High mobility group box (HMGB) proteins are architectural proteins whose HMG DNA binding domains confer significant preference for distorted DNA, such as 4-way junctions. HMO1 is one of 10 Saccharomyces cerevisiae HMGB proteins, and it is required for normal growth and plasmid maintenance and for regulating the susceptibility of yeast chromatin to nuclease. Using electrophoretic mobility shift assays, we have shown here that HMO1 binds 26-bp duplex DNA with K d ‫؍‬ 39.6 ؎ 5.0 nM and that its divergent box A domain participates in DNA interactions, albeit with low affinity. HMO1 has only modest preference for DNA with altered conformations, including DNA with nicks, gaps, overhangs, or loops, as well as for 4-way junction structures and supercoiled DNA. HMO1 binds 4-way junctions with half-maximal saturation of 19.6 ؎ 2.2 nM, with only a modest increase in affinity in the absence of magnesium ions (half-maximal saturation 6.1 ؎ 1.1 nM). Whereas the box A domain contributes modest structure-specific binding, the box B domain is required for high affinity binding. HMO1 bends DNA, as measured by DNA cyclization assays, facilitating cyclization of 136-, 105-, and 87-bp DNA, but not 75-bp DNA, and it has a significantly longer residence time on DNA minicircles compared with linear duplex DNA. The unique DNA binding properties of HMO1 are consistent with global roles in the maintenance of chromatin structure. High mobility group (HMG)1 proteins constitute a significant proportion of non-histone proteins of eukaryotic chromatin. They are abundant proteins that are grouped, in part based on their DNA binding characteristics, into three major classes, HMGA, HMGB, and HMGN (1-4). HMGB proteins contain one or more homologous repeats of the ϳ80-amino acid sequence HMG box and are classified into two families based on the abundance, function, and DNA specificity of this conserved region (1, 5, 6). The moderately sequence-specific family is typified by transcription factors such as sex-determining factor SRY and lymphoid enhancer factor LEF-1 (7, 8), whereas the non-sequence-specific family is represented by so-called architectural factors HMGB1/2 and the Saccharomyces cerevisiae non-histone chromosomal proteins 6A and 6B (NHP6A/B) (9).The tertiary structures of HMG boxes from sequence-specific and non-sequence-specific proteins have revealed an evolutionarily conserved, common global fold consisting of an L-shaped structure composed of three ␣-helices (10 -19). The HMG DNA binding domain, which interacts with ϳ10 bp of duplex, binds to the minor groove of DNA by partial intercalation of one or two surface-exposed, conserved hydrophobic residues into the base pair stack. Consequently, the DNA is greatly distorted, resulting in a sharp bend and helical underwinding (4,8,14,16,20). SRY and LEF-1 cause bending by insertion of helix I hydrophobic residues Ile and Met, respectively, into the base pair stack (Fig. 1) (8, 17). HMGB1 contains tandem HMG box domains referred to as box A and box B; DNA-intercalating residues are located...

Summary Decreased growth hormone (GH) and thyroid hormone (TH) signaling are associated with longevity and metabolic fitness. The mechanisms underlying these benefits are poorly understood, but may overlap with those of dietary restriction (DR), which imparts similar benefits. Recently we discovered that hydrogen sulfide (H2S) is increased upon DR and plays an essential role in mediating DR benefits across evolutionary boundaries. Here we found increased hepatic H2S production in long-lived mouse strains of reduced GH and/or TH action, and in a cell-autonomous manner upon serum withdrawal in vitro. Negative regulation of hepatic H2S production by GH and TH was additive and occurred via distinct mechanisms, namely direct transcriptional repression of the H2S-producing enzyme cystathionine γ-lyase (CGL) by TH, and substrate-level control of H2S production by GH. Mice lacking CGL failed to downregulate systemic T4 metabolism and circulating IGF-1, revealing an essential role for H2S in the regulation of key longevity-associated hormones.

The Saccharomyces cerevisiae high-mobility group protein HMO1 is composed of two DNA-binding domains termed box A and box B, of which only box B is predicted to adopt a HMG fold, and a lysine-rich C-terminal extension. To assess the interaction between individual domains and their contribution to DNA binding, several HMO1 variants were analyzed. Using circular dichroism spectroscopy, thermal stability was measured. While the melting temperatures of HMO1-boxA and HMO1-boxB are 57.2 and 47.2 degrees C, respectively, HMO1-boxBC, containing box B and the entire C-terminal tail, melts at 46.1 degrees C, suggesting little interaction between box B and the tail. In contrast, full-length HMO1 exhibits a single melting transition at 47.9 degrees C, indicating that interaction between box A and either box B or the tail destabilizes this domain. As HMO1-boxAB, lacking only the lysine-rich C-terminal segment, exhibits two melting transitions at 46.0 and 63.3 degrees C, we conclude that the destabilization of the box A domain seen in full-length HMO1 is due primarily to its interaction with the lysine-rich tail. Determination of DNA substrate specificity using electrophoretic mobility shift assays shows unexpectedly that the lysine-rich tail does not increase DNA binding affinity but instead is required for DNA bending by full-length HMO1; HMO1-boxBC, lacking the box A domain, also fails to bend DNA. In contrast, both HMO1 and HMO1-boxAB, but not the individual HMG domains, exhibit preferred binding to constrained DNA minicircles. Taken together, our data suggest that interactions between box A and the C-terminal tail induce a conformation that is required for DNA bending.

These studies suggest that NF-κB signaling is a key regulator of angiogenesis and growth of primary and metastatic thyroid cancer, and that IL-8 may be an important downstream mediator of NF-κB signaling in advanced thyroid cancer growth and progression.