Cooperative function of POU proteins and SOX proteins in glial cells

Glial cells of the oligodendrocyte lineage express several highly related POU proteins including Tst-1/Oct6/SCIP and Brn-1. Tst-1/Oct6/SCIP, but not Brn-1 efficiently cooperated with Sox10, the only SRY box protein so far identified in oligodendrocytes. Here we show that, in addition to Sox10, cells of the oligodendrocyte lineage contain significant amounts of the related SRY box proteins Sox4 and Sox11. During development, Sox11 was strongly expressed in the central nervous system. It was first detected in neural precursors throughout the neuroepithelium. During later stages of neural development, Sox11 was additionally expressed in areas of the brain in which neurons undergo differentiation. In agreement with its expression in neural precursors, Sox11 levels in cells of the oligodendrocyte lineage were high in precursors and down-regulated during terminal differentiation. Outside the nervous system, expression of Sox11 was also detected in the developing limbs, face, and kidneys. Structure function analysis revealed that Sox11 has a strong intrinsic transactivation capacity which is mediated by a transactivation domain in its carboxyl-terminal part. In addition, Sox11 efficiently synergized with Brn-1. Synergy was dependent on binding of both proteins to adjacent DNA elements, and required the presence of the respective transactivation domain in each protein. Our data suggest the existence of a specific code in which POU proteins require specific Sox proteins to exhibit cooperative effects in glial cells.     

Mouse Brn-3 family of POU transcription factors: a new aminoterminal domain is crucial for the oncogenic activity of Brn-3a

The class IV POU domain genes Brn-3a, -b and -c are differentially expressed during neural development and at least Brn-3a also in neuroectodermal tumors. In contrast to Brn-3b and Brn-3c, Brn-3a encodes two protein variants: Brn-3a(l) and Brn-3a(s). Brn-3a(s) lacks 84 aminoterminal residues but is otherwise identical to Brn-3a(l). Outside the well conserved carboxyterminal POU domains all three Brn-3 proteins (-a, -b and -c) diverge until the aminoterminal end where a new domain of about 100 amino acids is identified. This domain is conserved only between Brn-3 proteins and other class IV POU factors. Brn-3a(l) that contains the complete domain but not Brn-3a(s) that lacks 84 amino acids of it is able to tumorigenically transform primary fibroblasts. Brn-3b that lacks 40 amino acids of the new domain does itself not transform, but abolishes the oncogenic potential of Brn-3a(l) when transfected together. This demonstrates not only that Brn3-a(l) is a proto-oncogene and may well be causally involved in the generation of neuroectodermal tumors but also suggests that the intactness of the new aminoterminal domain described here is crucial for oncogenic activity. In addition, our data indicate that Brn-3b acts as an inhibitor of Brn-3a(l) activity.     

Comparison of the sequence-selective DNA binding by peptide dimers with covalent and noncovalent dimerization domains

Sequence-specific DNA binding proteins generally consist of more than two DNA-contacting regions to ensure the selectivity of recognition. The multiple DNA binding modules are connected either through the covalent linker or through the noncovalent dimerization domain. We have compared the DNA binding of peptide dimers with covalent and noncovalent dimerization domains to explore the potential advantage of each linkage on the sequence-specific DNA binding. Three sets of head-to-tail peptide dimers were synthesized by using the same basic region peptide to target the same DNA sequence; one dimer was assembled with a bridged biphenyl derivative as a covalent dimerization domain, and two other dimers were assembled with the cyclodextrin guest noncovalent dimerization domains. One of the noncovalent dimers was a heterodimer that consisted of cyclodextrin and guest peptides, while the other was a homodimer that consisted of peptides bearing both cyclodextrin and the guest molecule within the same chain. Both noncovalent dimers formed the specific DNA complexes within narrower ranges of peptide concentrations and showed higher sequence selectivity than the covalent dimer did. Among the three dimers, the noncovalent homodimer that can form an intramolecular inclusion complex showed the highest sequence selectivity. Because the noncovalent homodimer with the higher stability of the circular intramolecular inclusion complex exhibited the higher sequence selectivity, it was concluded that an equilibrium involving a conformational transition of a monomeric peptide effectively reduced the stability of its nonspecific binding complex, hence increasing the efficacy of cooperative dimer formation at the specific DNA sequence.     

Comparison of the DNA binding characteristics of the related zinc finger proteins WT1 and EGR1

The interactions of the related zinc finger proteins WT1 and EGR1 with DNA have been investigated using a quantitative binding assay. A recombinant peptide containing the four zinc fingers of WT1 binds to the dodecamer DNA sequence GCG-TGG-GCG-TGT with an apparent dissociation constant (Kd) of (1.14 +/- 0.09) x 10(-9) M under conditions of 0.1 M KCl, pH 7.5, at 22 degrees C. Under the same conditions, a recombinant peptide containing the three zinc fingers of EGR1 binds to the dodecamer sequence, the first nine bases comprising the EGR consensus binding site, with an apparent Kd of (3.55 +/- 0.24) x 10(-9) M. The nature of the equilibrium binding of each peptide to DNA was investigated as a function of temperature, pH, monovalent salt concentration, and divalent salt concentration. The interaction of WT1 with DNA is an entropy-driven process, while the formation of the EGR1-DNA complex is favored by enthalpy and entropy. The DNA binding activities of both proteins have broad pH optima centered at pH 8.0. The binding of both proteins to DNA shows similar sensitivity to ionic strength, with approximately 7.7 +/- 0.8 ion pairs formed in the EGR1-DNA complex and 9.2 +/- 1.8 ion pairs formed in the WT1-DNA complex. Results of measuring the effects of point mutations in the DNA binding site on the affinity of WT1 and EGR1 indicates a significant difference in the optimal binding sites: for EGR1, the highest affinity binding site has the sequence GNG-(T/G)GG-G(T/C)G, while for WT1 the highest affinity binding site has the sequence G(T/C)G-(T/G)GG-GAG-(T/C)G(T/C).     

Structural studies on a sulfated polysaccharide from an Arthrobacter sp. by NMR spectroscopy and methylation analysis

Interleukin-4 (IL-4) is a pleiotropic immunomodulatory cytokine secreted by T helper 2 cells. The IL-4 promoter contains multiple sites with DNA sequences homologous to the IL-2 NF-AT binding site. One of these sites--the P2 site--located between -173 and -150 was previously found to be flanked by two octamer-like motifs. NF-ATp/c and octamer proteins were suggested to bind to this region and to cooperatively activate the promoter activity (Chuvpilo et al., 1993). To precisely analyze the P2-binding factors we used antibodies against NF-ATp, NF-ATc, Fos, Jun, Oct-1 and Oct-2 in EMSA. We show here that nuclear extracts from T-cells form two P2-binding complexes--a PMA/ionomycin-inducible and a constitutive one. The PMA/ionomycin-inducible complex contains NF-ATp/c, Fos and Jun. No octamer binding factors could be detected in either of the two complexes. Analysis of the precise DNA contact points of the two complexes showed that both complexes are formed in the center of the NF-AT consensus site. No DNA contact points could be detected in the octamer-like motif site. Furthermore, purified recombinant POU domains of Oct-1 and Oct-2 failed to bind to the P2 site, suggesting that this site is not an independent octamer-binding site. Therefore, the DNA sequence at -173 to -150 of the IL-4 promoter is a binding site for NF-ATp/c and AP-1. Octamer proteins are unlikely to cooperate with NF-ATp/c at this site.     

Dimerization specificity of P22 and 434 repressors is determined by multiple polypeptide segments

The repressor protein of bacteriophage P22 binds to DNA as a homodimer. This dimerization is absolutely required for DNA binding. Dimerization is mediated by interactions between amino acids in the carboxyl (C)-terminal domain. We have constructed a plasmid, p22CT-1, which directs the overproduction of just the C-terminal domain of the P22 repressor (P22CT-1). Addition of P22CT-1 to DNA-bound P22 repressor causes the dissociation of the complex. Cross-linking experiments show that P22CT-1 forms specific heterodimers with the intact P22 repressor protein, indicating that inhibition of P22 repressor DNA binding by P22CT-1 is mediated by the formation of DNA binding-inactive P22 repressor:P22CT-1 heterodimers. We have taken advantage of the highly conserved amino acid sequences within the C-terminal domains of the P22 and 434 repressors and have created chimeric proteins to help identify amino acid regions required for dimerization specificity. Our results indicate that the dimerization specificity region of these proteins is concentrated in three segments of amino acid sequence that are spread across the C-terminal domain of each of the two phage repressors. We also show that the set of amino acids that forms the cooperativity interface of the P22 repressor may be distinct from those that form its dimer interface. Furthermore, cooperativity studies of the wild-type and chimeric proteins suggest that the location of cooperativity interface in the 434 repressor may also be distinct from that of its dimerization interface. Interestingly, changes in the dimer interface decreases the ability of the 434 repressor to discriminate between its wild-type binding sites, O(R)1, O(R)2, and O(R)3. Since 434 repressor discrimination between these sites depends in large part on the ability of this protein to recognize sequence-specific differences in DNA structure and flexibility, this result indicates that the C-terminal domain is intimately involved in the recognition of sequence-dependent differences in DNA structure and flexibility.