A T cell nuclear factor resembling NF-AT binds to an NF-kappa B site and to the conserved lymphokine promoter sequence "cytokine-1".

Nuclear extracts from a nontransformed murine T lymphocyte clone contained two inducible factors that bound to a nuclear factor kappa B (NF-kappa B) site. One factor was NF-kappa B, and the other was differentiated from NF-kappa B by its mobility in the electrophoretic mobility shift assay and its lack of sensitivity to protein kinase C depletion. Competition and methylation interference assays showed that the binding site for the novel factor was limited to nucleotides in the 3' half of the kappa B site. This part of the kappa B site resembled sequences in the binding site for a second inducible nuclear factor of T cells, NF-AT, as well as a conserved sequence found in several lymphokine genes, termed "cytokine-1" (CK-1). Competition and methylation interference analysis showed that both NF-AT and CK-1 sequences bound a factor similar to the novel kappa B-binding factor and that binding involved a four-nucleotide sequence (TTCC) that the kappa B, CK-1, and NF-AT sites have in common. The complexes that form with each site have characteristics of NF-AT: they are induced upon T cell receptor stimulation, are sensitive to protein synthesis inhibitors and cyclosporin A, and are not sensitive to protein kinase C depletion. Thus, a factor or factors similar to NF-AT can bind to three distinct promoter sequences which occur commonly in several T cell activation genes. These results raise the possibility that related factors binding to kappa B, CK-1, and NF-AT sequences could play a role in the coordinate induction of T cell activation genes. In addition, our results suggest that kappa B and CK-1 sites represent potential cyclosporin-sensitive promoter elements by virtue of their ability to bind an NF-AT-like factor.     

The DNA Methyltransferase Dnmt1 Directly Interacts with the SET and RING Finger-associated (SRA) Domain of the Multifunctional Protein Uhrf1 to Facilitate Accession of the Catalytic Center to Hemi-methylated DNA

Dnmt1 is responsible for the maintenance DNA methylation during replication to propagate methylation patterns to the next generation. The replication foci targeting sequence (RFTS), which plugs the catalytic pocket, is necessary for recruitment of Dnmt1 to the replication site. In the present study we found that the DNA methylation activity of Dnmt1 was DNA length-dependent and scarcely methylated 12-bp short hemi-methylated DNA. Contrarily, the RFTS-deleted Dnmt1 and Dnmt1 mutants that destroyed the hydrogen bonds between the RFTS and catalytic domain showed significant DNA methylation activity even toward 12-bp hemi-methylated DNA. The DNA methylation activity of the RFTS-deleted Dnmt1 toward 12-bp hemi-methylated DNA was strongly inhibited on the addition of RFTS, but to a lesser extent by Dnmt1 harboring the mutations that impair the hydrogen bond formation. The SRA domain of Uhrf1, which is a prerequisite factor for maintenance methylation and selectively binds to hemi-methylated DNA, stimulated the DNA methylation activity of Dnmt1. The SRA to Dnmt1 concentration ratio was the determinant for the maximum stimulation. In addition, a mutant SRA, which had lost the DNA binding activity but was able to bind to Dnmt1, stimulated the DNA methylation activity of Dnmt1. The results indicate that the DNA methylation activity of Dnmt1 was stimulated on the direct interaction of the SRA and Dnmt1. The SRA facilitated acceptance of the 12-bp fluorocytosine-containing DNA by the catalytic center. We propose that the SRA removes the RFTS plug from the catalytic pocket to facilitate DNA acceptance by the catalytic center.     

Thrombin-induced p65 homodimer binding to downstream NF-kappa B site of the promoter mediates endothelial ICAM-1 expression and neutrophil adhesion

We investigated the mechanisms by which proinflammatory mediator, thrombin, released during intravascular coagulation and tissue injury, induces ICAM-1 (CD54) expression in endothelial cells. Stimulation of HUVEC with thrombin resulted in dose- and time-dependent increases in ICAM-1 mRNA and cell surface expression and in ICAM-1-dependent endothelial adhesivity toward polymorphonuclear leukocytes. Transient transfection of endothelial cells with ICAM-1 promoter luciferase reporter gene (ICAM-1LUC) constructs indicated that deletion of upstream NF-kappa B site (-533 bases from translation start site) had no effect on thrombin responsiveness, whereas mutation/deletion of downstream NF-kappa B site (-223 bases from the translation start site) prevented the activation of ICAM-1 promoter, indicating that the downstream NF-kappa B site is critical for thrombin inducibility. NF-kappa B-directed luciferase activity increased approximately 3-fold when cells transfected with the plasmid pNF-kappa BLUC containing five copies of consensus NF-kappa B site linked to a minimal adenovirus E1B promoter-luciferase gene were exposed to thrombin, indicating that activation of NF-kappa B was essential for thrombin response. Gel supershift assays demonstrated that thrombin induced binding of NF-kappa Bp65 (Rel A) to downstream NF-kappa B site of the ICAM-1 promoter. Thrombin receptor activation peptide, a 14-amino-acid peptide representing the new NH2 terminus of proteolytically activated receptor-1, mimicked thrombin's action in inducing ICAM-1 expression. These data indicate that thrombin activates endothelial ICAM-1 expression and polymorphonuclear leukocyte adhesion by NF-kappa Bp65 binding to the downstream NF-kappa B site of ICAM-1 promoter after proteolytically activated receptor-1 activation.     

Probing the structure of gal operator-repressor complexes. Conformation change in DNA

The gal operon is regulated by binding of Gal repressor to two operator loci, OE and OI, which are separated by 114 base pairs (bp). We have probed the actual operator DNA segments with and without Gal repressor occupation by characterizing the regions protected by repressor from DNase I digestion and dimethyl sulfate methylation. The segments which are protected from DNase I digestion in both OE and OI are about 22 bp long and seem to include 2-3 extra bp on either side of a 16-bp similar sequence containing an approximate dyad symmetry, with a consensus half-symmetry sequence GTG(G/T)AA-C. Repressor occupation hinders the reactivity of the consensus guanines in the four half-symmetry sequences, as shown by retardation of methylation at the N-7 positions by dimethyl sulfate owing to repressor binding. The protected guanines are symmetrically located. Since a dimeric Gal repressor affects symmetrically located bases, it is consistent with the notion that each half-operator is occupied by a repressor subunit. Because the N-7 positions of methylation of guanines lie in the major grooves and the protected guanines are located at positions 1, 3, 8 and the rotational 1', 3', and 8' in the 16-bp dyad symmetry, we suggest that Gal repressor establishes direct contacts with bases at 1, 3, 1', and 3' through two major grooves lying on one face of an operator helix and prevents reactivity of the guanines at 8 and 8' of a third major groove on the opposite face by changing the DNA helical structure at this position. Contacts at other positions are also discussed.     

The methylation of C/EBP β gene promoter and regulated by GATA-2 protein

Mammalian genomes are punctuated by DNA sequences containing an atypically high frequency of CpG sites (CpG islands; CGIs) that are associated with the majority of annotated gene promoters. Methylated C bases of CpG sites inhibit the expression of downstream genes. During the differentiation of 3T3-L1 preadipocytes, the CCAAT/enhancer-binding protein (C/EBP) β gene plays an important role. We studied the CpG island methylation status of the C/EBP β promoter and its relationship with the GATA-2 protein. We used computer analysis to determine that the C/EBP β promoter sequence is rich in CGIs, and observed that two of seven methylated C bases were demethylated during the preadipocyte differentiation using bisulfite sequencing PCR (BSP). This corresponded with the onset of notable C/EBP β gene expression. Immunofluorescence and molecular docking showed that the GATA-2 protein binds the C/EBP β promoter in front of the first demethylated CpG site. We also found that expression of GATA-2 and C/EBP β proteins is negatively correlated. These results indicate that the methylated C bases in the C/EBP β promoter relate to expression of the C/EBP β gene, and that its demethylation is linked with GATA-2 protein association.     

A lipopolysaccharide-induced DNA-binding protein for a class II gene in B cells is distinct from NF-kappa B.

Class II (Ia) major histocompatibility complex molecules are cell surface proteins normally expressed by a limited subset of cells of the immune system. These molecules regulate the activation of T cells and are required for the presentation of antigens and the initiation of immune responses. The expression of Ia in B cells is determined by both the developmental stage of the B cell and by certain external stimuli. It has been demonstrated previously that treatment of B cells with lipopolysaccharide (LPS) results in increased surface expression of Ia protein. However, we have confirmed that LPS treatment results in a significant decrease in mRNA encoding the Ia proteins which persists for at least 18 h. Within the upstream regulatory region of A alpha k, an NF-kappa B-like binding site is present. We have identified an LPS-induced DNA-binding protein in extracts from athymic mice whose spleens consist predominantly of B cells. Binding activity is present in low levels in unstimulated spleen cells and is increased by LPS treatment. This protein binds to two sites in a regulatory region of the Ia A alpha k gene, one of which contains the NF-kappa B-like binding site. DNA fragments containing these sites cross-compete for protein binding. Analysis by DNase I footprinting identified a target binding sequence, named the LPS-responsive element. Although this target sequence contains an NF-kappa B-like binding site, competition with a mutant oligonucleotide demonstrated that bases critical for NF-kappa B binding are not required for binding of the LPS-inducible protein. Therefore, we hypothesized that this inducible protein represents a new mediator of LPS action, distinct from NF-kappa B, and may be one mechanism to account for the decrease in mRNA encoding the Ia proteins.     

Sequence requirement for specific interaction of an enhancer binding protein (EBP1) with DNA.

Short DNA sequence motifs have been identified in viral and cellular enhancers which represent the binding sites for a variety of trans- acting factors. One such HeLa cell factor, EBP1, has been purified and shown to bind to sequences in the SV40 enhancer. The PRDII element in the human beta-interferon gene regulatory element (IRE) shows strong sequence similarity to the EBP1 binding site in the SV40 enhancer. We demonstrate here that EBP1 binds to its sites in the SV40 enhancer and IRE in a similar manner, making base specific contacts over one complete turn of the DNA double helix. Mutational analysis of the EBP1 sites in the IRE and SV40 enhancer has identified the DNA sequence requirements necessary for specific EBP1/DNA complex formation. In addition, 34 DNA sequences related to the EBP1 binding site were analysed for their ability to bind EBP1. Sequences constituting high affinity binding sites possess the sequence 5'-GG(N)6CC-3'. Single base pair changes in the region between the conserved Gs and Cs can generally be tolerated although it is clear that these intervening bases contribute to binding affinity. Mutations in the recognition site which could lead to gross structural changes in the DNA abolish EBP1 binding.