Eukaryotic transcriptional regulation often involves regulatory elements separated from the cognate genes by long distances, whereas appropriately positioned insulator or enhancer-blocking elements shield promoters from illegitimate enhancer action. Four proteins have been identified in Drosophila mediating enhancer blocking-Su(Hw), Zw5, BEAF32 and GAGA factor. In vertebrates, the single protein CTCF, with 11 highly conserved zinc fingers, confers enhancer blocking in all known chromatin insulators. Here, we characterize an orthologous CTCF factor in Drosophila with a similar domain structure, binding site specificity and transcriptional repression activity as in vertebrates. In addition, we demonstrate that one of the insulators (Fab-8) in the Drosophila Abdominal-B locus mediates enhancer blocking by dCTCF. Therefore, the enhancer-blocking protein CTCF and, most probably, the mechanism of enhancer blocking mediated by this remarkably versatile factor are conserved from Drosophila to humans.

The interaction of leukocyte integrin ␣ M ␤ 2 (CD11b/ CD18, Mac-1) with fibrinogen has been implicated in the inflammatory response by contributing to leukocyte adhesion to the endothelium and subsequent transmigration. Previously, it has been demonstrated that a peptide, P1, corresponding to residues 190 -202 in the ␥-chain of fibrinogen, binds to ␣ M ␤ 2 and blocks the interaction of fibrinogen with the receptor and that Asp

The murine Igh locus has a 3 regulatory region (3 RR) containing four enhancers (hs3A, hs1,2, hs3B, and hs4) at DNase I-hypersensitive sites. The 3 RR exerts long-range effects on class switch recombination (CSR) to several isotypes through its control of germ line transcription. By measuring levels of acetylated histones H3 and H4 and of dimethylated H3 (K4) with chromatin immunoprecipitation assays, we found that early in B-cell development, chromatin encompassing the enhancers of the 3 RR began to attain stepwise modifications typical of an open conformation. The hs4 enhancer was associated with active chromatin initially in pro-and pre-B cells and then together with hs3A, hs1,2, and hs3B in B and plasma cells. The immunoglobulin heavy chain locus (Igh) (see Fig. 1) is arguably the most complex mammalian locus. B-cell development is marked by the progressive expression of Igh genes (via VDJ recombination, somatic hypermutation, and class switch recombination [CSR]). These regulated processes, some involving substantial DNA rearrangements and deletions, are required for the generation of antibody diversity (reviewed in references 47 and 57). Spanning ϳ3 Mb, the murine Igh locus contains a limited number of enhancers, including the intronic enhancer (E), which has been implicated in V to DJ joining and chain expression (reviewed in reference 47), and a complex 3Ј regulatory region (3Ј RR) located immediately downstream of C␣, the last constant region gene. The human Igh locus contains two 3Ј RRs, each located downstream of one of the duplicated C␣ genes (9, 49, 62).The murine 3Ј RR spans ϳ28 kb downstream of C␣ and comprises four enhancers, each associated with DNase I hypersensitivity (hs3A, hs1,2, hs3B, and hs4) (reviewed in reference 33). hs3A and hs3B are located at the termini of a 25-kb inverted repeat flanking hs1,2 (7, 66), and these three enhancers acquire DNase I hypersensitivity at later stages of B-cell development (66). The hs4 enhancer is the only 3Ј enhancer with activity and DNase I hypersensitivity throughout B-cell development (19, 48), although no role for hs4 or any of the 3Ј enhancers in early B-cell development has been shown.Targeted deletions of the 3Ј RR enhancers have shown that hs3A and hs1,2 are individually dispensable for B-cell development and Igh expression (46), while the combination of hs3B and hs4 is critical for the process of CSR (63). The 3Ј RR has also been proposed to regulate Igh expression in plasma cells (23,40,48,63,70). In accord with earlier suggestions that it functions as a locus control region (43), the 3Ј RR shows synergistic activity and position-independent regulation when tested in transgenic model systems (8,33,59). However, copynumber-dependent expression has not been observed (8). In addition to a function within the Igh locus, the 3Ј RR is likely to be responsible for dysregulation of c-myc expression resulting from certain Igh::c-myc translocations in mouse plasmacytoma cells as well as in human Burkitt's lymphoma and multiple myeloma cells (30,36,43).Th...

CTCF is a highly conserved, multifunctional zinc finger protein involved in critical aspects of gene regulation including transcription regulation, chromatin insulation, genomic imprinting, X-chromosome inactivation, and higher order chromatin organization. Such multifunctional properties of CTCF suggest an essential role in development. Indeed, a previous report on maternal depletion of CTCF suggested that CTCF is essential for pre-implantation development. To distinguish between the effects of maternal and zygotic expression of CTCF, we studied pre-implantation development in mice harboring a complete loss of function Ctcf knockout allele. Although we demonstrated that homozygous deletion of Ctcf is early embryonically lethal, in contrast to previous observations, we showed that the Ctcf nullizygous embryos developed up to the blastocyst stage (E3.5) followed by peri-implantation lethality (E4.5–E5.5). Moreover, one-cell stage Ctcf nullizygous embryos cultured ex vivo developed to the 16–32 cell stage with no obvious abnormalities. Using a single embryo assay that allowed both genotype and mRNA expression analyses of the same embryo, we demonstrated that pre-implantation development of the Ctcf nullizygous embryos was associated with the retention of the maternal wild type Ctcf mRNA. Loss of this stable maternal transcript was temporally associated with loss of CTCF protein expression, apoptosis of the developing embryo, and failure to further develop an inner cell mass and trophoectoderm ex vivo. This indicates that CTCF expression is critical to early embryogenesis and loss of its expression rapidly leads to apoptosis at a very early developmental stage. This is the first study documenting the presence of the stable maternal Ctcf transcript in the blastocyst stage embryos. Furthermore, in the presence of maternal CTCF, zygotic CTCF expression does not seem to be required for pre-implantation development.

CTCF is a candidate tumor suppressor gene encoding a multifunctional transcription factor. Surprisingly for a tumor suppressor, the levels of CTCF in breast cancer cell lines and tumors were found elevated compared with breast cell lines with finite life span and normal breast tissues. In this study, we aimed to investigate the possible cause for this increase in CTCF content and in particular to test the hypothesis that up-regulation of CTCF may be linked to resistance of breast cancer cells to apoptosis. For this purpose, apoptotic cell death was monitored following alterations of CTCF levels induced by transient transfection and conditional knockdown of CTCF in various cell lines. We observed apoptotic cell death in all breast cancer cell lines examined following CTCF down-regulation. In addition, overexpression of CTCF partially protected cells from apoptosis induced by overexpression of Bax or treatment with sodium butyrate. To elucidate possible mechanisms of this phenomenon, we used a proteomics approach and observed that levels of the proapoptotic protein, Bax, were increased following CTCF down-regulation in MCF7 cells. Taken together, these results suggest that in some cellular contexts CTCF shows antiapoptotic characteristics, most likely exerting its functions through regulation of apoptotic genes. We hypothesize that CTCF overexpression may have evolved as a compensatory mechanism to protect breast cancer cells from apoptosis, thus providing selective survival advantages to these cells. The observations reported in this study may lead to development of therapies based on selective reduction of CTCF in breast cancer cells.

The crystal structure of the fibrinogen gamma-module (residues gamma143-411) [Yee, V. C., et al. (1997) Structure 5, 125-138] revealed an unusual feature. Namely, residues gamma381-390 in the functionally important COOH-terminal region form a beta-strand that is inserted into an antiparallel beta-sheet of the central domain (gamma192-286), while the rest (gamma393-411) seems to be flexible. To clarify the structural and functional importance of this beta-strand insert, we analyzed the folding status of the plasmin-derived fibrinogen fragment D(3) and several truncated variants of the gamma-module expressed in Escherichia coli. It was found that D(3), in which most of the COOH-terminal domain of the gamma-module (gamma287-379) is removed proteolytically, retains a gamma374-405 peptide that seems to be associated noncovalently with the bulk of the molecule via its beta-strand insert region. A study of the denaturation-renaturation process of D(3) suggested that without this peptide its truncated gamma-module remains folded but is destabilized. This was confirmed directly with the truncated recombinant variants of the gamma-module, including residues gamma148-392, gamma148-373, and gamma148-286. They all were folded, but those devoid of the beta-strand insert were destabilized. The results indicate that although the beta-strand insert contributes to the stabilization of the gamma-module, it can be removed without destroying the compact structure of the latter. On the basis of this finding and some other observations, we propose a mechanism for the function-related conformational changes in the fibrin(ogen) gamma-modules.

BackgroundA common aberration in cancer is the activation of germline-specific proteins. The DNA-binding proteins among them could generate novel chromatin states, not found in normal cells. The germline-specific transcription factor BORIS/CTCFL, a paralog of chromatin architecture protein CTCF, is often erroneously activated in cancers and rewires the epigenome for the germline-like transcription program. Another common feature of malignancies is the changed expression and epigenetic states of genomic repeats, which could alter the transcription of neighboring genes and cause somatic mutations upon transposition. The role of BORIS in transposable elements and other repeats has never been assessed.ResultsThe investigation of BORIS and CTCF binding to DNA repeats in the K562 cancer cells dependent on BORIS for self-renewal by ChIP-chip and ChIP-seq revealed three classes of occupancy by these proteins: elements cohabited by BORIS and CTCF, CTCF-only bound, or BORIS-only bound. The CTCF-only enrichment is characteristic for evolutionary old and inactive repeat classes, while BORIS and CTCF co-binding predominately occurs at uncharacterized tandem repeats. These repeats form staggered cluster binding sites, which are a prerequisite for CTCF and BORIS co-binding. At the same time, BORIS preferentially occupies a specific subset of the evolutionary young, transcribed, and mobile genomic repeat family, SVA. Unlike CTCF, BORIS prominently binds to the VNTR region of the SVA repeats in vivo. This suggests a role of BORIS in SVA expression regulation. RNA-seq analysis indicates that BORIS largely serves as a repressor of SVA expression, alongside DNA and histone methylation, with the exception of promoter capture by SVA.ConclusionsThus, BORIS directly binds to, and regulates SVA repeats, which are essentially movable CpG islands, via clusters of BORIS binding sites. This finding uncovers a new function of the global germline-specific transcriptional regulator BORIS in regulating and repressing the newest class of transposable elements that are actively transposed in human genome when activated. This function of BORIS in cancer cells is likely a reflection of its roles in the germline.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0084-2) contains supplementary material, which is available to authorized users.

Condensin complexes are essential for chromosome condensation and segregation in mitosis, while condensin dysfunction, among other pathways leading to chromosomal bridging in mitosis, may play a role in tumor genomic instability, including recently discovered chromotripsis. To characterize potential double-strand breaks specifically occurring in late anaphase, human chromosomes depleted of condensin were analyzed by γ-H2AX ChIP followed by high-throughput sequencing (ChIP-seq). In condensin-depleted cells, the nonrepeated parts of the genome were shown to contain distinct γ-H2AX enrichment zones 75% of which overlapped with known hemizygous deletions in cancers. Furthermore, some tandemly repeated DNA sequences, analyzed separately from the rest of the genome, showed significant γ-H2AX enrichment in condensin-depleted anaphases. The most commonly occurring targets of such enrichment included simple repeats, centromeric satellites, and rDNA. The two latter categories indicate that acrocentric human chromosomes are especially susceptible to breaks upon condensin deficiency. The genomic regions that are specifically destabilized upon condensin dysfunction may constitute a condensin-specific chromosome destabilization pattern.