Orientation-dependent Dxz4 contacts shape the 3D structure of the inactive X chromosome.

The mammalian inactive X chromosome (Xi) condenses into a bipartite structure with two superdomains of frequent long-range contacts, separated by a hinge region. Using Hi-C in edited mouse cells with allelic deletions or inversions within the hinge, here we show that the conserved Dxz4 locus is necessary to maintain this bipartite structure. Dxz4 orientation controls the distribution of contacts on the Xi, as shown by a massive reversal in long-range contacts after Dxz4 inversion. Despite an increase in CTCF binding and chromatin accessibility on the Xi in Dxz4-edited cells, only minor changes in TAD structure and gene expression were detected, in accordance with multiple epigenetic mechanisms ensuring X silencing. We propose that Dxz4 represents a structural platform for frequent long-range contacts with multiple loci in a direction dictated by the orientation of its bank of CTCF motifs, which may work as a ratchet to form the distinctive bipartite structure of the condensed Xi.

Escape from X inactivation.

Although the process of X inactivation in mammalian cells silences the majority of genes on the inactivated X chromosome, some genes escape this chromosome-wide silencing. Genes that escape X inactivation present a unique opportunity to study the process of silencing and the mechanisms that protect some genes from being turned off. In this review, we will discuss evolutionary aspects of escape from X inactivation, in relation to the divergence of the sex chromosomes. Molecular characteristics, expression, and epigenetic modifications of genes that escape will be presented, including their developmental regulation and the implications of chromatin domains along the X chromosome in modeling the escape process.

Cell growth inhibition by the multifunctional multivalent zinc-finger factor CTCF.

The 11-zinc finger protein CCTC-binding factor (CTCF) employs different sets of zinc fingers to form distinct complexes with varying CTCF- target sequences (CTSs) that mediate the repression or activation of gene expression and the creation of hormone-responsive gene silencers and of diverse vertebrate enhancer-blocking elements (chromatin insulators). To determine how these varying effects would integrate in vivo, we engineered a variety of expression systems to study effects of CTCF on cell growth. Here we show that ectopic expression of CTCF in many cell types inhibits cell clonogenicity by causing profound growth retardation without apoptosis. In asynchronous cultures, the cell-cycle profile of CTCF-expressing cells remained unaltered, which suggested that progression through the cycle was slowed at multiple points. Although conditionally induced CTCF caused the S-phase block, CTCF can also arrest cell division. Viable CTCF-expressing cells could be maintained without dividing for several days. While MYC is the well-characterized CTCF target, the inhibitory effects of CTCF on cell growth could not be ascribed solely to repression of MYC, suggesting that additional CTS-driven genes involved in growth-regulatory circuits, such as p19ARF, are likely to contribute to CTCF-induced growth arrest. These findings indicate that CTCF may regulate cell-cycle progression at multiple steps within the cycle, and add to the growing evidence for the function of CTCF as a tumor suppressor gene.

CTCF-binding sites flank CTG/CAG repeats and form a methylation-sensitive insulator at the DM1 locus.

An expansion of a CTG repeat at the DM1 locus causes myotonic dystrophy (DM) by altering the expression of the two adjacent genes, DMPK and SIX5, and through a toxic effect of the repeat-containing RNA. Here we identify two CTCF-binding sites that flank the CTG repeat and form an insulator element between DMPK and SIX5. Methylation of these sites prevents binding of CTCF, indicating that the DM1 locus methylation in congenital DM would disrupt insulator function. Furthermore, CTCF-binding sites are associated with CTG/CAG repeats at several other loci. We suggest a general role for CTG/CAG repeats as components of insulator elements at multiple sites in the human genome.

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.

[The use of low-frequency magnetotherapy and EHF puncture in the combined treatment of arterial hypertension in vibration-induced disease]. / Primenenie nizkochastotnoi magnitoterapii i KVCh-punktury v kompleksnom lechenii arterial'noi gipertenzii pri vibratsionnoi bolezni.

Combination of EHF therapy + magnetotherapy + drugs results in faster and persistent hypotensive and analgetic effect compared to standard drug therapy, potentiates action of vascular drugs on cerebral and peripheral circulation, reduces dose of hypotensive drugs in patients with arterial hypertension and vibration disease.

Negative transcriptional regulation mediated by thyroid hormone response element 144 requires binding of the multivalent factor CTCF to a novel target DNA sequence.

DNA target sites for a "multivalent" 11-zinc-finger CCTC-binding factor (CTCF) are unusually long ( approximately 50 base pairs) and remarkably different. In conjunction with the thyroid receptor (TR), CTCF binding to the lysozyme gene transcriptional silencer mediates the thyroid hormone response element (TRE)-dependent transcriptional repression. We tested whether other TREs, which in addition to the presence of a TR binding site require neighboring sequences for transcriptional function, might also contain a previously unrecognized binding site(s) for CTCF. One such candidate DNA region, previously isolated by Bigler and Eisenman (Bigler, J., and Eisenman, R. N. (1995) EMBO J. 14, 5710-5723), is the TRE-containing genomic element 144. We have identified a new CTCF target sequence that is adjacent to the TR binding site within the 144 fragment. Comparison of CTCF recognition nucleotides in the lysozyme silencer and in the 144 sequences revealed both similarities and differences. Several C-terminal CTCF zinc fingers contribute differently to binding each of these sequences. Mutations that eliminate CTCF binding impair 144-mediated negative transcriptional regulation. Thus, the 144 element provides an additional example of a functionally significant composite "TRE plus CTCF binding site" regulatory element suggesting an important role for CTCF in cooperation with the steroid/thyroid superfamily of nuclear receptors to mediate TRE-dependent transcriptional repression.

Characterization of the chicken CTCF genomic locus, and initial study of the cell cycle-regulated promoter of the gene.

CTCF is a multifunctional transcription factor encoded by a novel candidate tumor suppressor gene (Filippova, G. N., Lindblom, A., Meinke, L. J., Klenova, E. M., Neiman, P. E., Collins, S. J., Doggett, N. D., and Lobanenkov, V. V. (1998) Genes Chromosomes Cancer 22, 26-36). We characterized genomic organization of the chicken CTCF (chCTCF) gene, and studied the chCTCF promoter. Genomic locus of chCTCF contains a GC-rich untranslated exon separated from seven coding exons by a long intron. The 2-kilobase pair region upstream of the major transcription start site contains a CpG island marked by a "Not-knot" that includes sequence motifs characteristic of a TATA-less promoter of housekeeping genes. When fused upstream of a reporter chloramphenicol acetyltransferase gene, it acts as a strong transcriptional promoter in transient transfection experiments. The minimal 180-base pair chCTCF promoter region that is fully sufficient to confer high level transcriptional activity to the reporter contains high affinity binding element for the transcription factor YY1. This element is strictly conserved in chicken, mouse, and human CTCF genes. Mutations in the core nucleotides of the YY1 element reduce transcriptional activity of the minimal chCTCF promoter, indicating that the conserved YY1-binding sequence is critical for transcriptional regulation of vertebrate CTCF genes. We also noted in the chCTCF promoter several elements previously characterized in cell cycle-regulated genes, including the "cell cycle-dependent element" and "cell cycle gene homology region" motifs shown to be important for S/G2-specific up-regulation of cdc25C, cdc2, cyclin A, and Plk (polo-like kinase) gene promoters. Presence of the cell cycle-dependent element/cell cycle gene homology region element suggested that chCTCF expression may be cell cycle-regulated. We show that both levels of the endogenous chCTCF mRNA, and the activity of the stably transfected chCTCF promoter constructs, increase in S/G2 cells.

A widely expressed transcription factor with multiple DNA sequence specificity, CTCF, is localized at chromosome segment 16q22.1 within one of the smallest regions of overlap for common deletions in breast and prostate cancers.

The cellular protooncogene MYC encodes a nuclear transcription factor that is involved in regulating important cellular functions, including cell cycle progression, differentiation, and apoptosis. Dysregulated MYC expression appears critical to the development of various types of malignancies, and thus factors involved in regulating MYC expression may also play a key role in the pathogenesis of certain cancers. We have cloned one such MYC regulatory factor, termed CTCF, which is a highly evolutionarily conserved-11-zinc finger transcriptional factor possessing multiple DNA sequence specificity. CTCF binds to a number of important regulatory regions within the 5' noncoding sequence of the human MYC oncogene, and it can regulate its transcription in several experimental systems. CTCF mRNA is expressed in cells of multiple different lineages. Enforced ectopic expression of CTCF inhibits cell growth in culture. Southern blot analyses and fluorescence in situ hybridization (FISH) with normal human metaphase chromosomes showed that the human CTCF is a single-copy gene situated at chromosome locus 16q22. Cytogenetic studies have pointed out that chromosome abnormalities (deletions) at this locus frequently occur in many different human malignancies, suggesting the presence of one or more tumor suppressor genes in the region. To narrow down their localization, several loss of heterozygosity (LOH) studies of chromosome arm 16q in sporadic breast and prostate cancers have been carried out to define the most recurrent and smallest region(s) of overlap (SRO) for commonly deleted chromosome arm 16q material. For CTCF to be considered as a candidate tumor suppressor gene associated with tumorigenesis, it should localize within one of the SROs at 16q. Fine-mapping of CTCF has enabled us to assign the CTCF gene to about a 2 centiMorgan (cM) interval of 16q22.1 between the somatic cell hybrid breakpoints CY130(D) and CY4, which is between markers D16S186 (16AC16-101) and D16S496 (AFM214zg5). This relatively small region, containing the CTCF gene, overlaps the most frequently observed SROs for common chromosomal deletions found in sporadic breast and prostate tumors. In one of four analyzed paired DNA samples from primary breast cancer patients, we have detected a tumor-specific rearrangement of CTCF exons encoding the 11-zinc-finger domain. Therefore, taken together with other CTCF properties, localization of CTCF to a narrow cancer-associated chromosome region suggests that CTCF is a novel candidate tumor suppressor gene at 16q22.1.

Mechanisms of cycloheximide-induced apoptosis in liver cells.

Cycloheximide in sublethal doses caused apoptosis in liver cells in vivo, inducing c-myc, c-fos, c-jun and p53 genes and accumulation of sphingosine, a toxic product of the sphingomyelin cycle. These data support the hypothesis that continuous synthesis of labile protective proteins is required to restrain apoptosis in liver; sphingosine might be important in mediating cycloheximide-induced apoptosis as an endogenous modulator of protein kinase C activity.

Negative protein 1, which is required for function of the chicken lysozyme gene silencer in conjunction with hormone receptors, is identical to the multivalent zinc finger repressor CTCF.

The transcriptional repressor negative protein 1 (NeP1) binds specifically to the F1 element of the chicken lysozyme gene silencer and mediates synergistic repression by v-ERBA, thyroid hormone receptor, or retinoic acid receptor. Another protein, CCCTC-binding factor (CTCF), specifically binds to 50-bp-long sequences that contain repetitive CCCTC elements in the vicinity of vertebrate c-myc genes. Previously cloned chicken, mouse, and human CTCF cDNAs encode a highly conserved 11-Zn-finger protein. Here, NeP1 was purified and DNA bases critical for NeP1-F1 interaction were determined. NeP1 is found to bind a 50-bp stretch of nucleotides without any obvious sequence similarity to known CTCF binding sequences. Despite this remarkable difference, these two proteins are identical. They have the same molecular weight, and NeP1 contains peptide sequences which are identical to sequences in CTCF. Moreover, NeP1 and CTCF specifically recognize each other's binding DNA sequence and induce identical conformational alterations in the F1 DNA. Therefore, we propose to replace the name NeP1 with CTCF. To analyze the puzzling sequence divergence in CTCF binding sites, we studied the DNA binding of 12 CTCF deletions with serially truncated Zn fingers. While fingers 4 to 11 are indispensable for CTCF binding to the human c-myc P2 promoter site A, a completely different combination of fingers, namely, 1 to 8 or 5 to 11, was sufficient to bind the lysozyme silencer site F1. Thus, CTCF is a true multivalent factor with multiple repressive functions and multiple sequence specificities.

An exceptionally conserved transcriptional repressor, CTCF, employs different combinations of zinc fingers to bind diverged promoter sequences of avian and mammalian c-myc oncogenes.

We have isolated and analyzed human CTCF cDNA clones and show here that the ubiquitously expressed 11-zinc-finger factor CTCF is an exceptionally highly conserved protein displaying 93% identity between avian and human amino acid sequences. It binds specifically to regulatory sequences in the promoter-proximal regions of chicken, mouse, and human c-myc oncogenes. CTCF contains two transcription repressor domains transferable to a heterologous DNA binding domain. One CTCF binding site, conserved in mouse and human c-myc genes, is found immediately downstream of the major P2 promoter at a sequence which maps precisely within the region of RNA polymerase II pausing and release. Gel shift assays of nuclear extracts from mouse and human cells show that CTCF is the predominant factor binding to this sequence. Mutational analysis of the P2-proximal CTCF binding site and transient-cotransfection experiments demonstrate that CTCF is a transcriptional repressor of the human c-myc gene. Although there is 100% sequence identity in the DNA binding domains of the avian and human CTCF proteins, the regulatory sequences recognized by CTCF in chicken and human c-myc promoters are clearly diverged. Mutating the contact nucleotides confirms that CTCF binding to the human c-myc P2 promoter requires a number of unique contact DNA bases that are absent in the chicken c-myc CTCF binding site. Moreover, proteolytic-protection assays indicate that several more CTCF Zn fingers are involved in contacting the human CTCF binding site than the chicken site. Gel shift assays utilizing successively deleted Zn finger domains indicate that CTCF Zn fingers 2 to 7 are involved in binding to the chicken c-myc promoter, while fingers 3 to 11 mediate CTCF binding to the human promoter. This flexibility in Zn finger usage reveals CTCF to be a unique "multivalent" transcriptional factor and provides the first feasible explanation of how certain homologous genes (i.e., c-myc) of different vertebrate species are regulated by the same factor and maintain similar expression patterns despite significant promoter sequence divergence.

[Change in the level of sphingosine in rat liver nuclei and cells during oncogene superexpression induced by cycloheximide]. / Izmenenie urovnia sfingozina v iadrakh i kletkakh pecheni krys pri indutsirovannoi superékspressii onkogenov tsiklogeksimidom.

Changes in the sphingosine content in rat liver cells and nuclei have been studied with reference to the level of nuclear oncogene expression, induced by cycloheximide (0.1, 0.5 and 3.0 mg/kg). It has been found that only the sublethal (3 mg/kg) dose of cycloheximide which induces the superexpression of c-fos and c-myc oncogenes can promote sphingosine accumulation in the cell. At the moment of enhanced expression of nuclear oncogenes, the maximum content of free sphingosine exceeds the control level 1.5- and 3-fold in the cell and in the nuclei, respectively. The difference in the sphingosine accumulation patterns in the cell and in the nuclei testifies to the fact that sphingomyelin metabolism is more active in the nuclei than in the cell. Sphingosine accumulation in the nuclei is characterized by coordination of sphingomyelinase activity and changes in the sphingomyelin content. A comparative analysis of activities of enzymes of sphingomyelin (sphingomyelinase) and phosphatidyl inositol (phosphatidyl inositol kinase) cycles indicates that in the nuclei the activation of the sphingomyelin cycle forestalls the cycloheximide-induced activation of the phosphatidyl inositol cycle and the maximal accumulation of nuclear oncogene mRNAs. A model of activation of oncogene expression with participation of sphingosine inhibiting protein kinase C and activating casein kinase II, the key enzymes of the signal transduction system of cell proliferation and differentiation, is proposed.

[The effect of tumor necrosis factor on the free sphingosine level and sphingomyelinase in murine liver cells and nuclei]. / Vliianie faktora nekroza opukholi na uroven'svobodnogo sfingozina i aktivnost' sfingomielinazy v kletkakh i iadrakh pecheni myshei.

The effects of the human recombinant tumour necrosis factor (TNF) (10 and 40 mg/kg of body mass) on sphingomyelinase activity and sphingosine content in mouse (C57bl) liver cells and nuclei have been studied. Whereas sphingomyelinase is known to be a key enzyme of sphingomyelin metabolism, sphingosine, being a product of deep enzymatic hydrolysis of sphingomyelin, controls the activity of various phosphokinases. The primary response of liver cell to TNF consists in the inhibition of sphingomyelinase; its activation occurs at later periods: after 2 hours at 10 mg/kg TNF and after 4 hours at 40 mg/kg TNF. In the nucleus activation of sphingomyelinase is observed within the first 60 min after TNF administration. Sphingosine accumulation in mouse liver cells and nuclei coincides in time with sphingomyelinase stimulation. In the nuclei activation of the sphingomyelin cycle by TNF is far more pronounced than in the cells, being observed at early periods after TNF injection. A signal mechanism of TNF action on mouse liver cells and nuclei involving a TNF-specific receptor and sphingosine which may activate this receptor phosphorylation is discussed.

[Change in the level of endogenous sphingosine in cell nuclei from regenerating rat liver]. / Izmenenie urovnia éndogennogo sfingozina v iadrakh kletok regeneriruiushchei pecheni krys.

High-performance liquid chromatography was used to study the changes in the sphingosine content in regenerating rat liver cell nuclei during RNA and DNA synthesis. It was found that activation of nucleic acid synthesis was accompanied by sphingosine accumulation in cell nuclei in parallel with the induction of the sphingomyelin cycle consisting in the increasing activity of sphingomyelinase and alteration of the sphingomyelin and ceramide content. To clarify the mechanism of sphingosine involvement in replication and transcription, the ability of this product to interact with DNA and modify the activity of RNA-polymerase in vitro was studied. At 10(-4) M sphingosine prevented the interaction of acridine orange with DNA and activated the transcription enzymes. Several alternative mechanisms of sphingosine involvement in the control of nucleic acid synthesis are discussed.

[Comparison of lipid metabolism in rat liver cells and nuclei during cycloheximide-induced superinduction of nuclear oncogenes]. / Sravnenie metabolizma lipidov v iadrakh i kletkakh pecheni krys v usloviiakh TsGI-indutsirovannoi superékspressii iadernykh onkogenov.

Using a model of cycloheximide (CHI)-induced expression of nuclear oncogens, a comparative study of metabolism of the major lipid classes in rat liver nuclei and cells was carried out. A short-term activation of sphingomyelinase which preceded on a time scale the maximal accumulation of c-fos and c-myc transcripts was observed both in the cells and in the nuclei. In contrast with the whole cell, the level of phospholipase C activity in the nuclei did not change under conditions of oncogene activation. It was found that the maximal expression of nuclear oncogens coincided in time with cyclic changes in the content of practically all phospholipids and neutral lipids with simultaneous activation of their synthesis both in the cells and in the nuclei. However, in the nuclei the sphingomyelin metabolism activation was predominant. It is concluded that in the nucleus sphingomyelin and its metabolites may influence oncogene expression via nuclear protein kinase C.

[Turbidimetric method of determination of glycogen synthase activity in rabbit skeletal muscles]. / Turbidimetricheskii metod opredeleniia aktivnosti glikogensintazy skeletnykh myshts krolika.

A turbidimetric procedure is described, which involves the monitoring of changes in glycogen turbidity at wavelengths above 300 nm and continuous recording of rabbit skeletal muscle synthase activity. The recalculation coefficients were found to be equal to 1.69 +/- 0.08 mM UDP per unit of optical density at 360 nm and to 2.03 +/- 0.01 mM UDP per unit of optical density at 400 nm. The procedure allows a kinetic analysis of the enzyme within a broad range of concentrations and under various conditions. The glycogen synthase activity did not depend on the buffer capacity when 10-100 mM Tris-HCL buffer, pN 7.8, was used. The rate of the enzymatic reaction was correlated with the enzyme concentration within the range of 5 to 50 micrograms/ml. The curve for glycogen synthase saturation with UDPG is described by the Michaelis-Menten equation, when either 0.04-0.08 mM glucose-6-phosphate for for the D-form were used in mixtures containing 5 mM MgCl2 for the D-form were used in mixtures containing 5 mM MgCl2 and 10 mM Na2SO4. The turbidimetric and spectrophotometric procedures yielded similar results.

[Expression of oncogenes in the rat liver under conditions of template biosynthesis uncoupling by sublethal doses of cycloheximide]. / Ekspressiia onkogenov v pecheni krys v usloviiakh razobshcheniia matrichnykh biosintezov subletal'nymi dozami tsiklogeksimida.

Injection of sublethal doses of cycloheximide (CHI) to rats allowed to reveal three stages in the dynamics of protein synthesis: 1) suppression stage (0-6 hrs), 2) regeneration stage (6-12 hrs), 3) stimulation stage (6-12 hrs). RNA-polymerases are activated when protein synthesis is inhibited. The stimulation stage precedes the activation of DNA replication. This model of DNA replication induced by CHI is specified by the expression of various cell oncogenes (c-fos, c-mys, p53, c-Ha-ras, c-sis, c-src). The investigated oncogenes may be divided into 4 groups according to the character of their expression. 1. Oncogenes (c-fos, c-myc) are switched on step-by-step 1 hour after CHI injection, the superexpression of the oncogenes being comparatively short. Maximum expression of c-fos and c-myc oncogenes is registered after 2-3 hours, respectively. 2./p53 oncogene expression increases within a few hours' after CHI injection and manifests itself at all three stages of protein synthesis till DNA replication. 3. c-Ha-ras oncogene is expressed at a high level in control and experimental animals. 4. Expression of c-sis and c-src oncogenes are absent both before and after CHI injection. Sublethal doses of CHI have the same effect on oncogene expression as the lethal ones.