The Drosophila GAGA factor self-oligomerizes both in vivo and in vitro. GAGA oligomerization depends on the presence of the N-terminal POZ domain and the formation of dimers, tetramers, and oligomers of high stoichiometry is observed in vitro. GAGA oligomers bind DNA with high affinity and specificity. As a consequence of its multimeric character, the interaction of GAGA with DNA fragments carrying several GAGA binding sites is multivalent and of higher affinity than its interaction with fragments containing single short sites. A single GAGA oligomer is capable of binding adjacent GAGA binding sites spaced by as many as 20 base pairs. GAGA oligomers are functionally active, being transcriptionally competent in vitro. GAGA-dependent transcription activation depends strongly on the number of GAGA binding sites present in the promoter. The POZ domain is not necessary for in vitro transcription but, in its absence, no synergism is observed on increasing the number of binding sites contained within the promoter. These results are discussed in view of the distribution of GAGA binding sites that, most frequently, form clusters of relatively short sites spaced by small variable distances.

The chromatin high mobility group protein 1 (HMGB1) is a very abundant and conserved protein that is structured into two HMG box domains plus a highly acidic C-terminal domain. From the ability to bind DNA nonspecifically and to interact with various proteins, several functions in DNA-related processes have been assigned to HMGB1. Nevertheless, its functional role remains the subject of controversy. Using a phage display approach we have shown that HMGB1 can recognize several peptide motifs. A computer search of the protein data bases found peptide homologies with proteins already known to interact with HMGB1, like p53, and have allowed us to identify new potential candidates. Among them, transcriptional activators like the heterogeneous nuclear ribonucleoprotein K (hnRNP K), repressors like methyl-CpG binding protein 2 (MeCP2), and co-repressors like the retinoblastoma susceptibility protein (pRb) and Groucho-related gene proteins 1 (Grg1) and 5 (Grg5) can be found. A detailed analysis of the interaction of Grg1 with HMGB1 confirmed that the binding region contained the sequence homologous to one of the peptides identified. Our results have led us to propose that HMGB1 may play a central role in the stabilization and/or assembly of several multifunctional complexes through protein-protein interactions.In the eukaryotic cell nucleus of all vertebrate cell types, HMGB1 1 (formerly named HMG1, see Ref. 1 for a revised nomenclature) is one of the most abundant non-histone proteins. HMGB1 has been shown to be essential because knockout mice die 24 h after birth (2). HMGB1 is highly conserved, particularly in mammals and to a lesser extent throughout the animal kingdom. HMGB1 is structured into three domains, two basic HMG boxes (HMG domains A and B) and a highly acidic C-terminal domain, which confer an overall dipolar appearance to this protein (see Refs. 3-5 for reviews). Each of the HMG boxes is formed by two short and one long ␣-helix that upon folding produce an L-or V-shaped three-dimensional domain structure (6 -8). Whereas the acidic C-terminal domain is presumably involved in the modulation of HMGB1 activity, the HMG box domains allow the protein to bind to linear DNA with moderate affinity and to highly structured (3-and 4-way junction DNA, cruciform DNA) or distorted DNA (bent or kink DNA, bulged DNA, cisplatin-modified DNA) with higher affinity, but always without sequence specificity. The concave surface of the L-or V-shaped HMG box domain contacts the DNA in the minor groove in two slightly different ways introducing important modifications in the structure of DNA, in particular a strong bend (reviewed in Ref. 5). Presumably, these features will be of relevance for the biological functions in which HMGB1 has been involved (DNA repair, recombination, replication, and transcription).The activity of HMGB1 is not solely mediated by its ability to bind to DNA. Indeed, HMGB1 and the related HMGB2 protein can interact through their HMG box domains with a broad range of proteins ranging from nuclear cell prote...

PhoB is a two-component response regulator that activates transcription by interacting with the r 70 subunit of the E. coli RNA polymerase in promoters in which the À35 r 70 -recognition element is replaced by the pho box. The crystal structure of a transcription initiation subcomplex that includes the r 4 domain of r 70 fused with the RNA polymerase b subunit flap tip helix, the PhoB effector domain and the pho box DNA reveals how r 4 recognizes the upstream pho box repeat. As with the À35 element, r 4 achieves this recognition through the N-terminal portion of its DNA recognition helix, but contact with the DNA major groove is less extensive. Unexpectedly, the same recognition helix contacts the transactivation loop and helices a2 and a3 of PhoB. This result shows a simple and elegant mechanism for polymerase recruitment to pho box promoters in which the lost À35 element contacts are compensated by new ones with the activator. In addition, r 4 is reoriented, thereby suggesting a remodelling mechanism for transcription initiation.

We present evidence that transcription factor TFIID, known for its central role in transcription by RNA polymerase II, is also involved in RNA polymerase III transcription of the human U6 snRNA gene. Recombinant human TFIID, expressed either via a vaccinia virus vector in HeLa cells or in Escherichia coli, affects U6 transcription in three different in vitro assays. First, TFIID‐containing fractions stimulate U6 transcription in reactions containing rate‐limiting amounts of HeLa nuclear extract. Second, TFIID addition relieves transcriptional exclusion between two competing U6 templates. Third, TFIID can replace one of two heat labile fractions essential for U6 transcription. Thus, at least one basal transcription factor is involved in transcription by two different RNA polymerases.

In this paper, we have analysed the conformational behaviour shown by the homopurine–homopyrimidine alternating d(GA.CT)22 sequence cloned into SV40. Our results show that, in the presence of zinc ions, the d(GA.CT)22 sequence adopts an altered secondary DNA structure (*H‐DNA) which differs from either B‐DNA or H‐DNA. Formation of *H‐DNA is facilitated by negative supercoiling and does not appear to require base protonation, since it is induced at neutral pH by approximately 0.4 mM ZnCl2. The patterns of OsO4 and DEPC modification obtained in the presence of zinc are compatible with a homopurine–homopurine–homopyridimine triplex, though other structural models for *H‐DNA are also possible. The hypersensitivity to S1‐cleavage of the d(GA.CT)22 sequence is reinterpreted in terms of the equilibria between the B‐, H‐ and *H‐forms of the sequence. These results reveal the high degree of structural polymorphism shown by homopurine‐homopyrimidine sequences. Its biological relevance is discussed.

The high mobility group (HMG) box domain has defined a family of proteins, mostly transcription factors, that specifically interacts with DNA on the minor groove and sharply bends it. The founding member of the family, HMG1, does not specifically recognize regular B-DNA but is recruited to DNA by interaction with other transcription factors and TATA box-binding protein (TBP). However, conflicting effects of HMG1 on transcription have been reported. We show that the interaction between HMG1 and TBP is species-specific. This interaction in turn affects the interaction of TBP with transcription factor (TF) IIB and is competed by TFIIA. A primary binding site was mapped to the H2 ␣-helix in the highly conserved core domain of human TBP. On HMG1, the primary binding site was only in the HMG box A, and HMG box A was also sufficient to interact with native TFIID. Both HMG boxes efficiently repressed transcription in vitro as fusions to the Gal4-DNA binding domain. Additionally, HMG box B showed a weak level of activation at very low amounts. These results suggest a general involvement of HMG1 at the early stages of polymerase II transcription that may result in subtle activation or repression of individual genes. High mobility group protein 1 (HMG1)1 is an abundant, highly conserved nuclear protein found in practically all eukaryotes. It is structured in three domains, one C-terminal highly acidic domain and two basic domains, A and B. The structures of the A and B domains have been solved in solution by NMR (1, 2). Both domains adopt a very similar L-shaped structure, formed by two short and one long ␣-helix, that is known as the HMG box domain. An increasing number of proteins containing one or more HMG box domains have been described; the HMG box domains for which the structure has been solved, such as the ones in LEF-1 or SRY, are very similar to the HMG box domains of HMG1 (3, 4). However, whereas SRY and LEF-1 interact with DNA in the minor groove with a certain sequence specificity, HMG1 (and the related protein HMG2) does not interact specifically with regular B-DNA. Nevertheless, HMG1 shows a clear preference for binding angled structures in the DNA without any sequence specificity, such as cisplatin-modified DNA, bulged DNA, or four-way DNA junctions (5, 6). Structure-specific DNA recognition has also been observed for the HMG box domains of several other proteins such as UBF and SRY (7,8).The HMG boxes of HMG1 are also a place for protein-protein interactions. Both HMG1 domains A and B have been reported to interact with the POU domains of Oct2 and HOXD9 (9, 10), and full-length HMG1 has been shown to interact with TBP (11) and recently with p53 (12) and steroid hormone receptors (13).The fact that many members of the HMG box family are transcription factors (14), along with the interactions of HMG1/2 with several transcription factors, has suggested a role for HMG1/2 in transcription. In this respect, enhancement of progesterone receptor binding to specific DNA sequences (15), reversible repression of basal...

Linker histone H1 is an important structural component of chromatin that stabilizes the nucleosome and compacts the nucleofilament into higher-order structures. The biology of histone H1 remains, however, poorly understood. Here we show that Drosophila histone H1 (dH1) prevents genome instability as indicated by the increased γH2Av (H2AvS137P) content and the high incidence of DNA breaks and sister-chromatid exchanges observed in dH1-depleted cells. Increased γH2Av occurs preferentially at heterochromatic elements, which are upregulated upon dH1 depletion, and is due to the abnormal accumulation of DNA:RNA hybrids (R-loops). R-loops accumulation is readily detectable in G1-phase, whereas γH2Av increases mainly during DNA replication. These defects induce JNK-mediated apoptosis and are specific of dH1 depletion since they are not observed when heterochromatin silencing is relieved by HP1a depletion. Altogether, our results suggest that histone H1 prevents R-loops-induced DNA damage in heterochromatin and unveil its essential contribution to maintenance of genome stability.

The human U1 and U6 genes have similar basal promoter structures. A first analysis of the factor requirements for the transcription of a human U1 gene by RNA polymerase II in vitro has been undertaken, and these requirements compared with those of human U6 gene transcription by RNA polymerase III in the same extracts. Fractions containing PSE‐binding protein (PBP) are shown to be essential for transcription of both genes, and further evidence that PBP itself is required for U1 as well as U6 transcription is presented. On the other hand, the two genes have distinct requirements for TATA‐binding protein (TBP). On the basis of chromatographic and functional properties, the TBP, or TBP complex, required for U1 transcription appears to differ from previously described complexes required for RNA polymerase I, II or III transcription. The different TBP requirements of the U1 and U6 promoters are reflected by specific association with either TFIIB or TFIIIB respectively, thus providing a basis for differential RNA polymerase selection.