to the rDNA promoter. ChIP of Flag-tagged TAF_I110 and UBF from HeLa cells expressing wildtype Flag-hTAF_I110 (light blue bars) or mutant Flag-hTAF_I110/T852 (dark blue bars). Precipitated DNA was analyzed by qPCR using primers that amplify the rDNA promoter. Bars denote the mean value (± SD) from two biological replicates. (D) Mutation of T852 does not prevent loss of SL1 from the rDNA promoter during mitosis. Left panel: ChIP of UBF, SL1 (TBP, TAF_I110, TAF_I68), and Pol I (RPA116) from G₁/S phase-arrested (light blue bars) or M-phase (red bars) HeLa cells expressing Flag-tagged hTAF_I110/WT. Right panel: ChIPs from G₁/S- and M-phase HeLa cells expressing Flag-tagged hTAF_I110/T852A (dark blue and red bars). Precipitated DNA was analyzed by qPCR using primers that amplify the rDNA promoter. Bars denote the mean value (± SD) from two biological replicates. (E) Nucleolar transcription is repressed in mitotic cells regardless of whether they express wildtype (WT) or mutant hTAF_I110 (T852A). Cells were labeled for 20 min with FUrd and nucleolar transcripts were visualized with antibodies against BrdU. NORs were immunostained with anti-UBF antibodies. Mitotic cells are encircled. Representative images out of 200 analyzed cells are shown. Scale bar, 10 μm. For quantification of the fluorescence signals, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005246#pgen.1005246.s002" target="_blank">S2B Fig</a>.</p