A novel nuclear inhibitor I-92 regulates DNA binding activity of octamer binding protein p92 during the cell cycle.

Nuclear DNA binding protein p92 is a sequence specific octamer binding protein with identical molecular weight as the ubiquitous octamer binding protein Oct-1. It binds to octamer related sequences from the enhancer of human papillomavirus type 18. The activity and intracellular distribution of p92 is regulated by extracellular signals. In serum starved Hela-fibroblast hybrid cells p92 is localized to the cytosol. Serum stimulation leads to nuclear import of p92. In fractions of asynchronously growing cells, which were separated according to cell cycle phases into G1, S, and G2 populations by centrifugal elutriation, p92 DNA binding is confined to S phase. In binding site blots however, p92 DNA binding activity is also present in G1 and G2. In G1 and G2 DNA binding activity of p92 is masked by a novel nuclear inhibitor I-92. The cyclic association of p92 with its inhibitor I-92 provides a new mechanism of regulating S phase dependent activity of a sequence specific DNA binding protein.     

Single strand binding protein specific for the polyoma early-coding strand of PEA1 (AP1) regulatory sequence.

We have shown that nuclear and cytosolic proteins from embryonal carcinoma F9 cells are able to bind to the early-coding strand of polyoma enhancer A domain. As demonstrated by mobility shift specific competition experiments, DNase I footprinting, depurination and depyrimidation interference, and proteolytic clipping performed with single stranded oligonucleotides, some of these proteins bind specifically to the early-coding PEA1 (AP1) motif. In addition, 'Southwestern' analysis has made possible the identification of a 46 KD nuclear protein that binds to this sequence. These cellular proteins did not bind to the complementary single strand as demonstrated by mobility shift analysis, nor did they bind to RNA synthesized in vitro by using the complementary strand as template. They were also shown to be different from their corresponding double strand binding factors. This new dimension in the functional flexibility and complexity of the polyoma enhancer suggests new properties of the classic regulating sequences that could provide additional modulation of regulating activities.     

Characterization of constitutive and inducible transcription factors binding to the P2 NF-AT site in the human interleukin-4 promoter

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.© 1997 Elsevier Science B.V. All rights reserved.     

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.     

Structure and function of the enhancer 3'' to the human A gamma globin gene.

An enhancer is located immediately 3' to the A gamma globin gene. We have used DNase I footprinting to map the sites of interaction of nuclear proteins with the DNA sequences of this enhancer. Eight footprints were discovered, distributed over 600 base pairs of DNA. Three of these contain a consensus binding site for the erythroid specific factor GATA-I. Each of these GATA-1 sites had an enhancer activity when inserted into a reporter plasmid and tested in human erythroleukemia cells. Other footprints within the enhancer contained consensus binding sequences for the ubiquitous, positive regulatory proteins AP2 and CBP-1. An Sp1-like recognition sequence was also identified. Synthetic oligonucleotides encompassing two of the footprints generated a slowly migrating complex in gel mobility shift assays. The same complex forms on a fragment of the human gamma globin gene promoter extending from -260 to -200. The DNaseI footprint of this protein complex with the enhancer overlapped a sequence, AGGAGGA, found within the binding site for a protein that interacts with the chicken beta globin promoter and enhancer, termed the stage selector element. We propose that this complex of proteins may be involved in the human gamma globin promoter-enhancer interaction.     

The constitutively expressed octamer binding protein OTF-1 and a novel octamer binding protein expressed specifically in cervical cells bind to an octamer-related sequence in the human papillomavirus 16 enhancer.

A novel octamer binding protein expressed specifically in cervical cells but not in other cell types has been identified. This protein differs in size and sequence specificity from the constitutively expressed octamer binding protein OTF-1. In particular it binds with higher affinity to a sequence in the human papillomavirus 16 (HPV) upstream regulatory region which has a seven out of eight base pair match compared to the consensus octamer motif. This is the first example of a tissue specific protein which has been observed to bind to the papillomavirus enhancer. The possible role of this protein in producing the observed tissue specific activity of the enhancer and in cervical carcinogenesis induced by HPV is discussed.     

Kinetic analysis of the interactions of human papillomavirus E6 oncoproteins with the ubiquitin ligase E6AP using surface plasmon resonance

Cervical cancers evolve from lesions generated by genital human papillomaviruses (HPV). "Low-risk" genital HPVs cause benign proliferations whereas "high-risk" types have the potential to progress into cancer. High-risk HPV E6 oncoproteins interact with the ubiquitin ligase E6AP and target several cellular proteins, including p53 and proteins of the MAGI family, towards ubiquitin-mediated degradation. E6AP, like other E6 binding proteins such as E6BP, IRF-3 and paxillin, interacts with E6 via a consensus leucine-charged motif. Here we have investigated the kinetics of the interactions of a 15-mer peptide containing the LxxvarphiLsh motif of E6AP with E6. For this we have developed a Biacore assay based on antibody-capture on the sensor surface of GST- and/or MBP-E6AP peptide constructs followed by E6 protein injection. Our experiments show that E6 oncoproteins from four major high-risk (16, 18, 33 and 58) HPV types bind to E6AP with equilibrium dissociation constants in the low micromolar range. The kinetic dissociation parameters of these interactions are remarkably similar. On the other hand, low-risk HPV 11 E6 does not interact with E6AP even at relatively high concentrations. We also show that the two zinc-binding domains of E6 are required for E6AP recognition. Finally, we have analysed the binding properties of site-directed mutants of the E6AP-derived peptide. We demonstrate the importance for binding of conserved aliphatic side-chains and the moderate role of the global negative charge of the peptide. This work provides the first quantitative data on an HPV E6-mediated interaction, which support the current models of E6AP-mediated degradation.     

Divergent RNA binding specificity of yeast Puf2p

PUF proteins bind mRNAs and regulate their translation, stability, and localization. Each PUF protein binds a selective group of mRNAs, enabling their coordinate control. We focus here on the specificity of Puf2p and Puf1p of Saccharomyces cerevisiae, which copurify with overlapping groups of mRNAs. We applied an RNA-adapted version of the DRIM algorithm to identify putative binding sequences for both proteins. We first identified a novel motif in the 3' UTRs of mRNAs previously shown to associate with Puf2p. This motif consisted of two UAAU tetranucleotides separated by a 3-nt linker sequence, which we refer to as the dual UAAU motif. The dual UAAU motif was necessary for binding to Puf2p, as judged by gel shift, yeast three-hybrid, and coimmunoprecipitation from yeast lysates. The UAAU tetranucleotides are required for optimal binding, while the identity and length of the linker sequences are less critical. Puf1p also binds the dual UAAU sequence, consistent with the prior observation that it associates with similar populations of mRNAs. In contrast, three other canonical yeast PUF proteins fail to bind the Puf2p recognition site. The dual UAAU motif is distinct from previously known PUF protein binding sites, which invariably possess a UGU trinucleotide. This study expands the repertoire of cis elements bound by PUF proteins and suggests new modes by which PUF proteins recognize their mRNA targets.