In contrast to its behavior as naked DNA, the MMTV promoter assembled in minichromosomes can be activated synergistically by the progesterone receptor and NF1 in a process involving ATP-dependent chromatin remodeling. The DNA-binding domain of NF1 is required and sufficient for stable occupancy of all receptor-binding sites and for functional synergism. Activation of purified minichromosomes is observed in the absence of SWI/SNF and can be enhanced by recombinant ISWI. Receptor binding to minichromosomes recruits ISWI and NURF38, but not brahma. We propose a two-step synergism in which the receptor triggers a chromatin remodeling event that facilitates access of NF1, which in turn stabilizes an open nucleosomal conformation required for efficient binding of further receptor molecules and full transactivation.

Sp3 is a ubiquitous transcription factor closely related to Sp1. Previous analyses showed that, unlike Sp1, Sp3 fails to activate transcription in certain promoter settings. This is due to the presence of an inhibitory domain located between the second glutamine-rich activation domain and the DNA-binding domain. To further analyze the transcriptional properties of Sp3, we have expressed and purified recombinant Sp3 and Sp1 as epitope-tagged proteins from stable transfected insect cells. We found that Sp3 does act as a strong activator similar to Sp1 in an in vitro transcription assay using Sp1/Sp3-depleted HeLa nuclear extract. However, on the same promoter Sp3 is almost inactive when transfected into cells. Mutational studies demonstrate that a single lysine residue is responsible for the low transcriptional activity of Sp3 in vivo. We show that Sp3, but not a mutant of Sp3 that lacks this lysine residue, is highly acetylated in vivo. Our results strongly suggest that the transcriptional activity of Sp3 is regulated by acetylation. The consequences of acetylation for the activity of Sp3 are discussed.

The transcription factor IID (TFIID) complex is highly conserved between the Drosophila and mammalian systems. A mammalian homolog has been described for all the Drosophila TATA box-binding protein-associated factors (TAFs), with the exception of dTAF(II)150. We previously reported the identification of CIF, an essential cofactor for TFIID-dependent transcription from promoters containing initiator (Inr) elements. Here we describe the molecular cloning of CIF150, the human homolog of dTAF(II)150, and present biochemical evidence that this factor is involved in Inr activity. CIF150 is capable of mediating TFIID-dependent Inr activity in a complementation assay, and a protein fraction lacking Inr activity lacks detectable amounts of CIF150. Despite the striking similarity to dTAF(II)150, CIF150 does not appear to be associated with human TFIID. However, in vitro binding assays revealed a specific and direct interaction between CIF150 and hTAF(II)135. This interaction might be structurally important for the functional interaction between CIF150 and human TFIID, since CIF150 stabilizes TFIID binding to a core promoter.

Minichromosomes assembled on the mouse mammary tumor virus (MMTV) promoter in vitro exhibit positioned nucleosomes, one of which covers the binding sites for progesterone receptor (PR) and nuclear factor 1 (NF1). Incorporation of histone H1 into MMTV minichromosomes improves the stability of this nucleosome and decreases basal transcription from the MMTV promoter, as well as its response to either PR or NF1. However, histone H1-containing minichromosomes display better PR binding and support a more ef®cient synergism between PR and NF1, leading to enhanced transcription initiation. A mutant MMTV promoter lacking positioned nucleosomes does not display enhanced transcriptional synergism in the presence of H1. Binding of PR leads to phosphorylation of H1, which leaves the promoter upon transcription initiation. Thus, H1 assumes a complex and dynamic role in the regulation of the MMTV promoter.