Interactions between BTB domain of CP190 and two adjacent regions in Su(Hw) are required for the insulator complex formation.

The best-studied Drosophila insulator complex consists of two BTB-containing proteins, the Mod(mdg4)-67.2 isoform and CP190, which are recruited cooperatively to chromatin through interactions with the DNA-binding architectural protein Su(Hw). While Mod(mdg4)-67.2 interacts only with Su(Hw), CP190 interacts with many other architectural proteins. In spite of the fact that CP190 is critical for the activity of Su(Hw) insulators, interaction between these proteins has not been studied yet. Therefore, we have performed a detailed analysis of domains involved in the interaction between the Su(Hw) and CP190. The results show that the BTB domain of CP190 interacts with two adjacent regions at the N-terminus of Su(Hw). Deletion of either region in Su(Hw) only weakly affected recruiting of CP190 to the Su(Hw) sites in the presence of Mod(mdg4)-67.2. Deletion of both regions in Su(Hw) prevents its interaction with CP190. Using mutations in vivo, we found that interactions with Su(Hw) and Mod(mdg4)-67.2 are essential for recruiting of CP190 to the Su(Hw) genomic sites.

Functional Dissection of the Blocking and Bypass Activities of the Fab-8 Boundary in the Drosophila Bithorax Complex.

Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C) homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS) regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass) for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence) fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B) transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw)) sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors.

Transvection in Drosophila: trans-interaction between yellow enhancers and promoter is strongly suppressed by a cis-promoter only in certain genomic regions.

Transvection is a phenomenon of interallelic communication whereby enhancers of one allele can activate a promoter located on the homologous chromosome. It has been shown for many independent genes that enhancers preferentially act on the cis-linked promoter, but deletion of this promoter allows the enhancers to act in trans. Here, we tested whether this cis-preference in the enhancer-promoter interaction could be reconstituted outside of the natural position of a gene. The yellow gene was chosen as a model system. Transgenic flies were generated that carried the yellow gene modified by the inclusion of the strategically placed recognition sites for the Cre and Flp recombinases. To facilitate transvection, an endogenous Su(Hw) insulator (1A2) or gypsy insulator was placed behind the yellow gene. Independent action of the recombinases produced a pair of derivative alleles, one containing the promoter-driven yellow gene, and the other, the enhancers and promoter that failed to produce a functional yellow protein. As a result, we observed strong transvection in many genomic regions, suggesting that a complete cis-preference of the enhancer-promoter interactions is mainly restricted to genes in their natural loci.

Effective blocking of the white enhancer requires cooperation between two main mechanisms suggested for the insulator function.

Chromatin insulators block the action of transcriptional enhancers when interposed between an enhancer and a promoter. In this study, we examined the role of chromatin loops formed by two unrelated insulators, gypsy and Fab-7, in their enhancer-blocking activity. To test for this activity, we selected the white reporter gene that is activated by the eye-specific enhancer. The results showed that one copy of the gypsy or Fab-7 insulator failed to block the eye enhancer in most of genomic sites, whereas a chromatin loop formed by two gypsy insulators flanking either the eye enhancer or the reporter completely blocked white stimulation by the enhancer. However, strong enhancer blocking was achieved due not only to chromatin loop formation but also to the direct interaction of the gypsy insulator with the eye enhancer, which was confirmed by the 3C assay. In particular, it was observed that Mod(mdg4)-67.2, a component of the gypsy insulator, interacted with the Zeste protein, which is critical for the eye enhancer-white promoter communication. These results suggest that efficient enhancer blocking depends on the combination of two factors: chromatin loop formation by paired insulators, which generates physical constraints for enhancer-promoter communication, and the direct interaction of proteins recruited to an insulator and to the enhancer-promoter pair.

Insulators form gene loops by interacting with promoters in Drosophila.

Chromatin insulators are regulatory elements involved in the modulation of enhancer-promoter communication. The 1A2 and Wari insulators are located immediately downstream of the Drosophila yellow and white genes, respectively. Using an assay based on the yeast GAL4 activator, we have found that both insulators are able to interact with their target promoters in transgenic lines, forming gene loops. The existence of an insulator-promoter loop is confirmed by the fact that insulator proteins could be detected on the promoter only in the presence of an insulator in the transgene. The upstream promoter regions, which are required for long-distance stimulation by enhancers, are not essential for promoter-insulator interactions. Both insulators support basal activity of the yellow and white promoters in eyes. Thus, the ability of insulators to interact with promoters might play an important role in the regulation of basal gene transcription.

Selective interactions of boundaries with upstream region of Abd-B promoter in Drosophila bithorax complex and role of dCTCF in this process.

Expression of the genes Ubx, abd-A, and Abd-B of the bithorax complex depends on its cis-regulatory region, which is divided into discrete functional domains (iab). Boundary/insulator elements, named Mcp, Fab-6, Fab-7 and Fab-8 (PTS/F8), have been identified at the borders of the iab domains. Recently, binding sites for a Drosophila homolog of the vertebrate insulator protein CTCF have been identified in Mcp, Fab-6 and Fab-8 and also in several regions that correspond to predicted boundaries, Fab-3 and Fab-4 in particular. Taking into account the inability of the yeast GAL4 activator to stimulate the white promoter when the activator and the promoter are separated by a 5-kb yellow gene, we have tested functional interactions between the boundaries. The results show that all dCTCF-containing boundaries interact with each other. However, inactivation of dCTCF binding sites in Mcp, Fab-6 and PTS/F8 only partially reduces their ability to interact, suggesting the presence of additional protein(s) supporting distant interactions between the boundaries. Interestingly, only Fab-6, Fab-7 (which contains no dCTCF binding sites) and PTS/F8 interact with the upstream region of the Abd-B promoter. Thus, the boundaries might be involved in supporting the specific interactions between iab enhancers and promoters of the bithorax complex.

E(y)2/Sus1 is required for blocking PRE silencing by the Wari insulator in Drosophila melanogaster.

Chromatin insulators affect interactions between promoters and enhancers/silencers and function as barriers to the spread of repressive chromatin. Recently, we have found an insulator, named Wari, located on the 3' side of the white gene. Here, we show that the previously identified 368-bp core of this insulator is sufficient for blocking Polycomb response element-mediated silencing. Although Wari does not contain binding sites for known insulator proteins, the E(y)2 and CP190 proteins bind to Wari as well as to the Su(Hw)-containing insulators in vivo. It may well be that these proteins are recruited to the insulator by as yet unidentified DNA-binding protein. Partial inactivation of E(y)2 in a weak e(y)2 ( u1 ) mutation impairs only the anti-silencing but not the enhancer-blocking activity of the Wari insulator. Thus, the E(y)2 protein in different Drosophila insulators serves to protect gene expression from silencing.

Study of the functional interaction between Mcp insulators from the Drosophila bithorax complex: effects of insulator pairing on enhancer-promoter communication.

Boundary elements have been found in the Abd-B 3' cis-regulatory region, which is subdivided into a series of iab domains. Previously, a 340-bp insulator-like element, M(340), was identified in one such 755-bp Mcp fragment linked to the PcG-dependent silencer. In this study, we identified a 210-bp core that was sufficient for pairing of sequence-remote Mcp elements. In two-gene transgenic constructs with two Mcp insulators (or their cores) surrounding yellow, the upstream yeast GAL4 sites were able to activate the distal white only if the insulators were in the opposite orientations (head-to-head or tail-to-tail), which is consistent with the looping/bypass model. The same was true for the efficiency of the cognate eye enhancer, while yellow thus isolated in the loop from its enhancers was blocked more strongly. These results indicate that the relative placement and orientation of insulator-like elements can determine proper enhancer-promoter communication.

Red flag on the white reporter: a versatile insulator abuts the white gene in Drosophila and is omnipresent in mini-white constructs.

Much of the research on insulators in Drosophila has been done with transgenic constructs using the white gene (mini-white) as reporter. Hereby we report that the sequence between the white and CG32795 genes in Drosophila melanogaster contains an insulator of a novel kind. Its functional core is within a 368 bp segment almost contiguous to the white 3'UTR, hence we name it as Wari (white-abutting resident insulator). Though Wari contains no binding sites for known insulator proteins and does not require Su(Hw) or Mod(mdg4) for its activity, it can equally well interact with another copy of Wari and with unrelated Su(Hw)-dependent insulators, gypsy or 1A2. In its natural downstream position, Wari reinforces enhancer blocking by any of the three insulators placed between the enhancer and the promoter; again, Wari-Wari, Wari-gypsy or 1A2-Wari pairing results in mutual neutralization (insulator bypass) when they precede the promoter. The distressing issue is that this element hides in all mini-white constructs employed worldwide to study various insulators and other regulatory elements as well as long-range genomic interactions, and its versatile effects could have seriously influenced the results and conclusions of many works.

New properties of Drosophila fab-7 insulator.

In the Abd-B 3' cis-regulatory region, which is subdivided into a series of iab domains, boundary elements have previously been detected, including the Fab-7 element providing for the autonomous functioning of the iab-6 and iab-7 cis-regulatory domains. Here, it has been shown that a single copy of the 860-bp Fab-7 insulator effectively blocks the yellow and white enhancers. The eye and testis enhancers can stimulate the white promoter across the pair of Fab-7, which is indicative of a functional interaction between the insulators. Unexpectedly, Fab-7 has proved to lose the enhancer-blocking activity when placed near the white promoter. It seems likely that Fab-7 strengthens the relatively weak white promoter, which leads to the efficient enhancer-promoter interaction and insulator bypass.

The Mcp element from the bithorax complex contains an insulator that is capable of pairwise interactions and can facilitate enhancer-promoter communication.

Chromatin insulators, or boundary elements, appear to control eukaryotic gene expression by regulating interactions between enhancers and promoters. Boundaries have been identified in the 3' cis-regulatory region of Abd-B, which is subdivided into a series of separate iab domains. Boundary elements such as Mcp, Fab-7, and Fab-8 and adjacent silencers flank the iab domains and restrict the activity of the iab enhancers. We have identified an insulator in the 755-bp Mcp fragment that is linked to the previously characterized Polycomb response element (PRE) and silences the adjacent genes. This insulator blocks the enhancers of the yellow and white genes and protects them from PRE-mediated repression. The interaction between the Mcp elements, each containing the insulator and PRE, allows the eye enhancer to activate the white promoter over the repressed yellow domain. The same level of white activation was observed when the Mcp element combined with the insulator alone was interposed between the eye enhancer and the promoter, suggesting that the insulator is responsible for the interaction between the Mcp elements.

Pairing between gypsy insulators facilitates the enhancer action in trans throughout the Drosophila genome.

The Suppressor of the Hairy wing [Su(Hw)] binding region within the gypsy retrotransposon is the best known chromatin insulator in Drosophila melanogaster. According to previous data, two copies of the gypsy insulator inserted between an enhancer and a promoter neutralize each other's actions, which is indicative of an interaction between the protein complexes bound to the insulators. We have investigated the role of pairing between the gypsy insulators located on homologous chromosomes in trans interaction between yellow enhancers and a promoter. It has been shown that trans activation of the yellow promoter strongly depends on the site of the transposon insertion, which is evidence for a role of surrounding chromatin in homologous pairing. The presence of the gypsy insulators in both homologous chromosomes even at a distance of 9 kb downstream from the promoter dramatically improves the trans activation of yellow. Moreover, the gypsy insulators have proved to stabilize trans activation between distantly located enhancers and a promoter. These data suggest that gypsy insulator pairing is involved in communication between loci in the Drosophila genome.

Role of Su(Hw) zinc finger 10 and interaction with CP190 and Mod(mdg4) proteins in recruiting the Su(Hw) complex to chromatin sites in Drosophila.

Su(Hw) belongs to the class of proteins that organize chromosome architecture and boundaries/insulators between regulatory domains. This protein contains a cluster of 12 zinc finger domains most of which are responsible for binding to three different modules in the consensus site. Su(Hw) forms a complex with CP190 and Mod(mdg4)-67.2 proteins that binds to well-known Drosophila insulators. To understand how Su(Hw) performs its activities and binds to specific sites in chromatin, we have examined the previously described su(Hw)f mutation that disrupts the 10th zinc finger (ZF10) responsible for Su(Hw) binding to the upstream module. The results have shown that Su(Hw)f loses the ability to interact with CP190 in the absence of DNA. In contrast, complete deletion of ZF10 does not prevent the interaction between Su(Hw)Δ10 and CP190. Having studied insulator complex formation in different mutant backgrounds, we conclude that both association with CP190 and Mod(mdg4)-67.2 partners and proper organization of DNA binding site are essential for the efficient recruitment of the Su(Hw) complex to chromatin insulators.

Multiple interactions are involved in a highly specific association of the Mod(mdg4)-67.2 isoform with the Su(Hw) sites in Drosophila.

The best-studied Drosophila insulator complex consists of two BTB-containing proteins, the Mod(mdg4)-67.2 isoform and CP190, which are recruited to the chromatin through interactions with the DNA-binding Su(Hw) protein. It was shown previously that Mod(mdg4)-67.2 is critical for the enhancer-blocking activity of the Su(Hw) insulators and it differs from more than 30 other Mod(mdg4) isoforms by the C-terminal domain required for a specific interaction with Su(Hw) only. The mechanism of the highly specific association between Mod(mdg4)-67.2 and Su(Hw) is not well understood. Therefore, we have performed a detailed analysis of domains involved in the interaction of Mod(mdg4)-67.2 with Su(Hw) and CP190. We found that the N-terminal region of Su(Hw) interacts with the glutamine-rich domain common to all the Mod(mdg4) isoforms. The unique C-terminal part of Mod(mdg4)-67.2 contains the Su(Hw)-interacting domain and the FLYWCH domain that facilitates a specific association between Mod(mdg4)-67.2 and the CP190/Su(Hw) complex. Finally, interaction between the BTB domain of Mod(mdg4)-67.2 and the M domain of CP190 has been demonstrated. By using transgenic lines expressing different protein variants, we have shown that all the newly identified interactions are to a greater or lesser extent redundant, which increases the reliability in the formation of the protein complexes.

Mechanisms of endothelial dysfunction after ionized radiation: selective impairment of the nitric oxide component of endothelium-dependent vasodilation.

(1) Gamma radiation impairs vascular function, leading to the depression of endothelium-dependent vasodilatation. Loss of the nitric oxide (NO) pathway has been implicated, but little is known about radiation effects on other endothelial mediators. (2) This study investigated the mechanisms of endothelial dysfunction in rabbits subjected to whole-body irradiation from a cobalt(60) source. (3) The endothelium-dependent relaxation of rabbit aorta evoked by acetylcholine (ACh) or A23187 was impaired in a dose-dependent manner by irradiation at 2 Gy or above. Inhibition was evident 9 days post-irradiation and persisted over the 30 day experimental period. (4) Endothelium-independent responses to glyceryl trinitrate (GTN), sodium nitroprusside (SNP) and 3-morpholino-sydnonimine (SIN-1) were suppressed over a similar dose range at 7-9 days post-irradiation, but recovered fully by 30 days post-irradiation. (5) In healthy vessels, ACh-induced relaxation was inhibited by L-N(omega)-nitroarginine (L-NA; 3 x 10(-4) M) and charybdotoxin (10(-8) M) plus apamin (10(-6) M) but resistant to indomethacin, indicating the involvement of NO and endothelium-derived hyperpolarizing factor (EDHF). Supporting this, ACh caused smooth muscle hyperpolarization that was reduced by L-NA and charybdotoxin plus apamin. (6) In irradiated vessels, responses to ACh were insensitive to L-NA but abolished by charybdotoxin plus apamin, indicating selective loss of NO-mediated relaxation. (7) In animals treated shortly after irradiation with the antioxidant, alpha-tocopherol acetate, the NO-dependent relaxation was restored without effect on the EDHF-dependent component. (8) The results imply that radiation selectively impairs the NO pathway as a consequence of oxidative stress, while EDHF is able to maintain endothelium-dependent relaxation at a reduced level.

PKC-δ isozyme gene silencing restores vascular function in diabetic rat.

Abstract Background: Endothelium and K+ channel functionality in smooth muscle cells (SMCs) regulates vascular function and is exposed to damage in diabetes. The regulatory enzyme protein kinase C (PKC) is known to play a key role in vascular tone regulation in health and disease. In this study, we evaluated the effect of PKC-δ gene silencing using small interfering RNAs (siRNAs) on endothelial dysfunction and acquired potassium channelopathy in vascular SMCs in diabetes. Methods: The experimental design comprised diabetes induction by streptozotocin (65 mg/kg) in rats, RNA interference, isolated aortic ring contractile recordings, whole-cell patch-clamp technique, measurements of reactive oxygen species (ROS), and real-time polymerase chain reaction technique. Animals were killed by cervical dislocation following ketamine (45 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) anesthesia administration on the third month of diabetes and on the seventh day after intravenous injection of siRNAs. Results: The aortas of diabetic rats demonstrated depressed endothelium-dependent relaxation and integral SMCs outward K+ currents as compared with those of controls. On the seventh day, PKC-δ gene silencing effectively restored K+ currents and increased the amplitude of vascular relaxation up to control levels. An increased level of PKC-δ mRNA in diabetic aortas appeared to be reduced after targeted PKC-δ gene silencing. Similarly, the level of ROS production that was increased in diabetes came back to control values after siRNAs administration. Conclusions: The silencing of PKC-δ gene expression using siRNAs led to restoration of vasodilator potential in rats with diabetes mellitus. It is likely that the siRNA technique can be a good therapeutic tool to normalize vascular function in diabetes.

Highly conserved ENY2/Sus1 protein binds to Drosophila CTCF and is required for barrier activity.

Chromatin insulators affect interactions between promoters and enhancers/silencers and function as barriers for the spreading of repressive chromatin. Drosophila insulator protein dCTCF marks active promoters and boundaries of many histone H3K27 trimethylation domains associated with repressed chromatin. In particular, dCTCF binds to such boundaries between the parasegment-specific regulatory domains of the Bithorax complex. Here we demonstrate that the evolutionarily conserved protein ENY2 is recruited to the zinc-finger domain of dCTCF and is required for the barrier activity of dCTCF-dependent insulators in transgenic lines. Inactivation of ENY2 by RNAi in BG3 cells leads to the spreading of H3K27 trimethylation and Pc protein at several dCTCF boundaries. The results suggest that evolutionarily conserved ENY2 is responsible for barrier activity mediated by the dCTCF protein.

Transcription through enhancers suppresses their activity in Drosophila.

BACKGROUND: Enhancer elements determine the level of target gene transcription in a tissue-specific manner, providing for individual patterns of gene expression in different cells. Knowledge of the mechanisms controlling enhancer action is crucial for understanding global regulation of transcription. In particular, enhancers are often localized within transcribed regions of the genome. A number of experiments suggest that transcription can have both positive and negative effects on regulatory elements. In this study, we performed direct tests for the effect of transcription on enhancer activity. RESULTS: Using a transgenic reporter system, we investigated the relationship between the presence of pass-through transcription and the activity of Drosophila enhancers controlling the expression of the white and yellow genes. The results show that transcription from different promoters affects the activity of enhancers, counteracting their ability to activate the target genes. As expected, the presence of a transcriptional terminator between the inhibiting promoter and the affected enhancer strongly reduces the suppression. Moreover, transcription leads to dislodging of the Zeste protein that is responsible for the enhancer-dependent regulation of the white gene, suggesting a 'transcription interference' mechanism for this regulation. CONCLUSIONS: Our findings suggest a role for pass-through transcription in negative regulation of enhancer activity.

New properties of Drosophila scs and scs' insulators.

Insulators are defined as a class of regulatory elements that delimit independent transcriptional domains within eukaryotic genomes. The first insulators to be identified were scs and scs', which flank the domain including two heat shock 70 genes. Zw5 and BEAF bind to scs and scs', respectively, and are responsible for the interaction between these insulators. Using the regulatory regions of yellow and white reporter genes, we have found that the interaction between scs and scs' improves the enhancer-blocking activity of the weak scs' insulator. The sequences of scs and scs' insulators include the promoters of genes that are strongly active in S2 cells but not in the eyes, in which the enhancer-blocking activity of these insulators has been extensively examined. Only the promoter of the Cad87A gene located at the end of the scs insulator drives white expression in the eyes, and the white enhancer can slightly stimulate this promoter. The scs insulator contains polyadenylation signals that may be important for preventing transcription through the insulator. As shown previously, scs and scs' can insulate transcription of the white transgene from the enhancing effects of the surrounding genome, a phenomenon known as the chromosomal position effect (CPE). After analyzing many independent transgenic lines, we have concluded that transgenes carrying the scs insulator are rarely inserted into genomic regions that stimulate the white reporter expression in the eyes.

Functional interaction between the Fab-7 and Fab-8 boundaries and the upstream promoter region in the Drosophila Abd-B gene.

Boundary elements have been found in the regulatory region of the Drosophila melanogaster Abdominal-B (Abd-B) gene, which is subdivided into a series of iab domains. The best-studied Fab-7 and Fab-8 boundaries flank the iab-7 enhancer and isolate it from the four promoters regulating Abd-B expression. Recently binding sites for the Drosophila homolog of the vertebrate insulator protein CTCF (dCTCF) were identified in the Fab-8 boundary and upstream of Abd-B promoter A, with no binding of CTCF to the Fab-7 boundary being detected either in vivo or in vitro. Taking into account the inability of the yeast GAL4 activator to stimulate the white promoter when its binding sites are separated by a 5-kb yellow gene, we have tested the functional interactions between the Fab-7 and Fab-8 boundaries and between these boundaries and the upstream promoter A region containing a dCTCF binding site. It has been found that dCTCF binding sites are essential for pairing between two Fab-8 insulators. However, a strong functional interaction between the Fab-7 and Fab-8 boundaries suggests that additional, as yet unidentified proteins are involved in long-distance interactions between them. We have also shown that Fab-7 and Fab-8 boundaries effectively interact with the upstream region of the Abd-B promoter.