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

In vitro phosphorylation and acetylation of the murine pocket protein Rb2/p130.

The retinoblastoma protein (pRb) and the related proteins Rb2/p130 and 107 represent the "pocket protein" family of cell cycle regulators. A key function of these proteins is the cell cycle dependent modulation of E2F-regulated genes. The biological activity of these proteins is controlled by acetylation and phosphorylation in a cell cycle dependent manner. In this study we attempted to investigate the interdependence of acetylation and phosphorylation of Rb2/p130 in vitro. After having identified the acetyltransferase p300 among several acetyltransferases to be associated with Rb2/p130 during S-phase in NIH3T3 cells in vivo, we used this enzyme and the CDK4 protein kinase for in vitro modification of a variety of full length Rb2/p130 and truncated versions with mutations in the acetylatable lysine residues 1079, 128 and 130. Mutation of these residues results in the complete loss of Rb2/p130 acetylation. Replacement of lysines by arginines strongly inhibits phosphorylation of Rb2/p130 by CDK4; the inhibitory effect of replacement by glutamines is less pronounced. Preacetylation of Rb2/p130 strongly enhances CDK4-catalyzed phosphorylation, whereas deacetylation completely abolishes in vitro phosphorylation. In contrast, phosphorylation completely inhibits acetylation of Rb2/p130 by p300. These results suggest a mutual interdependence of modifications in a way that acetylation primes Rb2/p130 for phosphorylation and only dephosphorylated Rb2/p130 can be subject to acetylation. Human papillomavirus 16-E7 protein, which increases acetylation of Rb2/p130 by p300 strongly reduces phosphorylation of this protein by CDK4. This suggests that the balance between phosphorylation and acetylation of Rb2/p130 is essential for its biological function in cell cycle control

Regulation and Processing of Maize Histone Deacetylase Hda1 by Limited Proteolysis

A maize histone deacetylase gene was identified as a homolog of yeast Hda1. The predicted protein corresponds to a previously purified maize deacetylase that is active as a protein monomer with a molecular weight of 48,000 and is expressed in all tissues of germinating embryos. Hda1 is synthesized as an enzymatically inactive protein with an apparent molecular weight of 84,000 that is processed to the active 48-kD form by proteolytic removal of the C-terminal part, presumably via a 65-kD intermediate. The enzymatically inactive 84-kD protein also is part of a 300-kD protein complex of unknown function. The proteolytic cleavage of ZmHda1 is regulated during maize embryo germination in vivo. Expression of the recombinant full-length protein and the 48-kD form confirmed that only the smaller enzyme form is active as a histone deacetylase. In line with this finding, we show that the 48-kD protein is able to repress transcription efficiently in a reporter gene assay, whereas the full-length protein, including the C-terminal part, lacks full repression activity. This report on the processing of Hda1-p84 to enzymatically active Hda1-p48 demonstrates that proteolytic cleavage is a mechanism to regulate the function of Rpd3/Hda1-type histone deacetylases

Human papilloma virus 16-E7 protein reduces <i>in vitro</i> phosphorylation of Rb2/p130.

<p>Recombinant full length Rb2/p130 was used in CDK4 protein kinase assays in the presence and absence of HPV16-E7 and lysozyme, respectively. Samples were then subjected to SDS-PAGE with subsequent autoradiography. (<b>A</b>), Coomassie-Blue stained gel; (<b>B</b>), autoradiogram. Arrows mark the position of Rb2/p130, HPV16-E7 and lysozyme, respectively. M, positions of marker proteins indicated.</p

Expression of recombinant FLAG-tagged Rb2/p130 proteins using the baculovirus expression system.

<p>Schematic representation of the different full length and truncated versions of Rb2/p130. Mutations of acetylatable lysine residues that were mutated to arginine or glutamine are indicated. The approximate position of lysines 128, 130 and 1079 respectively, are marked by red lines. Molecular size of the truncated proteins is given in kD. C-terminus (CT); N-terminus (NT); pocket domain (P).</p

Effect of exchange of lysines 128, 130 and 1079 by arginines or glutamines on <i>in vitro</i> acetylation of Rb2/p130 by p300.

<p>(A) The different proteins were incubated with p300 and ¹⁴C-labelled acetylCoA; assay products were subjected to SDS-PAGE with subsequent autoradiography. Arrows indicate the position of Rb2/p130. The slower migrating band is p300. Histones served as a positive incorporation control. (B) Gels/autoradiograms of three independent experiments were analyzed by densitometry using Image-Quant software (Molecular Dynamics) for quantitation. Control (p130) is 100%.</p

<i>In vitro</i> phosphorylation of full length Rb2/p130 and truncated versions by CDK4.

<p>Recombinant full length Rb2/p130, the C-terminus (CT), the C-terminus + pocket domain (CT+P), the N-terminus (NT) and the N-terminus + pocket domain (NT+P) were subjected to CDK4 protein kinase assays using ³²P-labelled ATP. Samples were then subjected to SDS-PAGE with subsequent autoradiography. (<b>A</b>), Coomassie-Blue stained gel; (<b>B</b>), autoradiogram. Arrows in (<b>A</b>) mark the position of the recombinant Rb2/p130 proteins. Assay sample without ³²P-labelled ATP served as negative control. M, positions of marker proteins indicated.</p