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...

Transcriptional repressors often employ multiple activities, but the molecular mechanisms and physiological relevance of this functional diversity remain obscure. The Drosophila melanogaster Knirps repressor uses CtBP corepressor-dependent and -independent pathways. To separately analyze the components of Knirps repression activity, we elucidated the specific repression properties of CtBP and of Knirps subdomains. Like Knirps, CtBP represses adjacent transcriptional activators; but unlike Knirps, CtBP is unable to repress basal promoter elements. We determined that the ability of CtBP to recapitulate only a subset of Knirps activities is due to a quantitative, rather than qualitative, deficiency in repression activity. The CtBP-dependent portion of Knirps synergizes with the CtBP-independent repression activity to potently repress promoter elements from enhancer-or promoter-proximal positions. This result indicates that multiple repression activities are combined to exceed critical thresholds on target genes. CtBP mutant proteins unable to bind NAD fail to interact with DNA-bound factors. We show that DNA-binding Gal4-CtBP fusion proteins also require NAD binding for activity, indicating that NAD plays a role in repression at a step subsequent to CtBP recruitment to the promoter.Transcriptional repression is an essential feature of gene regulation widely utilized in development and other biological processes. Repression is effected by a variety of transcription factors and cofactors, which utilize different pathways to mediate their function. Repression mechanisms include direct competition between repressors and activators, interactions of the repressors with the basal transcriptional machinery, and recruitment of different chromatin modification activities (7,9,22,25). When and how these distinct mechanisms of repression are employed remains poorly understood.A variety of transcriptional repressors have been demonstrated to possess multiple repression activities. In a few cases, the distinct repression activities appear to block specific types of transcriptional activators. For example, the mammalian Zeb protein represses the muscle-specific activator MEF2C via a CtBP (C-terminal binding protein)-dependent domain and cEts, c-Myb, and other activators via a CtBP-independent activity (28). In other cases, such as that with the NRSF repressor, different repression activities have been found to be utilized at distinct promoters (19). Multiple repression activities might also be invoked to increase the overall level of repression activity at a given promoter, much as activators have been suggested to contact multiple promoter and cofactor targets to synergistically potentiate transcription.A broad functional distinction between Drosophila melanogaster transcriptional repressors based on their range of repression activity has been made (9). Short-range repressors (e.g., Knirps, Giant, Snail, and Krüppel) are active when bound close (Յ100 bp) to activators within enhancers, and in experimental settings, these protein...

CtBP corepressor proteins potentiate the activity of many metazoan transcriptional repressors. These proteins are homologous to prokaryotic D-2-hydroxyacid dehydrogenases, possessing an NAD/NADH binding fold and conserved active site residues. When expressed in Drosophila, a catalytic site mutant retains biological activity, however, we find that an NAD binding mutant lacks biological activity. The NAD mutant, similar to a dimerization mutant, is expressed at low levels, indicating that binding of NAD/NADH may affect CtBP stability. These data support the idea that the ancestral dehydrogenase activity is not required for CtBP function, and NAD binding may play a regulatory, rather than catalytic, role.

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