PCAF is a histone acetyltransferase that associates with p300/CBP and competes with E1A for access to them. While exogenous expression of PCAF potentiates both MyoD-directed transcription and myogenic differentiation, PCAF inactivation by anti-PCAF antibody microinjection prevents differentiation. MyoD interacts directly with both p300/CBP and PCAF, forming a multimeric protein complex on the promoter elements. Viral transforming factors that interfere with muscle differentiation disrupt this complex without affecting the MyoD-DNA interaction, indicating functional significance of the complex formation. Exogenous expression of PCAF or p300 promotes p21 expression and terminal cell-cycle arrest. Both of these activities are dependent on the histone acetyltransferase activity of PCAF, but not on that of p300. These results indicate that recruitment of histone acetyltransferase activity of PCAF by MyoD, through p300/CBP, is crucial for activation of the myogenic program.

Yu et al., 1992; reviewed by Olson et al., 1995). As Studi di Roma La Sapienza, 00161 Rome, Italy, 4 DCBOC/Path, regards MyoD, a dual role during myogenesis has been

Inhibition of herpes simplex virus (HSV) thymidine kinase (TK) gene transcription (pHSV-106, pML-BPV-TK4) by DNA methylation is an indirect effect, which occurs with a latency period of -8 hr after microinjection of the DNA into TK-rat 2 and mouse LTK-cells. We have strong evidence that chromatin formation is critical for the transition of the injected DNA from methylation insensitivity to methylation sensitivity. Chromatin was reconstituted in vitro by using methylated and mock-methylated HSV TK DNA and purified chicken histone octamers. After microinjection, the methylated chromatin was always biologically inactive, as tested by autoradiography of the cells after incubation with [3H] Preparation of Histone Octamers and Chromatin Reconstitution. Chicken nuclei were isolated as described (16,17).Nuclei (3 x 1010) suspended in 0.25 M sucrose solution were diluted with an equal volume of 0.25 M HCl/0.2 M NaCl solution. After 30 min on ice, the suspension was centrifuged at 10,000 x g for 20 min and the supernatant was dialyzed twice against water and then freeze-dried. Core histones were isolated by treatment of the freeze-dried material with 5% perchloric acid (0.1 ml/mg) and centrifugation at 10,000 x g for 15 min. The pellet was reextracted in 2 ml of 0.25 M HCl and dialyzed twice against 0.25 M HCl and three times against water. The freeze-dried core histones (free of histones H1 and H5) can be stored at -20°C.To reconstitute histone octamer (17), the freeze-dried core histones were suspended in 8 M urea/1% (vol/vol) 2-mercaptoethanol to a final concentration of 4 mg/ml. After incubation for 2 hr at room temperature, the suspension was dialyzed against 2 M NaCl/1% 2-mercaptoethanol/10 mM Tris HCl, pH 7.4. The probe was then applied to a column (Sephadex 6B; 1 m x 2.5 cm), and eluted with 2 M NaCl/0.1 mM phenylmethylsulfonyl fluoride/10 mM Tris'HCl, pH 7.4, and the fractions containing the octamers were collected by measuring the absorbance at 280 nm. Final concentration was -0.1 mg/ml.For chromatin reconstitution, 10 ,g of DNA was mixed with 10 ,ug of reconstituted octamers in a final volume of 100 Al and dialyzed twice against 100 mM KCl/20 mM NaCl/0.25 mM EDTA/10 mM 2-mercaptoethanol/0.1 mM phenylmethylsulfonyl fluoride/10 mM Tris HCl, pH 7.4, for 48 hr. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

p300 is a nuclear phosphoprotein likely to be involved in the control of cell growth. Here we show that SV40 large T antigen (Tag) forms a specific complex with p300. In various Tag‐expressing cell lines, the affinity of Tag for p300 was restricted to a newly identified unphosphorylated but ubiquitinated form of the protein. Further, Tag did not associate with p300 in an SV40 Tag‐producing cell line (REV2) in which the original transformed phenotype (SV52) is reverted. Biochemical studies demonstrate that both the phosphorylation and the ubiquitination profile of p300 are altered in REV2 with respect to the wild‐type fully transformed SV52 parental cells, wherein Tag‐p300 complexes are readily detected. In contrast to Tag, the adenovirus early expression product E1a interacts with both phosphorylated and unphosphorylated forms of p300. In addition, when REV2 cells were infected with adenovirus, E1a‐p300 complexes were detected, suggesting that the p300 expressed in REV2 has lost the affinity for Tag, but not for E1a. We then compared the ability of Tag and E1a to affect the transcription levels of the cAMP‐responsive promoter (CRE), which is modulated in vivo by p300, in REV2 cells. We found that Tag repressed the CRE promoter in all of the cell lines in which Tag‐p300 complexes were detected, but not in REV2 cells. In contrast, E1a efficiently inhibited CRE‐directed transcription in this cell line. The data thus indicate that the different specificities exhibited by Tag and E1a towards the various forms of p300 are reflected in vivo as a difference in the ability of these viral oncoproteins to modulate the expression of CRE‐containing genes.

The present work shows that histones are able to directly cross cell plasma membranes and mediate penetration of macromolecules covalently attached to them. Adding a mixture containing the five nucleosomal histones, H1, H2A, H2B, H3 and H4, as well as each of the last four individual histones to intact HeLa and Colo-205 cultured cells resulted in cell penetration and nuclear import of these externally added histones. This was observed by fluorescent and confocal microscopy using fixed and unfixed cells, showing that penetration was not due to the fixation process.