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.

Functional phosphorylation sites in the C-terminal region of the multivalent multifunctional transcriptional factor CTCF.

CTCF is a widely expressed and highly conserved multi-Zn-finger (ZF) nuclear factor. Binding to various CTCF target sites (CTSs) is mediated by combinatorial contributions of different ZFs. Different CTSs mediate distinct CTCF functions in transcriptional regulation, including promoter repression or activation and hormone-responsive gene silencing. In addition, the necessary and sufficient core sequences of diverse enhancer-blocking (insulator) elements, including CpG methylation-sensitive ones, have recently been pinpointed to CTSs. To determine whether a posttranslational modification may modulate CTCF functions, we studied CTCF phosphorylation. We demonstrated that most of the modifications that occur at the carboxy terminus in vivo can be reproduced in vitro with casein kinase II (CKII). Major modification sites map to four serines within the S(604)KKEDS(609)S(610)DS(612)E motif that is highly conserved in vertebrates. Specific mutations of these serines abrogate phosphorylation of CTCF in vivo and CKII-induced phosphorylation in vitro. In addition, we showed that completely preventing phosphorylation by substituting all serines within this site resulted in markedly enhanced repression of the CTS-bearing vertebrate c-myc promoters, but did not alter CTCF nuclear localization or in vitro DNA-binding characteristics assayed with c-myc CTSs. Moreover, these substitutions manifested a profound effect on negative cell growth regulation by wild-type CTCF. CKII may thus be responsible for attenuation of CTCF activity, either acting on its own or by providing the signal for phosphorylation by other kinases and for CTCF-interacting protein partners.

A novel sequence-specific DNA binding protein which interacts with three regularly spaced direct repeats of the CCCTC-motif in the 5'-flanking sequence of the chicken c-myc gene.

The chicken c-myc 5'-flanking sequence has previously been shown to bind multiple proteins present in undifferentiated and differentiated red blood cells. In this report the protein binding to one specific region within a hypersensitive site approximately 200 base pairs upstream of the start of transcription has been analysed in detail. Using a combination of a modified agarose gel retardation assay with O-phenanthroline-copper footprinting in situ, missing contact point and methylation interference techniques, two proteins were found to bind to overlapping sequences within 180-230 bp upstream of the start of transcription. One protein resembles the transcription factor Sp1, the other is a protein which binds to three regularly spaced repeats of the core sequence CCCTC. This CCCTC-binding factor was termed CTCF. It requires additional sequences outside the three recognition motifs for tight binding. CTCF was purified to near homogeneity by sequence-specific DNA chromatography. The approximate molecular weight of the CTCF was estimated to be 130,000. Removal of 110 bp sequence binding both CTCF and Sp1-like proteins leads to a 4 to 8-fold increase in transcription of stably transfected c-myc fusion constructs in chicken embryonic fibroblasts, suggesting that the CTCF is likely to be one of multiple nuclear factors involved in the transcriptional regulation of the chicken c-myc gene.

Are the high mobility group non-histone chromosomal proteins associated with 'active' chromatin?

When 10--20% of the DNA in thymus and liver nuclei is rendered acid-soluble by DNAase I digestion, under conditions which may be expected to specifically degrade away most of the transcribed sequences, very little of the high-mobility-group non-histone proteins are released from the nuclei. Therefore, high-mobility-group proteins are probably not specifically associated with this portion of the genome.

The isolation of three new high mobility group nuclear proteins.

In addition to the four high mobility group non-histone chromosomal proteins HMG 1, 2, 14 and 17 and histone H1, perchloric acid extracts of nuclei contain a number of other smaller low molecular weight proteins. Three of these proteins (HMG 18, 19A, and 19B) have been purified and characterized. Protein HMG 18 has high lysine and alanine contents, resembling histone H1. Proteins HMG 19A and 19B have high contents of basic and acidic amino acids and resemble HMG proteins 1, 2, 14 and 17. N-terminal sequence analyses of the proteins show that they are not degradation products of histones or the other HMG proteins. However, there are sequence similarities between HMG 18 and histone H5, and between HMG 19B and HMG 17, supporting the view that the HMG proteins and the lysine-rich histones are functionally related.

An improved large scale fractionation of high mobility group non-histone chromatin proteins.

1. Methodology is presented for the large scale preparation and fractionation of high mobility group proteins from calf thymus chromatin. The total high mobility group protein from approx. 1 kg calf thymus tissue can be separated into five fractions by CM-Sephadex C25 ion-exchange chromatography. High mobility group proteins 1 and 2 comprise two fo the fractions. From a third fraction two more chromatin proteins, protein 3 and 17, can be isolated by trichloroacetic acid precipitation and CM-cellulose chromatography at pH 5.5. 2. The four proteins thus purified are lysine-rich proteins. Proteins 1 and 2 are additionally characterised by their high contents of acidic amino acids, as described previously (Goodwin, G. H. and Johns, E. W. (1973) Eur. J. Biochem. 40, 215-219). Proteins 3 and 17, having lower contents of acidic amino acids, are basic proteins similar to the histones. All four proteins exhibit single N-terminal amino acids; glycine is the N-terminal group of proteins 1, 2 and 3; protein 17 has a proline N-terminal amino acid. The proteins are not highly phosphorylated nor are they associated with appreciable quantities of nucleic acid.

Studies on the degradation of high mobility group non-histone chromosomal proteins.

During the isolation of high mobility group non-histone proteins from calf thymus chromatin by methods described previously (e.g. Goodwin, G.H., Nicolas, R.H. and Johns, E.W. (1975) Biochim. Biophys, Acta. 405, 280--291) protein degradation occurs resulting in a number of proteins appearing in the chromatin extracts which are not present in high mobility group protein preparations in which proteolysis has been completely inhibited. These extra proteins, formerly numbered high mobility group proteins 3, 5, 6 and 8, are thus probably degradation products of other nuclear proteins, produced during the isolation procedure. From the amino acid analyses, tryptic peptides and N-terminal sequences, it is concluded that high mobility group protein 3 is probably a degradation product of high mobility group protein 1. The amino acid analysis of high mobility group protein 8 is very similar to that of the N-terminal half of histone H1 suggesting that high mobility group protein 8 is a degradation product of this histone.

Comparisons of the structures of the chromosomal high mobility group proteins HMG1 and HMG2 prepared under conditions of neutral and acidic pH.

The chromosomal proteins HMG1 and 2 have been prepared by salt extraction and phosphocellulose chromatography at neutral pH (Isackson, P.J., Debold, W.A. and Reeck, G.R. (1980) FEBS Lett. 119, 337-342) to minimize protein denaturation. The structures of these phosphocellulose-prepared high mobility group proteins have been compared with those of high mobility group proteins using the previously described acid-extraction conditions which fully denature the proteins. When compared in the same solvent conditions the acid-extracted proteins did not refold to give the same level of alpha-helical and tertiary folded structures as the phosphocellulose-prepared proteins, suggesting that acid treatment can cause some irreversible damage to the proteins. This finding was supported by changes in the structure observed when phosphocellulose-prepared HMG1 was neutralized after exposure to acid. Gel filtration studies reveal no differences in the size of the high mobility group proteins, phosphocellulose-prepared and acid-extracted proteins both being largely monomeric in solution. Little difference was detected in the DNA-binding properties of the two types of protein, nor was there any difference in the oxidation state of the cysteines. However, isoelectric focusing analysis revealed differences in the subfractions of HMG2 prepared by the two methods.

cDNA cloning of a homeobox-containing gene expressed in avian myeloblastic virus-transformed chicken monoblastic leukaemia cells.

By combining the polymerase chain reaction and differential library screening, a cDNA for an mRNA expressed in chicken avian myeloblastosis virus (AMV)-transformed monoblasts was isolated. This mRNA is not expressed in erythroblast or T-lymphoblast cell lines. Induced differentiation of the cells of the AMV-transformed BM2 line was associated with reduced levels of this transcript. The predicted protein product of Chox M was a homeodomain factor similar to murine Hox-4.3.

A myeloid-lineage-specific enhancer upstream of the mouse myeloperoxidase (MPO) gene.

The myeloperoxidase (MPO) gene is selectively expressed during haemopoiesis in the granulocytic lineage. Compared with the erythroid (beta-globin) and B-cell (immunoglobulin) lineages, little is known of the regulatory sequences and transcription factors involved in the regulation of genes specific for granulopoiesis. We have approached this issue by identifying a strong enhancer for the murine MPO gene. A candidate enhancer region was mapped by the detection of a strong DNase I hypersensitive site, -3.4 to -3.2 kb upstream of the MPO gene. A 301 bp fragment encompassing the DNase I site was shown to have strong enhancer function in a transient assay following transfection of a reporter gene into a MPO-expressing cell (WEHI 3BD+), but was inactive in lymphoid cells. Analysis of sub-fragments revealed that the whole 301 bp fragment is required for maximal enhancer function.

Isolation of cDNAs encoding chicken homologues of the yeast SNF2 and Drosophila Brahma proteins.

The SNF2/Brahma proteins are a class of DNA-dependent ATPases which activate gene expression by disrupting chromatin repression. They also cooperate with nuclear hormone receptors to activate transcription. Two cDNAs encoding chicken homologues of the SNF2/Brahma proteins have been isolated from chicken haematopoietic libraries. The encoded proteins closely resemble the human homologues, hBRM and BRG1, and the chicken homologues have therefore been termed cBRH and cBRG1. Homology is conserved in five characteristic domains: an N-terminal domain that binds the SNF11 protein, a conserved domain A of unknown function, a central ATPase domain, a domain that binds the retinoblastoma tumor suppressor protein Rb, and a C-terminal bromodomain of unknown function.

Molecular cloning of polybromo, a nuclear protein containing multiple domains including five bromodomains, a truncated HMG-box, and two repeats of a novel domain.

A number of transcription factors that act as adaptor proteins have been found to contain an 87 amino acid domain called the bromodomain. In a study to identify and characterise bromodomain proteins expressed in chicken cells, a novel gene has been isolated which encodes five repeats of the bromodomain. In addition, the encoded protein, termed polybromo, contains four other domains: an unusual truncated HMG box, two repeats of a novel domain which we term the BAH domain and a sequence related to a region within the regulatory domain of the DNA cytosine-5 methyltransferase enzyme. Polybromo was found to be related to a yeast protein U19102 which has two bromo domains, a BAH domain and the DNA methyltransferase-related sequence. Antibodies that were raised against polybromo were used in confocal microscopy analysis to show that the 180-kDa polybromo protein is located within the nucleus but excluded from the nucleolus. Gel filtration analysis of nuclear extracts demonstrate that polybromo is part of a large complex with a mass of approximately 2 million dalton.

The BAH domain, polybromo and the RSC chromatin remodelling complex.

The BAH (Bromo-adjacent homology) domain is a domain first identified in the vertebrate polybromo protein, a protein present in a large nuclear complex. Polybromo has two BAH domains, six bromodomains and an HMG-box. The BAH domain has been identified in a number of proteins involved in gene transcription and repression and is likely to be involved in protein-protein interactions. Polybromo resembles two related proteins in yeast, the Rsc1 and Rsc2 proteins, both having a BAH domain and two bromodomains as well as a DNA binding motif, the AT -hook. The Rsc1 and 2 proteins are components of the RSC (remodelling the structure of chromatin) complex and are required for transcriptional control. In this paper we review recent data on the function of the BAH and bromodomains in relation to polybromo and the Rsc proteins.

Isolation and characterization of the gene coding for murine high-mobility-group protein HMGI-C.

The HMGI-C protein is a nuclear factor expressed in human and rodent neoplastic cells which has been shown to be involved in the process of cell transformation. We have previously isolated the cDNA encoding murine HMGI-C and now we report the cloning and analysis of the mouse Hmgi-c gene. The gene is at least 50 kb long, contains five exons, and each of the three DNA-binding domains is encoded by a different exon. The location of exon-intron junctions was determined and shown to follow the GT-AG rule. The sequence revealed that the overall organization is similar to the gene encoding human HMGI(Y), the other member of the HMGI family, suggesting that HMGI genes probably evolved through gene duplication and exon shuffling events from an ancestral gene. A highly homologous pseudogene is also present in the mouse genome. Our results on Hmgi-c structure provide basic information to carry out further studies on the regulation of its expression.

A homology-based molecular model of the proline-rich homeodomain protein Prh, from haematopoietic cells.

A molecular structural model for the homeodomain of the haematopoietic protein Prh together with its DNA recognition sequence, has been built using the known crystal structure of the MAT alpha 2 homeodomain as a starting-point. The modelling procedure used main and side-chain optimisations by means of molecular mechanics/simulated annealing procedures to obtain stereochemically plausible geometries. The resulting structure has a number of specific interactions in both major and minor grooves of the DNA that serve to define the consensus binding sequence for Prh. In particular, the side-chain of glutamine 50 is postulated to be involved in hydrogen bonds to adjacent adenine and cytosine bases within the consensus sequence.

Detection of Sequence-Specific Protein-DNA Interactions by the DNA-Footprinting Technique.

The initiation of transcription of eukaryotic genes by RNA polymerases is controlled by complex interactions between nonhistone proteins and specific regulatory DNA sequences (promoters and enhancers) (1). In order to characterize and purify such transacting protein factors, a sensitive and accurate assay for sequence-specific DNA-binding proteins is the DNA-footprinting technique, which can be used to analyze the interaction of a complex mixture of proteins with a gene regulatory sequence(s) that is known to be important for the expression of that gene.

A method for silver staining HMG chromosomal proteins in polyacrylamide electrophoretic gels.

A method is presented for sensitive staining of the HMG14 and 17 proteins in polyacrylamide gels pre-stained with Coomassie Blue R250. The procedure involves binding negatively and positively charged polycyclic aromatic compounds to the proteins followed by staining with silver using the method of Wray et al. (1981).