Physical and functional interaction between two pluripotent proteins, the Y-box DNA/RNA-binding factor, YB-1, and the multivalent zinc finger factor, CTCF.

CTCF is a unique, highly conserved, and ubiquitously expressed 11 zinc finger (ZF) transcriptional factor with multiple DNA site specificities. It is able to bind to varying target sequences to perform different regulatory roles, including promoter activation or repression, creating hormone-responsive gene silencing elements, and functional block of enhancer-promoter interactions. Because different sets of ZFs are utilized to recognize different CTCF target DNA sites, each of the diverse DNA.CTCF complexes might engage different essential protein partners to define distinct functional readouts. To identify such proteins, we developed an affinity chromatography method based on matrix-immobilized purified recombinant CTCF. This approach resulted in isolation of several CTCF protein partners. One of these was identified as the multifunctional Y-box DNA/RNA-binding factor, YB-1, known to be involved in transcription, replication, and RNA processing. We examined CTCF/YB-1 interaction by reciprocal immunoprecipitation experiments with anti-CTCF and anti-YB-1 antibodies, and found that CTCF and YB-1 form complexes in vivo. We show that the bacterially expressed ZF domain of CTCF is fully sufficient to retain YB-1 in vitro. To assess possible functional significance of CTCF/YB-1 binding, we employed the very first identified by us, negatively regulated, target for CTCF (c-myc oncogene promoter) as a model in co-transfection assays with both CTCF and YB-1 expression vectors. Although expression of YB-1 alone had no effect, co-expression with CTCF resulted in a marked enhancement of CTCF-driven c-myc transcriptional repression. Thus our findings demonstrate, for the first time, the biological relevance of the CTCF/YB-1 interaction.

Molecular weight abnormalities of the CTCF transcription factor: CTCF migrates aberrantly in SDS-PAGE and the size of the expressed protein is affected by the UTRs and sequences within the coding region of the CTCF gene.

CTCF belongs to the Zn finger transcription factors family and binds to the promoter region of c-myc. CTCF is highly conserved between species, ubiquitous and localised in nuclei. The endogenous CTCF migrates as a 130 kDa (CTCF-130) protein on SDS-PAGE, however, the open reading frame (ORF) of the CTCF cDNA encodes only a 82 kDa protein (CTCF-82). In the present study we investigate this phenomenon and show with mass-spectra analysis that this occurs due to aberrant mobility of the CTCF protein. Another paradox is that our original cDNA, composed of the ORF and 3'-untranslated region (3'-UTR), produces a protein with the apparent molecular weight of 70 kDa (CTCF-70). This paradox has been found to be an effect of the UTRs and sequences within the coding region of the CTCF gene resulting in C-terminal truncation of CTCF-130. The potential attenuator has been identified and point-mutated. This restored the electrophoretic mobility of the CTCF protein to 130 kDa. CTCF-70, the aberrantly migrating CTCF N-terminus per se, is also detected in some cell types and therefore may have some biological implications. In particular, CTCF-70 interferes with CTCF-130 normal function, enhancing transactivation induced by CTCF-130 in COS6 cells. The mechanism of CTCF-70 action and other possible functions of CTCF-70 are discussed.

CTCF, a conserved nuclear factor required for optimal transcriptional activity of the chicken c-myc gene, is an 11-Zn-finger protein differentially expressed in multiple forms.

A novel sequence-specific DNA-binding protein, CTCF, which interacts with the chicken c-myc gene promoter, has been identified and partially characterized (V. V. Lobanenkov, R. H. Nicolas, V. V. Adler, H. Paterson, E. M. Klenova, A. V. Polotskaja, and G. H. Goodwin, Oncogene 5:1743-1753, 1990). In order to test directly whether binding of CTCF to one specific DNA region of the c-myc promoter is important for chicken c-myc transcription, we have determined which nucleotides within this GC-rich region are responsible for recognition of overlapping sites by CTCF and Sp1-like proteins. Using missing-contact analysis of all four nucleotides in both DNA strands and homogeneous CTCF protein purified by sequence-specific chromatography, we have identified three sets of nucleotides which contact either CTCF or two Sp1-like proteins binding within the same DNA region. Specific mutations of 3 of 15 purines required for CTCF binding were designed to eliminate binding of CTCF without altering the binding of other proteins. Electrophoretic mobility shift assay of nuclear extracts showed that the mutant DNA sequence did not bind CTCF but did bind two Sp1-like proteins. When introduced into a 3.3-kbp-long 5'-flanking noncoding c-myc sequence fused to a reporter CAT gene, the same mutation of the CTCF binding site resulted in 10- and 3-fold reductions, respectively, of transcription in two different (erythroid and myeloid) stably transfected chicken cell lines. Isolation and analysis of the CTCF cDNA encoding an 82-kDa form of CTCF protein shows that DNA-binding domain of CTCF is composed of 11 Zn fingers: 10 are of C2H2 class, and 1 is of C2HC class. CTCF was found to be abundant and conserved in cells of vertebrate species. We detected six major nuclear forms of CTCF protein differentially expressed in different chicken cell lines and tissues. We conclude that isoforms of 11-Zn-finger factor CTCF which are present in chicken hematopoietic HD3 and BM2 cells can act as a positive regulator of the chicken c-myc gene transcription. Possible functions of other CTCF forms are discussed.

Biochemical characterization of matrilysin. Activation conforms to the stepwise mechanisms proposed for other matrix metalloproteinases.

The latent precursor of matrilysin (EC 3.4.24.23; punctuated metalloproteinase (PUMP) was purified from transfected mouse myeloma cell conditioned medium and was found to contain one zinc atom per molecule which was essential for catalytic activity. Promatrilysin could be activated to the same specific activity by (4-aminophenyl)mercuric acetate, trypsin, and incubation at elevated temperatures (heat activation). Active matrilysin hydrolyzed the fluorescent substrate 2,4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond with a maximum value for kcat/Km of 1.3 x 10(4) M-1 s-1 at the pH optimum of 6.5 and pKa values of 4.60 and 8.65. Activity is inhibited by the tissue inhibitor of metalloproteinases-1 in a 1:1 stoichiometric interaction. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis in conjunction with N-terminal sequencing revealed that, as with all other matrix metalloproteinases similarly studied, promatrilysin activation was accompanied by the stepwise proteolytic removal of an M(r) 9000 propeptide from the N-terminus. The intermediates generated were dependent on the mode of activation used but, in all cases studied, activation terminated with an autocatalytic cleavage at E77-Y78 to yield the final M(r) 19,000 active matrilysin. From an analysis of the stability of the various intermediates, we propose that the sequence L13-K33 is particularly important in protecting the E77-Y78 site from autocatalytic cleavage, thereby maintaining the latency of the proenzyme.

Induction of differentiation of avian erythroblastosis virus-transformed erythroblasts by the protein kinase inhibitor H7: analysis of the transcription factor EF1.

The protein kinase inhibitor H7 [1-5(isoquinolinesulfonyl-2-methylpiperazine)] together with a temperature shift to 42 degrees C was found to reproducibly and efficiently induce differentiation of avian erythroblasts transformed with the avian erythroblastosis virus containing v-erbA and a temperature-sensitive v-erbB oncogene. Although a temperature shift to 42 degrees C without H7 results in some elevation of globin transcripts, much higher levels of transcripts accrue when cells are incubated at 42 degrees C with H7; under such conditions, 50-70% of the cells become benzidine positive. In order to investigate the mechanism by which the differentiation occurs, we have characterized and analyzed the levels of the erythroid transcription factor EF1, originally described as a factor binding to the beta H-globin promoter. Protein sequencing of EF1 shows that it is identical to the factor Eryf1. Using a peptide antibody and DNA-binding assays, we demonstrate that EF1 is present at high levels in the nucleus of undifferentiated HD3 cells, and, although there may be a small change when the cells are shifted to 42 degrees C, incubation of the cells with H7 at 42 degrees C does not result in a further elevation commensurate with the high levels of globin transcripts. It is concluded that v-erbA and v-erbB do not repress differentiation by limiting the levels of EF1.

Disulphide bond assignment in human tissue inhibitor of metalloproteinases (TIMP).

Disulphide bonds in human recombinant tissue inhibitor of metalloproteinases (TIMP) were assigned by resolving proteolytic digests of TIMP on reverse-phase h.p.l.c. and sequencing those peaks judged to contain disulphide bonds by virtue of a change in retention time on reduction. This procedure allowed the direct assignment of Cys-145-Cys-166 and the isolation of two other peptides containing two disulphide bonds each. Further peptide cleavage in conjunction with fast-atom-bombardment m.s. analysis permitted the assignments Cys-1-Cys-70, Cys-3-Cys-99, Cys-13-Cys-124 and Cys-127-Cys-174 from these peptides. The sixth bond Cys-132-Cys-137 was assigned by inference, as the native protein has no detectable free thiol groups.

Purification to homogeneity and partial amino acid sequence of a fragment which includes the methyl acceptor site of the human DNA repair protein for O6-methylguanine.

DNA repair by O6-methylguanine-DNA methyltransferase (O6-MT) is accomplished by removal by the enzyme of the methyl group from premutagenic O6-methylguanine-DNA, thereby restoring native guanine in DNA. The methyl group is transferred to an acceptor site cysteine thiol group in the enzyme, which causes the irreversible inactivation of O6-MT. We detected a variety of different forms of the methylated, inactivated enzyme in crude extracts of human spleen of molecular weights higher and lower than the usually observed 21-24kDa for the human O6-MT. Several apparent fragments of the methylated form of the protein were purified to homogeneity following reaction of partially-purified extract enzyme with O6-[3H-CH3]methylguanine-DNA substrate. One of these fragments yielded amino acid sequence information spanning fifteen residues, which was identified as probably belonging to human methyltransferase by virtue of both its significant sequence homology to three procaryote forms of O6-MT encoded by the ada, ogt (both from E. coli) and dat (B. subtilis) genes, and sequence position of the radiolabelled methyl group which matched the position of the conserved procaryote methyl acceptor site cysteine residue. Statistical prediction of secondary structure indicated good homologies between the human fragment and corresponding regions of the constitutive form of O6-MT in procaryotes (ogt and dat gene products), but not with the inducible ada protein, indicating the possibility that we had obtained partial amino acid sequence for a non-inducible form of the human enzyme. The identity of the fragment sequence as belonging to human methyltransferase was more recently confirmed by comparison with cDNA-derived amino acid sequence from the cloned human O6-MT gene from HeLa cells (1). The two sequences compared well, with only three out of fifteen amino acids being different (and two of them by only one nucleotide in each codon).

Dissociation of tissue inhibitor of metalloproteinases (TIMP) from enzyme complexes yields fully active inhibitor.

Recombinant human tissue inhibitor of metalloproteinases (TIMP) forms complexes with high-Mr active recombinant stromelysin that are stable over long periods under physiological conditions. TIMP-stromelysin complexes could be dissociated in the presence of EDTA at pH 3, releasing free TIMP and destroying stromelysin activity. The dissociated TIMP was apparently unmodified, in contrast with other known protein inhibitors of metalloproteinases and many classes of serine-proteinase inhibitor, which are slowly cleaved.

Chemical modification of proteins.

The two most widely used applications for chemical modification are in primary structure analysis and in the identification of essential groups involved in the binding and catalytic sites of proteins. The methods discussed here are those used frequently in primary structure analysis. Chemical modifications involving protein "active center" identification are the subject of a review by Pfleiderer (1).

An H1-like protein from the macronucleus of Euplotes eurystomus.

An H1-like protein has been purified from the macronucleus (MAC) of the hypotrichous ciliated protozoan, Euplotes eurystomus. It is present in amounts comparable to the inner histones and is extracted by treatment with 5% perchloric acid or 0.65 M NaCl, but not by 0.35 M NaCl. Treatment of soluble MAC chromatin with the ionic exchange resin AG 50W-X2 in 80 mM NaCl removes MAC H1 and yields H1-depleted chromatin. Mac H1 is lysine-rich and deficient in acidic amino acids. The stoichiometry of the H1 protein is reduced in mononucleosome preparations, consistent with its postulated interaction with linker DNA regions. Thermal denaturation and circular dichroism studies reveal that H1-depleted chromatin contains a larger portion of destabilized DNA than control chromatin. The molecular weight of Euplotes MAC H1 is significantly smaller than most reported H1 proteins. Comparisons are made with extracts of macronuclei from other hypotrichous ciliated protozoa and published reports of other lower eukaryotes.

D-glyceraldehyde-3-phosphate dehydrogenase. Complete amino-acid sequence of the enzyme from Bacillus stearothermophilus.

1. The complete amino acid sequence of D-glyceraldehyde-3-phosphate dehydrogenase from the moderate thermophile Bacillus stearothermophilus has been determined. 2. This has been achieved largely by the automated sequence analysis of large fragements derived by chemical cleavage with cyanogen bromide, BNPS-skatole [the product of reaction between N-bromosuccinimide and 2-(nitrophenyl-sulphenyl)-3-methylindole] and hydroxylamine and enzymic hydrolysis with trypsin at arginine residues. 3. The sequence is as follows: (See Text). It has been numbered to maximise homology with the four complete sequences of this enzyme from other sources. Hence the N-terminal residue is numberd 0 and two deletions and two insertions have been introduced. 4. The inability of the B. stearothermophilus apo-enzyme to transfer an acyl moiety from Cys-149 to Lys-183 oberved with muscle enzymes is explained by the replacement of lysine by arginine in the enzyme from the thermophilic organism. 5. The sequences of the S-loop regions, which form the core of the tetrameric enzyme, are similar to each other in B. stearothermophilus and Thermus aquaticus and differ from the highly conserved S-loops of three enzymes from mesophiles.