My major studies are designed to emphasize biological implications of unusual Multiple Sequence Specificity Possessed by Two Central 11 Zinc Finger DNA Binding Domains within 2 Full Length Proteins that Comprise “Mammalian CTCF CTCFL/BORIS Trans Factor Family” co-Evolved In Human (Epi)Genomes Together with both Loss and Gains of Dissimilar CTCF Target Sites (CTS) a.k.a. CTCF Binding Sites (CBS). The latter include but not limited to 1st) Mitotic Bookmarking of Promoter-Specific Interactions, and 2nd ) maintaining Trans-Generationally Heritable epigenetic marks placed irreversibly by CTCF BORIS hetero-dimers along haploid sperm chromatin.
Rat livers were disrupted in the TMS buffer (10 mM Tris-HCl, pH 7.6, 5 mM MgCl2, 0.25 M sucrose +... more Rat livers were disrupted in the TMS buffer (10 mM Tris-HCl, pH 7.6, 5 mM MgCl2, 0.25 M sucrose +70 microM beta-mercaptoethanol) and the nuclei were purified by sedimentation through 2.2 M sucrose with the same components as in TMS. Then the nuclei were resuspended and washed 3 times in TMS. After that the nuclei were resuspended to 1 mg DNA per ml in TM buffer (10 mM Tris-HCl, pH 7.6, 0.2 mM MgCl2) followed by centrifugation at low speed. About 60% of total nuclear DNP was recovered by this extraction. The protein/DNA ratio in the extracted chromatin fraction (DNPs) was about 1.1. The bulk of the non-extracted in TM residual chromatin fraction was released from the nuclear pellet after treatment with micrococcal nuclease. This matrix-associated chromatin fraction (DNPm) is significantly enriched in non-histone proteins as compared with the DNPs; hence the protein/DNA ratio of DNPm is at least two times higher than that of DNPs. The protein components of DNPs are represented by five histones containing negligible non-histone admixture. One of them was identified as protein A24, another--as non-dissociated from DNA in 0.6 M NaCl acid-soluble protein with m. w. of about 42,500. The possible structural features of these two distinguishable chromatin fractions are discussed.
Isolated cell nuclei were incubated with nucleases followed by extraction of chromatin with a low... more Isolated cell nuclei were incubated with nucleases followed by extraction of chromatin with a low salt buffer. With an increase of nuclear chromatin degradation with DNAse I or micrococcal nuclease, solubilization of deoxyribonucleoprotein (DNP) by a low salt buffer increases, reaching a maximum upon hydrolysis with 2-4% nuclear DNA and then decreases appreciably after extensive treatment with nucleases. Soluble fragmented chromatin aggregates in the course of treatment with DNAase. I. Addition to gel chromatin preparations of exogenous products of nuclease treatment of isolated nuclei leads to its aggregation. Pretreatment of nuclear chromatin with RNAase prevents solubilization of DNP by low ionic strength solutions. Some experimental data obtained with the use of severe nuclease treatment are discussed; for a correct interpretation of these data the aggregation of fragmented chromatin by products of its nuclease degradation should be taken into consideration.
Constitutive over-expression of myc oncogenes in the stem cell compartment of the bursa-of Fabric... more Constitutive over-expression of myc oncogenes in the stem cell compartment of the bursa-of Fabricius of chickens provides an in-vivo model system for analysis of the mechanisms of myc-induced, multi-staged neoplastic change. The biology, molecular biology and genetics of this system has recently been reviewed (Neiman 1994a). Briefly, the lymphoid population of embryonic follicles of the bursa is composed of large cycling lymphoblasts expressing surface IgM (Thompson et al. 1987a) This population includes a compartment of stem cells that can clonally reconstitute ablated bursal follicles in transplantation experiments (Pink et al. 1985). Deregulated expression of a myc oncogene within this stem cell compartment, either due to integration of an avian leukosis virus (ALV) near c-myc (Hayward et al. 1981) or due to ex-vivo infection of embryonic bursal lymphoblasts with a v-myc transducing myelocytomatosis virus (HB1)(Neiman et al. 1985; Thompson et al. 1987a), initiates neoplastic change in bursal follicles. This change is signaled by the replacement of the lymphoid cells of the follicle with a monomorphic population of pyroninophilic lymphoblasts producing a histologically distinctive lesion called a transformed follicle (TF)(Cooper et al. 1960; Neiman et al. 1980a, b; Baba and Humphries 1985).
Chromatin fractions from rat liver nuclei digested by nucleases were separated by differential so... more Chromatin fractions from rat liver nuclei digested by nucleases were separated by differential solubility into several fractions. Material solubilized during digestion (predominantly monomer nucleosomes and polynucleosomes) had the highest HMG14 + 17/DNA ratios but were not enriched in active gene sequences (albumin and c-Ha-ras1 genes). Material soluble in a low ionic strength buffer containing 0.2 mM MgCl2 (monomer nucleosomes and polynucleosomes) contained in addition to the histones, HMG14 and 17 plus a 41K non-histone protein. This fraction was depleted in active gene sequences and enriched in inactive sequences. The insoluble material was highly enriched in active sequences and had the lowest HMG14 + 17/DNA ratio. This fraction could be further fractionated into a histone-containing 2 M NaCl-soluble fraction and a 2 M NaCl-insoluble matrix-bound fraction, both of which were enriched in active sequences. The results show that the HMG proteins do not partition with active sequences during fractionation of chromatin. The 41K protein may be associated with inactive chromatin fraction.
Three 5'-end-labelled double-stranded linear DNA fragments of defined sequence were treat... more Three 5'-end-labelled double-stranded linear DNA fragments of defined sequence were treated with r-7, t-8-dihydroxy-t-9, 10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE). The DNA samples were then examined by gel electrophoresis both before and after denaturation and treatment with alkali. The extent of modification of deoxyguanosine (dG) residues was estimated from changes in electrophoretic mobility: at saturation less than 25% of the dG residues appeared to be modified by reaction with anti-BPDE. The determination of the sites of G-specific strand cleavage in a total of 0.5 kbp of DNA by sequencing gel electrophoresis showed that scission at dG residues is sequence specific and that whilst, for example, cleavage occurred at the central dG residues of all 5'-CGG-3' (21/21), of all 5'-TGG-3' (14/14), of all 5'-TGT-3' (7/7) and of all 5'-CGT-3' (5/5) sequences examined, it did not occur in any of the 5'-GGA-3' (0/12) or 5'-GGC-3' (0/15) sequences and only occurred rarely in the 5'-GGG-3' (1/48) and 5'-GGT-3' (2/11) sequences. No cleavage was found at internal dG residues within poly(dG)9 or poly(dG)18 sequences. The data may permit prediction of the sites of strand scission in DNA molecules of known sequence that have been modified by diol-epoxides of polycyclic hydrocarbons.
The interaction of nuclear sequence-specific DNA-binding proteins from definitive chicken erythro... more The interaction of nuclear sequence-specific DNA-binding proteins from definitive chicken erythrocytes, thymus and proliferating transformed erythroid precursor (HD3) cells with the 700-base-pair (700-bp) DNA 5'-flanking region of the chicken c-myc gene was investigated by in vitro footprint analysis. The major HD3 protein-binding activity binds to a site (site V) 200 bp upstream from the 'cap' site but, after further fractionation, a second distinct binding activity is detected to a site (site VIII) which contains both the 'CAAT' and 'SP1-binding' consensus sequences. Protein from thymus and erythrocyte cells which express c-myc at lower levels, bind to seven and eight sites respectively. In common with HD3 cell protein, they both bind to site VIII and, although binding to the sequence at site V is also detected, the footprint protection pattern is sufficiently different (site V') to suggest the involvement of different proteins in terminally differentiated and proliferating cells. The DNA-binding activities were partially fractionated by high-performance liquid chromatography gel filtration and include an erythrocyte-specific protein which binds to a c-myc gene poly(dG) homopolymer sequence similar to that found upstream of the chicken beta A-globin gene.
Isolated cell nuclei were incubated with nucleases followed by extraction of chromatin with a low... more Isolated cell nuclei were incubated with nucleases followed by extraction of chromatin with a low salt buffer. With an increase of nuclear chromatin degradation with DNAse I or micrococcal nuclease, solubilization of deoxyribonucleoprotein (DNP) by a low salt buffer increases, reaching a maximum upon hydrolysis with 2-4% nuclear DNA and then decreases appreciably after extensive treatment with nucleases. Soluble fragmented chromatin aggregates in the course of treatment with DNAase. I. Addition to gel chromatin preparations of exogenous products of nuclease treatment of isolated nuclei leads to its aggregation. Pretreatment of nuclear chromatin with RNAase prevents solubilization of DNP by low ionic strength solutions. Some experimental data obtained with the use of severe nuclease treatment are discussed; for a correct interpretation of these data the aggregation of fragmented chromatin by products of its nuclease degradation should be taken into consideration.
Rat livers were disrupted in the TMS buffer (10 mM Tris-HCl, pH 7.6, 5 mM MgCl2, 0.25 M sucrose +... more Rat livers were disrupted in the TMS buffer (10 mM Tris-HCl, pH 7.6, 5 mM MgCl2, 0.25 M sucrose +70 microM beta-mercaptoethanol) and the nuclei were purified by sedimentation through 2.2 M sucrose with the same components as in TMS. Then the nuclei were resuspended and washed 3 times in TMS. After that the nuclei were resuspended to 1 mg DNA per ml in TM buffer (10 mM Tris-HCl, pH 7.6, 0.2 mM MgCl2) followed by centrifugation at low speed. About 60% of total nuclear DNP was recovered by this extraction. The protein/DNA ratio in the extracted chromatin fraction (DNPs) was about 1.1. The bulk of the non-extracted in TM residual chromatin fraction was released from the nuclear pellet after treatment with micrococcal nuclease. This matrix-associated chromatin fraction (DNPm) is significantly enriched in non-histone proteins as compared with the DNPs; hence the protein/DNA ratio of DNPm is at least two times higher than that of DNPs. The protein components of DNPs are represented by five histones containing negligible non-histone admixture. One of them was identified as protein A24, another--as non-dissociated from DNA in 0.6 M NaCl acid-soluble protein with m. w. of about 42,500. The possible structural features of these two distinguishable chromatin fractions are discussed.
Isolated cell nuclei were incubated with nucleases followed by extraction of chromatin with a low... more Isolated cell nuclei were incubated with nucleases followed by extraction of chromatin with a low salt buffer. With an increase of nuclear chromatin degradation with DNAse I or micrococcal nuclease, solubilization of deoxyribonucleoprotein (DNP) by a low salt buffer increases, reaching a maximum upon hydrolysis with 2-4% nuclear DNA and then decreases appreciably after extensive treatment with nucleases. Soluble fragmented chromatin aggregates in the course of treatment with DNAase. I. Addition to gel chromatin preparations of exogenous products of nuclease treatment of isolated nuclei leads to its aggregation. Pretreatment of nuclear chromatin with RNAase prevents solubilization of DNP by low ionic strength solutions. Some experimental data obtained with the use of severe nuclease treatment are discussed; for a correct interpretation of these data the aggregation of fragmented chromatin by products of its nuclease degradation should be taken into consideration.
Constitutive over-expression of myc oncogenes in the stem cell compartment of the bursa-of Fabric... more Constitutive over-expression of myc oncogenes in the stem cell compartment of the bursa-of Fabricius of chickens provides an in-vivo model system for analysis of the mechanisms of myc-induced, multi-staged neoplastic change. The biology, molecular biology and genetics of this system has recently been reviewed (Neiman 1994a). Briefly, the lymphoid population of embryonic follicles of the bursa is composed of large cycling lymphoblasts expressing surface IgM (Thompson et al. 1987a) This population includes a compartment of stem cells that can clonally reconstitute ablated bursal follicles in transplantation experiments (Pink et al. 1985). Deregulated expression of a myc oncogene within this stem cell compartment, either due to integration of an avian leukosis virus (ALV) near c-myc (Hayward et al. 1981) or due to ex-vivo infection of embryonic bursal lymphoblasts with a v-myc transducing myelocytomatosis virus (HB1)(Neiman et al. 1985; Thompson et al. 1987a), initiates neoplastic change in bursal follicles. This change is signaled by the replacement of the lymphoid cells of the follicle with a monomorphic population of pyroninophilic lymphoblasts producing a histologically distinctive lesion called a transformed follicle (TF)(Cooper et al. 1960; Neiman et al. 1980a, b; Baba and Humphries 1985).
Chromatin fractions from rat liver nuclei digested by nucleases were separated by differential so... more Chromatin fractions from rat liver nuclei digested by nucleases were separated by differential solubility into several fractions. Material solubilized during digestion (predominantly monomer nucleosomes and polynucleosomes) had the highest HMG14 + 17/DNA ratios but were not enriched in active gene sequences (albumin and c-Ha-ras1 genes). Material soluble in a low ionic strength buffer containing 0.2 mM MgCl2 (monomer nucleosomes and polynucleosomes) contained in addition to the histones, HMG14 and 17 plus a 41K non-histone protein. This fraction was depleted in active gene sequences and enriched in inactive sequences. The insoluble material was highly enriched in active sequences and had the lowest HMG14 + 17/DNA ratio. This fraction could be further fractionated into a histone-containing 2 M NaCl-soluble fraction and a 2 M NaCl-insoluble matrix-bound fraction, both of which were enriched in active sequences. The results show that the HMG proteins do not partition with active sequences during fractionation of chromatin. The 41K protein may be associated with inactive chromatin fraction.
Three 5'-end-labelled double-stranded linear DNA fragments of defined sequence were treat... more Three 5'-end-labelled double-stranded linear DNA fragments of defined sequence were treated with r-7, t-8-dihydroxy-t-9, 10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE). The DNA samples were then examined by gel electrophoresis both before and after denaturation and treatment with alkali. The extent of modification of deoxyguanosine (dG) residues was estimated from changes in electrophoretic mobility: at saturation less than 25% of the dG residues appeared to be modified by reaction with anti-BPDE. The determination of the sites of G-specific strand cleavage in a total of 0.5 kbp of DNA by sequencing gel electrophoresis showed that scission at dG residues is sequence specific and that whilst, for example, cleavage occurred at the central dG residues of all 5'-CGG-3' (21/21), of all 5'-TGG-3' (14/14), of all 5'-TGT-3' (7/7) and of all 5'-CGT-3' (5/5) sequences examined, it did not occur in any of the 5'-GGA-3' (0/12) or 5'-GGC-3' (0/15) sequences and only occurred rarely in the 5'-GGG-3' (1/48) and 5'-GGT-3' (2/11) sequences. No cleavage was found at internal dG residues within poly(dG)9 or poly(dG)18 sequences. The data may permit prediction of the sites of strand scission in DNA molecules of known sequence that have been modified by diol-epoxides of polycyclic hydrocarbons.
The interaction of nuclear sequence-specific DNA-binding proteins from definitive chicken erythro... more The interaction of nuclear sequence-specific DNA-binding proteins from definitive chicken erythrocytes, thymus and proliferating transformed erythroid precursor (HD3) cells with the 700-base-pair (700-bp) DNA 5'-flanking region of the chicken c-myc gene was investigated by in vitro footprint analysis. The major HD3 protein-binding activity binds to a site (site V) 200 bp upstream from the 'cap' site but, after further fractionation, a second distinct binding activity is detected to a site (site VIII) which contains both the 'CAAT' and 'SP1-binding' consensus sequences. Protein from thymus and erythrocyte cells which express c-myc at lower levels, bind to seven and eight sites respectively. In common with HD3 cell protein, they both bind to site VIII and, although binding to the sequence at site V is also detected, the footprint protection pattern is sufficiently different (site V') to suggest the involvement of different proteins in terminally differentiated and proliferating cells. The DNA-binding activities were partially fractionated by high-performance liquid chromatography gel filtration and include an erythrocyte-specific protein which binds to a c-myc gene poly(dG) homopolymer sequence similar to that found upstream of the chicken beta A-globin gene.
Isolated cell nuclei were incubated with nucleases followed by extraction of chromatin with a low... more Isolated cell nuclei were incubated with nucleases followed by extraction of chromatin with a low salt buffer. With an increase of nuclear chromatin degradation with DNAse I or micrococcal nuclease, solubilization of deoxyribonucleoprotein (DNP) by a low salt buffer increases, reaching a maximum upon hydrolysis with 2-4% nuclear DNA and then decreases appreciably after extensive treatment with nucleases. Soluble fragmented chromatin aggregates in the course of treatment with DNAase. I. Addition to gel chromatin preparations of exogenous products of nuclease treatment of isolated nuclei leads to its aggregation. Pretreatment of nuclear chromatin with RNAase prevents solubilization of DNP by low ionic strength solutions. Some experimental data obtained with the use of severe nuclease treatment are discussed; for a correct interpretation of these data the aggregation of fragmented chromatin by products of its nuclease degradation should be taken into consideration.
Polynucleotide molecules encoding CTCF are isolated and purified and sequenced. The CTCF proteins... more Polynucleotide molecules encoding CTCF are isolated and purified and sequenced. The CTCF proteins and antibodies thereto can be used to identify mutant CTCFs in methods of diagnosis.
An isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence enc... more An isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding a human or a non-human BORIS, or a fragment of either of the foregoing; an isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence that is complementary to a nucleotide sequence encoding a human or a non-human BORIS, or a fragment of either of the foregoing; a vector comprising such an isolated or purified nucleic acid molecule; a cell comprising such a vector; an isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding a human or a non-human BORIS, or a fragment of either of the foregoing; a cell line that produces a monoclonal antibody that is specific for an aforementioned isolated or purified polypeptide molecule; and the monoclonal antibody produced by the cell line; methods of diagnosing a cancer or a predisposition to a cancer in a male or female mammal; a method of prognosticating a cancer in a mammal; a method of assessing the effectiveness of treatment of a cancer in a mammal; a method of treating a mammal prophylactically or therapeutically for a cancer, and a composition comprising a carrier and an inhibitor of BORIS.
Polynucleotide molecules encoding CTCF are isolated and purified and sequenced. The CTCF proteins... more Polynucleotide molecules encoding CTCF are isolated and purified and sequenced. The CTCF proteins and antibodies thereto can be used to identify mutant CTCFs in methods of diagnosis.
Pugacheva EM, Rivero-Hinojosa S, Espinoza CA, Mendez-Catala CF,
Kang S, Suzuki T, Kosaka-Suzuki N... more Pugacheva EM, Rivero-Hinojosa S, Espinoza CA, Mendez-Catala CF, Kang S, Suzuki T, Kosaka-Suzuki N, Robinson S, Nagarajan V, Ye Z, et al. Comparative analyses of CTCF and BORIS occupancies uncover two distinct classes of CTCF binding genomic regions. Genome Biol. 2015;16:161.
A comprehensive survey of the data on the nuclear factor I (NF-I) published by the end of 1987 is... more A comprehensive survey of the data on the nuclear factor I (NF-I) published by the end of 1987 is given. Among all the known DNA-binding chromosomal protein factors which are capable of specific interaction with certain nucleotide sequences, NF-I compels a particular attention as the first example of a conservative multifunctional factor being involved in the assembly of nuclease-hypersensitive chromatin structure within regulatory regions of active genes, in the trans-activation of RNA polymerase II transcription, and also in the replication of the adenoviral genome and presumably in the replication of some other DNAs including the cellular one.
CCCTC-binding factor (CTCF) is a conserved, essential regulator of chromatin architecture contain... more CCCTC-binding factor (CTCF) is a conserved, essential regulator of chromatin architecture containing a unique array of 11 zinc fingers (ZFs). Gene duplication and sequence divergence during early amniote evolution generated the CTCF paralog Brother Of the Regulator of Imprinted Sites (BORIS), which has a DNA binding specificity identical to that of CTCF but divergent N-and C-termini. While healthy somatic tissues express only CTCF, CTCF and BORIS are normally co-expressed in meiotic and post-meiotic germ cells, and aberrant activation of BORIS occurs in tumors and some cancer cell lines. This has led to a model in which CTCF and BORIS compete for binding to some but not all genomic target sites; however, regulation of CTCF and BORIS genomic co-occupancy is not well understood. We recently addressed this issue, finding evidence for two major classes of CTCF target sequences, some of which contain single CTCF target sites (1xCTSes) and others containing two adjacent CTCF motifs (2xCTSes). The functional and chromatin structural features of 2xCTSes are distinct from those of 1xCTS-containing regions bound by a CTCF monomer. We suggest that these previously overlooked classes of CTCF binding regions may have different roles in regulating diverse chromatin-based phenomena, and may impact our understanding of heritable epigenetic regulation in cancer cells and normal germ cells.
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Papers by Victor Lobanenkov
Kang S, Suzuki T, Kosaka-Suzuki N, Robinson S, Nagarajan V, Ye Z,
et al. Comparative analyses of CTCF and BORIS occupancies uncover
two distinct classes of CTCF binding genomic regions. Genome Biol.
2015;16:161.