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PCAF histone acetylase plays a role in regulation of transcription, cell cycle progression, and differentiation. Here, we show that PCAF is found in a complex consisting of more than 20 distinct polypeptides. Strikingly, some polypeptides are identical to TBP-associated factors (TAFs), which are subunits of TFIID. Like TFIID, histone fold-containing factors are present within the PCAF complex. The histone H3- and H2B-like subunits within the PCAF complex are identical to those within TFIID, namely, hTAF(II)31 and hTAF(II)20/15, respectively. The PCAF complex has a novel histone H4-like subunit with similarity to hTAF(II)80 that interacts with the histone H3-like domain of hTAF(II)31. Moreover, the PCAF complex has a novel subunit with WD40 repeats having a similarity to hTAF(II)100.

Histone acetylation is thought to have a role in transcription. To gain insight into the role of histone acetylation in retinoid-dependent transcription, we studied the effects of trichostatin A (TSA), a specific inhibitor of histone deacetylase, on P19 embryonal carcinoma cells. We show that coaddition of TSA and retinoic acid (RA) markedly enhances neuronal differentiation in these cells, although TSA alone does not induce differentiation but causes extensive apoptosis. Consistent with the cooperative effect of TSA and RA, coaddition of the two agents synergistically enhanced transcription from stably integrated RA-responsive promoters. The transcriptional synergy by TSA and RA required the RA-responsive element and a functional retinoid X receptor (RXR)/retinoic acid receptor (RAR) heterodimer, both obligatory for RA-dependent transcription. Furthermore, TSA led to promoter activation by an RXR-selective ligand that was otherwise inactive in transcription. In addition, TSA enhanced transcription from a minimum basal promoter, independently of the RA-responsive element. Finally, we show that TSA alone or in combination with RA increases in vivo endonuclease sensitivity within the RA-responsive promoter, suggesting that TSA treatment might alter a local chromatin environment to enhance RXR/RAR heterodimer action. Thus, these results indicate that histone acetylation influences activity of the heterodimer, which is in line with the observed interaction between the RXR/RAR heterodimer and a histone acetylase presented elsewhere.

Type I (alpha,beta) and type II (gamma) IFNs elicit antiproliferative and antiviral activities through two distinct transcription pathways involving 1) IRF family proteins and ISGF3, and 2) STAT1. We have employed a dominant negative strategy to study the role of IRF family proteins in eliciting the biologic activities of IFN. A truncated IRF protein retaining the DNA-binding domain (DBD) of ICSBP (a member of the IRF family) was stably transfected into U937 monocytic cells. Clones expressing DBD had markedly reduced ISRE-binding activity and were defective in expressing several type I IFN-inducible genes. STAT1 was one such type I IFN-inducible gene whose expression was also inhibited in DBD clones. As a result, the expression of several IFN-gamma-inducible genes was also inhibited in these clones, indicating functional coupling of the type I and type II IFN transcription pathways. Furthermore, DBD clones grew more slowly than control clones and were refractory to antiproliferative effects of both types of IFNs. We found that IFN treatment of U937 cells leads to a G1 arrest and an increase in underphosphorylated retinoblastoma gene product. However, IFN treatment did not change the cell cycle profile, nor retinoblastoma gene product phosphorylation state in DBD clones. These data indicate that expression of DBD disrupts cell cycle regulatory mechanisms. Combined with the previously noted failure of DBD clones to elicit antiviral activity, the present work shows that IRF family proteins play an integral part in growth control activities of IFNs.

p300/CBP is a transcriptional adaptor that integrates signals from many sequence-specific activators via direct interactions. Various cellular and viral factors target p300/CBP to modulate transcription and/or cell cycle progression. One such factor, the cellular p300/CBP associated factor (PCAF), possesses intrinsic histone acetyltransferase activity. Here, we demonstrate that p300/CBP is not only a transcriptional adaptor but also a histone acetyltransferase. p300/CBP represents a novel class of acetyltransferases in that it does not have the conserved motif found among various other acetyltransferases. p300/CBP acetylates all four core histones in nucleosomes. These observations suggest that p300/CBP acetylates nucleosomes in concert with PCAF.

The adenoviral oncoprotein E1A induces progression through the cell cycle by binding to the products of the p300/CBP and retinoblastoma gene families. A new cellular p300/CBP-associated factor (P/CAF) having intrinsic histone acetylase activity has been identified that competes with E1A. Exogenous expression of P/CAF in HeLa cells inhibits cell-cycle progression and counteracts the mitogenic activity of E1A. E1A disturbs the normal cellular interaction between p300/CBP and its associated histone acetylase.

Alu interspersed repetitive elements possess internal RNA polymerase III promoters that are transcribed in vitro and in transfected mouse cells but are nearly silent in human HeLa cells. Transcriptional repression of these elements is to some extent reversible, as pol III-dependent Alu expression can be induced with herpes simplex or adenovirus. To assess whether sequence-specific DNA binding proteins might contribute to Alu transcriptional silencing, we examined the internucleosomal spacer region surrounding the B box of the Alu pol III promoter in HeLa cell nuclei for evidence of proteins bound at specific sites in vivo. We identified a DNase I-hypersensitive site 5' to the B box and a DNase I-resistant region 3' to the B box in nuclei. An Alu-specific repressor binds to a 5-bp inverted repeat motif overlapping the 5' end of the TFIIIC binding site and may inhibit pol III transcription through competitive displacement. The level of Alu-specific pol III repressor activity is significantly reduced in adenovirus-infected HeLa cells, suggesting that the repressor may contribute to Alu transcriptional silencing in vivo. The 3' DNase I-resistant region coincided with a binding site for the pol II transcription factor YY1 in vitro. YY1 is one of the major proteins in HeLa cells having binding specificity for Alu elements. YY1 bound to tandem arrays of genomic Alu elements may play a role in chromatin organization and silencing.

When bacteriophage lambdapga18 DNA is transcribed in a purified in vitro system by E. coli RNA polymerase (nucleoside triphosphate: RNA nucleotidyl-transferase, EC 2.7.7.6), several major transcripts are synthesized. We have investigated transcriptional termination of one of these transcripts, the 4S, or "oop" RNA. Analysis by two-dimensional "fingerprinting" of T1 oligonucleotides reveals that transcription of the 4S RNA terminates at a specific site on the lambdapga18 DNA template, t-L with an efficiency of approximately 80%, i.e. 20% of transcripts are extended into larger RNAs. Addition of the E. coli protein rho to our transcription reactions has two effects: a) the efficiency of termination at the t-L site is increased to 100%; b) the number of 4S transcripts synthesized is increased by greater than 5-fold. Rho appears to stimulate 4S RNA synthesis by facilitating more rapid release of RNA polymerase from the t-L' termination site.