A novel DNA-binding motif abuts the zinc finger domain of insect nuclear hormone receptor FTZ-F1 and mouse embryonal long terminal repeat-binding protein.

Fruit fly FTZ-F1, silkworm BmFTZ-F1, and mouse embryonal long terminal repeat-binding protein are members of the nuclear hormone receptor superfamily, which recognizes the same sequence, 5'-PyCAAGGPyCPu-3'. Among these proteins, a 30-amino-acid basic region abutting the C-terminal end of the zinc finger motif, designated the FTZ-F1 box, is conserved. Gel mobility shift competition by various mutant peptides of the DNA-binding region revealed that the FTZ-F1 box as well as the zinc finger motif is involved in the high-affinity binding of FTZ-F1 to its target site. Using a gel mobility shift matrix competition assay, we demonstrated that the FTZ-F1 box governs the recognition of the first three bases, while the zinc finger region recognizes the remaining part of the binding sequence. We also showed that the DNA-binding region of FTZ-F1 recognizes and binds to DNA as a monomer. Occurrence of the FTZ-F1 box sequence in other members of the nuclear hormone receptor superfamily raises the possibility that these receptors constitute a unique subfamily which binds to DNA as a monomer.     

A plant DNA-binding protein that recognizes 5-methylcytosine residues.

A novel, 5-methylcytosine-specific, DNA-binding protein, DBP-m, has been identified in nuclear extracts of peas. DBP-m specifically recognizes 5-methylcytosine residues in DNA without appreciable DNA sequence specificity, unlike a mammalian DNA-binding protein (MDBP), which recognizes 5-methylcytosine residues but only in a related family of 14-base-pair sequences.     

Segments of the POU domain influence one another''s DNA-binding specificity.

The ubiquitously expressed mammalian POU-domain protein Oct-1 specifically recognizes two classes of cis-acting regulatory elements that bear little sequence similarity, the octamer motif ATGCAAAT and the TAATGARAT motif. The related pituitary-specific POU protein Pit-1 also recognizes these two motifs but, unlike Oct-1, binds preferentially to the TAATGARAT motif. Yet in our assay, Pit-1 still binds octamer elements better than does the octamer motif-binding protein Oct-3. The POU domain is responsible for recognizing these diverse regulatory sequences through multiple DNA contacts that include the two POU subdomains, the POU-specific region, and the POU homeodomain. The DNA-binding properties of 10 chimeric POU domains, in which different POU-domain segments are derived from either Oct-1 or Pit-1, reveal a high degree of structural plasticity; these hybrid proteins all bind DNA well and frequently bind particular sites better than does either of the parental POU domains. In these chimeric POU domains, the POU-specific A and B boxes and the hypervariable POU linker can influence DNA-binding specificity. The surprising result is that the influence a particular segment has on DNA-binding specificity can be greatly affected by the origin of other segments of the POU domain and the sequence of the binding site. Thus, the broad but selective DNA-binding specificity of Oct-1 is conferred both by multiple DNA contacts and by dynamic interactions within the DNA-bound POU domain.     

A Family of Novel DNA-Binding Nuclear Proteins Having Polypyrimidine Tract-Binding Motif and Arginine/Serine-Rich Motif

NP220 is a DNA-binding nuclear protein originally cloned from human cell lines. Human NP220 (hNP220) has an arginine/serine-rich motif found in non-small nuclear RNP splicing factors (SR proteins) and shares three domains (MH1, MH2 and MH3) with an acidic nuclear matrix protein, matrin 3. The MH2 domain repeats three times and has homology to the polypyrimidine tract-binding motif of heterogeneous nuclear RNP I/L. NP220 also has a DNA-binding domain and nine repeats of the sequence LVTVDEVIEEEDL (acidic repeat). We have now isolated mouse equivalents of NP220 (mNP220s) and found that NP220s form a family of proteins with four members produced by alternative splicing of a common pre-mRNA. Two longer forms (NP220α and NP220β) have all functional domains mentioned above while two shorter forms (NP220γ and NP220δ) lack the DNA-binding domain and the acidic repeat. The structural aspects of NP220s are distinct from that of the SR proteins but rather resemble U2AF and Tra2 which activate a specific 3′-splicing site of specific genes in response to differentiation-dependent signals.     

YY1's longer DNA-binding motifs

The DNA-binding sites of YY1 located within two newly identified downstream genes of YY1, Peg3 (GGCGCCATnTT) and Xist (CCGCCATnTT), are longer than the known motif of YY1 (CGCCATnTT). Gel shift assays indicated that these DNA-binding sites are previously unnoticed, longer motifs of YY1. Independent DNA-binding motif studies further confirmed that YY1 recognizes a longer sequence (GCCGCCATTTTG) as its consensus motif. This longer motif exhibited higher affinity to the YY1 protein than the known motif. Another DNA-binding motif study was also performed using a protein containing three amino acid substitutions in the first zinc finger unit of YY1, mimicking the zinc finger domain of pho (a drosophila homologue of YY1). The substitutions cause the weakening of DNA-binding specificity at both 5'- and 3'-sides of the longer motif, yielding a much shorter sequence (GCCAT) as a consensus motif. This indicates that the intact first finger unit is required for recognition of the longer motif of YY1. Also, this shortening suggests that the DNA recognition by YY1 is mediated through the concerted, but not modular, contribution by its four zinc finger units.     

Mbh 1: a novel gelsolin/severin-related protein which binds actin in vitro and exhibits nuclear localization in vivo.

We describe the characterization of a novel cDNA, mbh1 (myc basic motif homolog-1), which was found during a search for candidate factors which might interact with the c-Myc oncoprotein. Embedded within the amino acid sequence encoded by mbh1 is a region distantly related to the basic/helix-loop-helix (B/HLH) DNA-binding motif and a potential nuclear localization signal. Mbh1 encodes a polypeptide structurally similar to the actin-severing proteins gelsolin and severin. Translation of mbh1 RNA in rabbit reticulocyte extracts produces an approximately 45 kd protein capable of binding actin-coupled agarose beads in vitro in a Ca2(+)-dependent manner. Antiserum raised to a trpE/mbh1 bacterial fusion protein recognizes an approximately 45 kb protein in murine 3T3 fibroblasts, suggesting that the cDNA encodes the complete Mbh1 protein. Examination of Mbh1 localization in 3T3 fibroblasts by indirect immunofluorescence reveals a larger cell population showing diffuse staining, and a smaller population exhibiting a distinct nuclear stain. Western analysis corroborates this intracellular localization and indicates that total cellular levels and localization of Mbh1 are not affected by the cell growth state. The data suggest that Mbh1 may play a role in regulating cytoplasmic and/or nuclear architecture through potential interactions with actin.     

Methylated DNA-binding protein from human placenta recognizes specific methylated sites on several prokaryotic DNAs.

Methylated DNA-binding protein (MDBP) from human placenta recognizes specific DNA sequences containing 5-methylcytosine (m5C) residues. Comparisons of binding of various prokaryotic DNAs to MDBP indicate that m5CpG is present in the recognition sites for this protein but is only part of the recognition sequence. Specific binding to MDBP was observed for bacteriophage XP12 DNA, which naturally contains approximately 1/3 of its residues as m5C, and for Micrococcus luteus DNA, M13mp8 replicative form (RF) DNA, and pBR322 when these three DNAs were methylated at CpG sites by human DNA methyltransferase. Five DNA regions binding to MDBP have been localized by DNase I footprinting or restriction mapping in methylated pBR322 and M13mp8 RF DNAs. A comparison of their sequences reveals a common 5'-m5CGRm5CG-3' element or closely related sequence in which one of the m5C residues may be replaced by a T. In addition to this motif, one upstream and one downstream m5CpG as well as other common residues over an approximately 20-bp long region may be recognized by MDBP.     

Purification and characterization of Ku-2, an octamer-binding protein related to the autoantigen Ku

The octamer motif (ATTTGCAT) is an important regulatory element in eukaryotic gene expression. A previously unidentified protein that recognizes this motif has been isolated from the human B cell line, Daudi. The protein, which we term Ku-2, bears a close resemblance to the DNA-binding autoantigen Ku. Like Ku, it is a heterodimer with subunits of 83 and 72 kDa; antisera raised against either subunit of Ku cross-react with Ku-2. Two peptides have been sequenced and show a strong similarity to regions in the corresponding subunits of Ku. The sequences are not identical, however, suggesting that Ku-2 may be a B cell homologue of Ku. Both Ku and Ku-2 bind to the termini of DNA duplexes, but Ku-2 also binds to an internal octamer motif. It is not known whether Ku shares the latter property or whether the octamer binding is a consequence of sequence differences between the two proteins. Ku-2 does not react with antisera against the POU domain of the octamer-binding protein Oct-2, indicating that the DNA binding domains of the two proteins are dissimilar despite the ability of both to bind to the octamer motif. We discuss the evidence for the existence of a family of octamer-binding proteins related to Ku.     

Detecting DNA-binding helix-turn-helix structural motifs using sequence and structure information

In this work, we analyse the potential for using structural knowledge to improve the detection of the DNA-binding helix–turn–helix (HTH) motif from sequence. Starting from a set of DNA-binding protein structures that include a functional HTH motif and have no apparent sequence similarity to each other, two different libraries of hidden Markov models (HMMs) were built. One library included sequence models of whole DNA-binding domains, which incorporate the HTH motif, the second library included shorter models of ‘partial’ domains, representing only the fraction of the domain that corresponds to the functionally relevant HTH motif itself. The libraries were scanned against a dataset of protein sequences, some containing the HTH motifs, others not. HMM predictions were compared with the results obtained from a previously published structure-based method and subsequently combined with it. The combined method proved more effective than either of the single-featured approaches, showing that information carried by motif sequences and motif structures are to some extent complementary and can successfully be used together for the detection of DNA-binding HTHs in proteins of unknown function.     

GT-2: a transcription factor with twin autonomous DNA-binding domains of closely related but different target sequence specificity.

A triplet of adjacent, highly similar GT motifs in the phyA promoter of rice functions to support maximal expression of this gene. We have obtained a recombinant clone that encodes a full-length nuclear protein, designated GT-2, which binds specifically to these target sequences. This novel protein contains acidic, basic and proline- + glutamine-rich regions, as well as two autonomous DNA-binding domains, one NH2-terminal and the other COOH-terminal, that discriminate with high resolution between the three GT motifs. A duplicated sequence of 75 amino acids, present once in each DNA-binding domain, appears likely to mediate DNA target element recognition. Each copy of this duplicated protein sequence is predicted to form three amphipathic alpha-helices separated from each other by two short loops. The absence of sequence similarity to other known proteins suggests that this predicted structural unit, which we term the trihelix motif, might be representative of a new class of DNA-binding proteins.     

Site-specific DNA binding of nuclear factor I: analyses of cellular binding sites.

Nuclear factor I is a cellular site-specific DNA-binding protein required for the efficient in vitro replication of adenovirus DNA. We have characterized human DNA sequences to which nuclear factor I binds. Three nuclear factor I binding sites (FIB sites), isolated from HeLa cell DNA, each contain the sequence TGG(N)6-7GCCAA. Comparison with other known and putative FIB sites suggests that this sequence is important for the binding of nuclear factor I. Nuclear factor I protects a 25- to 30-base-pair region surrounding this sequence from digestion by DNase I. Methylation protection studies suggest that nuclear factor I interacts with guanine residues within the TGG(N)6-7GCCAA consensus sequence. One binding site (FIB-2) contained a restriction endonuclease HaeIII cleavage site (GGCC) at the 5' end of the GCCAA motif. Digestion of FIB-2 with HaeIII abolished the binding of nuclear factor I. Southern blot analyses indicate that the cellular FIB sites described here are present within single-copy DNA in the HeLa cell genome.     

Regulated expression of nuclear protein(s) in myogenic cells that binds to a conserved 3'' untranslated region in pro alpha 1 (I) collagen cDNA.

We describe the identification and DNA-binding properties of nuclear proteins from rat L6 myoblasts which recognize an interspecies conserved 3' untranslated segment of pro alpha 1 (I) collagen cDNA. Levels of the two pro alpha 1 (I) collagen RNAs, present in L6 myoblasts, decreased drastically between 54 and 75 h after induction of myotube formation in serum-free medium. Both mRNAs contained a conserved sequence segment of 135 nucleotides (termed tame sequence) in the 3' untranslated region that had 96% homology to the human and murine pro alpha 1 (I) collagen genes. The cDNA of this tame sequence was specifically recognized by nuclear protein(s) from L6 myoblasts, as judged by gel retardation assays and DNase I footprints. The tame-binding protein(s) was able to recognize its target sequence on double-stranded DNA but bound also to the appropriate single-stranded oligonucleotide. Protein that bound to the tame sequence was undetectable in nuclear extracts of L6 myotubes that did not accumulate the two collagen mRNAs. Therefore, the activity of this nuclear protein seems to be linked to accumulation of the sequences that it recognizes in vitro. The collagen RNAs and the nuclear tame-binding proteins reappeared after a change of medium, which further suggests that the RNAs and the protein(s) are coordinately regulated.     

Mapping of Human DNA-binding Nuclear Protein (NP220) to Chromosome Band 2p13.1-p13.2 and Its Relation to Matrin 3

Human NP220 (hNP220) is a novel DNA-binding nuclear protein, which has an arginine/serine-rich motif and polypyrimidine tract-binding motif, and NP220s and matrin 3 are thought to form a novel family of nuclear proteins. We have determined a chromosomal localization of the cDNA encoding human NP220 to 2p13.1-p13.2 by using fluorescence in situ hybridization. Human matrin 3 cDNA was mapped to chromosomes 1p13.1-p21.1 and 5q31.3, demonstrating that these novel nuclear proteins with similar functions are on different chromosomes.