Publications by authors named "Olga Kyrchanova"

29 Publications

  • Page 1 of 1

Mechanism and functional role of the interaction between CP190 and the architectural protein Pita in Drosophila melanogaster.

Epigenetics Chromatin 2021 Mar 22;14(1):16. Epub 2021 Mar 22.

Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, 3 4/5 Vavilov St., Moscow, 119334, Russia.

Background: Pita is required for Drosophila development and binds specifically to a long motif in active promoters and insulators. Pita belongs to the Drosophila family of zinc-finger architectural proteins, which also includes Su(Hw) and the conserved among higher eukaryotes CTCF. The architectural proteins maintain the active state of regulatory elements and the long-distance interactions between them. In particular, Pita is involved in the formation of several boundaries between regulatory domains that controlled the expression of three hox genes in the Bithorax complex (BX-C). The CP190 protein is recruited to chromatin through interaction with the architectural proteins.

Results: Using in vitro pull-down analysis, we precisely mapped two unstructured regions of Pita that interact with the BTB domain of CP190. Then we constructed transgenic lines expressing the Pita protein of the wild-type and mutant variants lacking CP190-interacting regions. We have demonstrated that CP190-interacting region of the Pita can maintain nucleosome-free open chromatin and is critical for Pita-mediated enhancer blocking activity in BX-C. At the same time, interaction with CP190 is not required for the in vivo function of the mutant Pita protein, which binds to the same regions of the genome as the wild-type protein. Unexpectedly, we found that CP190 was still associated with the most of genome regions bound by the mutant Pita protein, which suggested that other architectural proteins were continuing to recruit CP190 to these regions.

Conclusions: The results directly demonstrate role of CP190 in insulation and support a model in which the regulatory elements are composed of combinations of binding sites that interact with several architectural proteins with similar functions.
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http://dx.doi.org/10.1186/s13072-021-00391-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7983404PMC
March 2021

Mapping of functional elements of the Fab-6 boundary involved in the regulation of the Abd-B hox gene in Drosophila melanogaster.

Sci Rep 2021 Feb 18;11(1):4156. Epub 2021 Feb 18.

Department of the Control of Genetic Processes, Institute of Gene Biology Russian Academy of Sciences, 34/5 Vavilov St., Moscow, Russia, 119334.

The autonomy of segment-specific regulatory domains in the Bithorax complex is conferred by boundary elements and associated Polycomb response elements (PREs). The Fab-6 boundary is located at the junction of the iab-5 and iab-6 domains. Previous studies mapped it to a nuclease hypersensitive region 1 (HS1), while the iab-6 PRE was mapped to a second hypersensitive region HS2 nearly 3 kb away. To analyze the role of HS1 and HS2 in boundary we generated deletions of HS1 or HS1 + HS2 that have attP site for boundary replacement experiments. The 1389 bp HS1 deletion can be rescued by a 529 bp core Fab-6 sequence that includes two CTCF sites. However, Fab-6 HS1 cannot rescue the HS1 + HS2 deletion or substitute for another BX-C boundary - Fab-7. For this it must be combined with a PRE, either Fab-7 HS3, or Fab-6 HS2. These findings suggest that the boundary function of Fab-6 HS1 must be bolstered by a second element that has PRE activity.
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http://dx.doi.org/10.1038/s41598-021-83734-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7892861PMC
February 2021

Mechanisms of Enhancer-Promoter Interactions in Higher Eukaryotes.

Int J Mol Sci 2021 Jan 12;22(2). Epub 2021 Jan 12.

Department of the Control of Genetic Processes, Institute of Gene Biology Russian Academy of Sciences, 34/5 Vavilov St., 119334 Moscow, Russia.

In higher eukaryotes, enhancers determine the activation of developmental gene transcription in specific cell types and stages of embryogenesis. Enhancers transform the signals produced by various transcription factors within a given cell, activating the transcription of the targeted genes. Often, developmental genes can be associated with dozens of enhancers, some of which are located at large distances from the promoters that they regulate. Currently, the mechanisms underlying specific distance interactions between enhancers and promoters remain poorly understood. This review briefly describes the properties of enhancers and discusses the mechanisms of distance interactions and potential proteins involved in this process.
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http://dx.doi.org/10.3390/ijms22020671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828040PMC
January 2021

The insulator functions of the polydactyl C2H2 zinc finger protein CTCF: Necessity versus sufficiency.

Sci Adv 2020 03 25;6(13):eaaz3152. Epub 2020 Mar 25.

Department of the Control of Genetic Processes, Institute of Gene Biology Russian Academy of Sciences, 34/5 Vavilov St., Moscow 119334, Russia.

In mammals, a C2H2 zinc finger (C2H2) protein, CTCF, acts as the master regulator of chromosomal architecture and of the expression of Hox gene clusters. Like mammalian CTCF, the homolog, dCTCF, localizes to boundaries in the bithorax complex (BX-C). Here, we have determined the minimal requirements for the assembly of a functional boundary by dCTCF and two other C2H2 zinc finger proteins, Pita and Su(Hw). Although binding sites for these proteins are essential for the insulator activity of BX-C boundaries, these binding sites alone are insufficient to create a functional boundary. dCTCF cannot effectively bind to a single recognition sequence in chromatin or generate a functional insulator without the help of additional proteins. In addition, for boundary elements in BX-C at least four binding sites for dCTCF or the presence of additional DNA binding factors is required to generate a functional insulator.
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http://dx.doi.org/10.1126/sciadv.aaz3152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096168PMC
March 2020

Small Drosophila zinc finger C2H2 protein with an N-terminal zinc finger-associated domain demonstrates the architecture functions.

Biochim Biophys Acta Gene Regul Mech 2020 01 6;1863(1):194446. Epub 2019 Nov 6.

Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St., Moscow 119334, Russia. Electronic address:

Recently, the concept has arisen that a special class of architectural proteins exists, which are responsible not only for global chromosome architecture but also for the local regulation of enhancer-promoter interactions. Here, we describe a new architectural protein, with a total size of only 375 aa, which contains an N-terminal zinc finger-associated domain (ZAD) and a cluster of five zinc finger C2H2 domains at the C-terminus. This new protein, named ZAD and Architectural Function 1 protein (ZAF1 protein), is weakly and ubiquitously expressed, with the highest expression levels observed in oocytes and embryos. The cluster of C2H2 domains recognizes a specific 15-bp consensus site, located predominantly in promoters, near transcription start sites. The expression of ZAF1 by a tissue-specific promoter led to the complete blocking of the eye enhancer when clusters of ZAF1 binding sites flanked the eye enhancer in transgenic lines, suggesting that the loop formed by the ZAF1 protein leads to insulation. The ZAF1 protein also supported long-range interactions between the yeast GAL4 activator and the white promoter in transgenic Drosophila lines. A mutant protein lacking the ZAD failed to block the eye enhancer or to support distance interactions in transgenic lines. Taken together, these results suggest that ZAF1 is a minimal architectural protein that can be used to create a convenient model for studying the mechanisms of distance interactions.
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http://dx.doi.org/10.1016/j.bbagrm.2019.194446DOI Listing
January 2020

Distinct Elements Confer the Blocking and Bypass Functions of the Bithorax Boundary.

Genetics 2019 11 24;213(3):865-876. Epub 2019 Sep 24.

Department of Molecular Biology, Princeton University, New Jersey 08544.

Boundaries in the bithorax complex (BX-C) enable the regulatory domains that drive parasegment-specific expression of the three genes to function autonomously. The four regulatory domains (, , , and ) that control the expression of the () gene are located downstream of the transcription unit, and are delimited by the , , , and boundaries. These boundaries function to block cross talk between neighboring regulatory domains. In addition, three of the boundaries (, , and ) must also have bypass activity so that regulatory domains distal to the boundaries can contact the promoter. In the studies reported here, we have undertaken a functional dissection of the boundary using a boundary-replacement strategy. Our studies indicate that the boundary has two separable subelements. The distal subelement blocks cross talk, but cannot support bypass. The proximal subelement has only minimal blocking activity but is able to mediate bypass. A large multiprotein complex, the LBC (large boundary complex), binds to sequences in the proximal subelement and contributes to its bypass activity. The same LBC complex has been implicated in the bypass activity of the boundary.
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http://dx.doi.org/10.1534/genetics.119.302694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827379PMC
November 2019

Complete reconstitution of bypass and blocking functions in a minimal artificial insulator from complex.

Proc Natl Acad Sci U S A 2019 07 17;116(27):13462-13467. Epub 2019 Jun 17.

Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia;

Boundaries in the complex (BX-C) delimit autonomous regulatory domains that drive parasegment-specific expression of the genes , and The boundary is located between the and domains and has two key functions: blocking cross-talk between these domains and at the same time promoting communication (boundary bypass) between and the promoter. Using a replacement strategy, we found that multimerized binding sites for the architectural proteins Pita, Su(Hw), and dCTCF function as conventional insulators and block cross-talk between the and domains; however, they lack bypass activity, and is unable to regulate Here we show that an ∼200-bp sequence of dHS1 from the boundary rescues the bypass defects of these multimerized binding sites. The dHS1 sequence is bound in embryos by a large multiprotein complex, Late Boundary Complex (LBC), that contains the zinc finger proteins CLAMP and GAF. Using deletions and mutations in critical GAGAG motifs, we show that bypass activity correlates with the efficiency of recruitment of LBC components CLAMP and GAF to the artificial boundary. These results indicate that LBC orchestrates long-distance communication between the regulatory domain and the gene, while the Pita, Su(Hw), and dCTCF proteins function to block local cross-talk between the neighboring regulatory domains and .
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http://dx.doi.org/10.1073/pnas.1907190116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6613175PMC
July 2019

Boundaries mediate long-distance interactions between enhancers and promoters in the Drosophila Bithorax complex.

PLoS Genet 2018 12 12;14(12):e1007702. Epub 2018 Dec 12.

Department of the Control of Genetic Processes, Institute of Gene Biology Russian Academy of Sciences, Moscow, Russia.

Drosophila bithorax complex (BX-C) is one of the best model systems for studying the role of boundaries (insulators) in gene regulation. Expression of three homeotic genes, Ubx, abd-A, and Abd-B, is orchestrated by nine parasegment-specific regulatory domains. These domains are flanked by boundary elements, which function to block crosstalk between adjacent domains, ensuring that they can act autonomously. Paradoxically, seven of the BX-C regulatory domains are separated from their gene target by at least one boundary, and must "jump over" the intervening boundaries. To understand the jumping mechanism, the Mcp boundary was replaced with Fab-7 and Fab-8. Mcp is located between the iab-4 and iab-5 domains, and defines the border between the set of regulatory domains controlling abd-A and Abd-B. When Mcp is replaced by Fab-7 or Fab-8, they direct the iab-4 domain (which regulates abd-A) to inappropriately activate Abd-B in abdominal segment A4. For the Fab-8 replacement, ectopic induction was only observed when it was inserted in the same orientation as the endogenous Fab-8 boundary. A similar orientation dependence for bypass activity was observed when Fab-7 was replaced by Fab-8. Thus, boundaries perform two opposite functions in the context of BX-C-they block crosstalk between neighboring regulatory domains, but at the same time actively facilitate long distance communication between the regulatory domains and their respective target genes.
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http://dx.doi.org/10.1371/journal.pgen.1007702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6306242PMC
December 2018

The bithorax complex iab-7 Polycomb response element has a novel role in the functioning of the Fab-7 chromatin boundary.

PLoS Genet 2018 08 15;14(8):e1007442. Epub 2018 Aug 15.

Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia.

Expression of the three bithorax complex homeotic genes is orchestrated by nine parasegment-specific regulatory domains. Autonomy of each domain is conferred by boundary elements (insulators). Here, we have used an in situ replacement strategy to reanalyze the sequences required for the functioning of one of the best-characterized fly boundaries, Fab-7. It was initially identified by a deletion, Fab-71, that transformed parasegment (PS) 11 into a duplicate copy of PS12. Fab-71 deleted four nuclease hypersensitive sites, HS*, HS1, HS2, and HS3, located between the iab-6 and iab-7 regulatory domains. Transgenic and P-element excision experiments mapped the boundary to HS*+HS1+HS2, while HS3 was shown to be the iab-7 Polycomb response element (PRE). Recent replacement experiments showed that HS1 is both necessary and sufficient for boundary activity when HS3 is also present in the replacement construct. Surprisingly, while HS1+HS3 combination has full boundary activity, we discovered that HS1 alone has only minimal function. Moreover, when combined with HS3, only the distal half of HS1, dHS1, is needed. A ~1,000 kD multiprotein complex containing the GAF protein, called the LBC, binds to the dHS1 sequence and we show that mutations in dHS1, that disrupt LBC binding in nuclear extracts, eliminate boundary activity and GAF binding in vivo. HS3 has binding sites for GAF and Pho proteins that are required for PRE silencing. In contrast, HS3 boundary activity only requires the GAF binding sites. LBC binding with HS3 in nuclear extracts, and GAF association in vivo, depend upon the HS3 GAF sites, but not the Pho sites. Consistent with a role for the LBC in HS3 boundary activity, the boundary function of the dHS1+HS3mPho combination is lost when the flies are heterozygous for a mutation in the GAF gene. Taken together, these results reveal a novel function for the iab-7 PREs in chromosome architecture.
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http://dx.doi.org/10.1371/journal.pgen.1007442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6110506PMC
August 2018

The BEN Domain Protein Insensitive Binds to the Chromatin Boundary To Establish Proper Segmental Identity in .

Genetics 2018 10 6;210(2):573-585. Epub 2018 Aug 6.

Department of Molecular Biology, Princeton University, New Jersey 08544

Boundaries (insulators) in the bithorax complex (BX-C) delimit autonomous regulatory domains that orchestrate the parasegment (PS)-specific expression of the BX-C homeotic genes. The boundary separates the and regulatory domains, which control Abd-B expression in PS11 and PS12, respectively. This boundary is composed of multiple functionally redundant elements and has two key functions: it blocks cross talk between and and facilitates boundary bypass. Here, we show that two BEN domain protein complexes, Insensitive and Elba, bind to multiple sequences located in the nuclease hypersensitive regions. Two of these sequences are recognized by both Insv and Elba and correspond to a CCAATTGG palindrome. Elba also binds to a related CCAATAAG sequence, while Insv does not. However, the third Insv recognition sequences is ∼100 bp in length and contains the CCAATAAG sequence at one end. Both Insv and Elba are assembled into large complexes (∼420 and ∼265-290 kDa, respectively) in nuclear extracts. Using a sensitized genetic background, we show that the Insv protein is required for boundary function and that PS11 identity is not properly established in mutants. This is the first demonstration that a BEN domain protein is important for the functioning of an endogenous fly boundary.
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http://dx.doi.org/10.1534/genetics.118.301259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6216583PMC
October 2018

Opbp is a new architectural/insulator protein required for ribosomal gene expression.

Nucleic Acids Res 2017 Dec;45(21):12285-12300

Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilova St., Moscow 119334, Russia.

A special class of poorly characterized architectural proteins is required for chromatin topology and enhancer-promoter interactions. Here, we identify Opbp as a new Drosophila architectural protein, interacting with CP190 both in vivo and in vitro. Opbp binds to a very restrictive set of genomic regions, through a rare sequence specific motif. These sites are co-bound by CP190 in vivo, and generally located at bidirectional promoters of ribosomal protein genes. We show that Opbp is essential for viability, and loss of opbp function, or destruction of its motif, leads to reduced ribosomal protein gene expression, indicating a functional role in promoter activation. As characteristic of architectural/insulator proteins, the Opbp motif is sufficient for distance-dependent reporter gene activation and enhancer-blocking activity, suggesting an Opbp-mediated enhancer-promoter interaction. Rather than having a constitutive role, Opbp represents a new type of architectural protein with a very restricted, yet essential, function in regulation of housekeeping gene expression.
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http://dx.doi.org/10.1093/nar/gkx840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716193PMC
December 2017

Architectural protein Pita cooperates with dCTCF in organization of functional boundaries in Bithorax complex.

Development 2017 07 15;144(14):2663-2672. Epub 2017 Jun 15.

Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences,  Moscow 119334, Russia

Boundaries in the Bithorax complex (BX-C) of delimit autonomous regulatory domains that drive parasegment-specific expression of homeotic genes. BX-C boundaries have two crucial functions: they must block crosstalk between adjacent regulatory domains and at the same time facilitate boundary bypass. The C2H2 zinc-finger protein Pita binds to several BX-C boundaries, including and To study Pita functions, we have used a boundary replacement strategy by substituting modified DNAs for the boundary, which is located between the and regulatory domains. Multimerized Pita sites block crosstalk but fail to support regulation of (bypass). In the case of , we used a novel sensitized background to show that the two Pita-binding sites contribute to its boundary function. Although is from BX-C, it does not function appropriately when substituted for : it blocks crosstalk but does not support bypass. Mutation of the Pita site disrupts blocking activity and also eliminates dCTCF binding. In contrast, mutation of the dCTCF site does not affect Pita binding, and this mutant boundary retains partial function.
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http://dx.doi.org/10.1242/dev.149815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5536930PMC
July 2017

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

PLoS Genet 2016 07 18;12(7):e1006188. Epub 2016 Jul 18.

Department of Genetics, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia.

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.
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http://dx.doi.org/10.1371/journal.pgen.1006188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4948906PMC
July 2016

Architectural proteins Pita, Zw5,and ZIPIC contain homodimerization domain and support specific long-range interactions in Drosophila.

Nucleic Acids Res 2016 09 2;44(15):7228-41. Epub 2016 May 2.

Institute of Gene Biology, Russian Academy of Sciences, Vavilova str. 34/5, Moscow 119334, Russia

According to recent models, as yet poorly studied architectural proteins appear to be required for local regulation of enhancer-promoter interactions, as well as for global chromosome organization. Transcription factors ZIPIC, Pita and Zw5 belong to the class of chromatin insulator proteins and preferentially bind to promoters near the TSS and extensively colocalize with cohesin and condensin complexes. ZIPIC, Pita and Zw5 are structurally similar in containing the N-terminal zinc finger-associated domain (ZAD) and different numbers of C2H2-type zinc fingers at the C-terminus. Here we have shown that the ZAD domains of ZIPIC, Pita and Zw5 form homodimers. In Drosophila transgenic lines, these proteins are able to support long-distance interaction between GAL4 activator and the reporter gene promoter. However, no functional interaction between binding sites for different proteins has been revealed, suggesting that such interactions are highly specific. ZIPIC facilitates long-distance stimulation of the reporter gene by GAL4 activator in yeast model system. Many of the genomic binding sites of ZIPIC, Pita and Zw5 are located at the boundaries of topologically associated domains (TADs). Thus, ZAD-containing zinc-finger proteins can be attributed to the class of architectural proteins.
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http://dx.doi.org/10.1093/nar/gkw371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009728PMC
September 2016

Functional role of dimerization and CP190 interacting domains of CTCF protein in Drosophila melanogaster.

BMC Biol 2015 Aug 7;13:63. Epub 2015 Aug 7.

Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia.

Background: Insulators play a central role in gene regulation, chromosomal architecture and genome function in higher eukaryotes. To learn more about how insulators carry out their diverse functions, we have begun an analysis of the Drosophila CTCF (dCTCF). CTCF is one of the few insulator proteins known to be conserved from flies to man.

Results: In the studies reported here we have focused on the identification and characterization of two dCTCF protein interaction modules. The first mediates dCTCF multimerization, while the second mediates dCTCF-CP190 interactions. The multimerization domain maps in the N-terminus of the dCTCF protein and likely mediates the formation of tetrameric complexes. The CP190 interaction module encompasses a sequence ~200 amino acids long that spans the C-terminal and mediates interactions with the N-terminal BTB domain of the CP190 protein. Transgene rescue experiments showed that a dCTCF protein lacking sequences critical for CP190 interactions was almost as effective as wild type in rescuing the phenotypic effects of a dCTCF null allele. The mutation did, however, affect CP190 recruitment to specific Drosophila insulator elements and had a modest effect on dCTCF chromatin association. A protein lacking the N-terminal dCTCF multimerization domain incompletely rescued the zygotic and maternal effect lethality of the null and did not rescue the defects in Abd-B regulation evident in surviving adult dCTCF mutant flies. Finally, we show that elimination of maternally contributed dCTCF at the onset of embryogenesis has quite different effects on development and Abd-B regulation than is observed when the homozygous mutant animals develop in the presence of maternally derived dCTCF activity.

Conclusions: Our results indicate that dCTCF-CP190 interactions are less critical for the in vivo functions of the dCTCF protein than the N-terminal dCTCF-dCTCF interaction domain. We also show that the phenotypic consequences of dCTCF mutations differ depending upon when and how dCTCF activity is lost.
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http://dx.doi.org/10.1186/s12915-015-0168-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528719PMC
August 2015

The boundary paradox in the Bithorax complex.

Mech Dev 2015 Nov 26;138 Pt 2:122-132. Epub 2015 Jul 26.

Laboratory of Gene Expression Regulation in Development, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia; Department of Molecular Biology, Princeton University, Princeton, NJ, USA. Electronic address:

The parasegment-specific expression of the three Drosophila Bithorax complex homeotic genes is orchestrated by nine functionally autonomous regulatory domains. Functional autonomy depends upon special elements called boundaries or insulators that are located between each domain. The boundaries ensure the independent activity of each domain by blocking adventitious interactions with initiators, enhancers and silencers in the neighboring domains. However, this blocking activity poses a regulatory paradox--the Bithorax boundaries are also able to insulate promoters from regulatory interactions with enhancers and silencers and six of the nine Bithorax regulatory domains are separated from their target genes by at least one boundary element. Here we consider several mechanisms that have been suggested for how the Bithorax regulatory domains are able to bypass intervening boundary elements and direct the appropriate parasegment-specific temporal and spatial expression of their target gene.
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http://dx.doi.org/10.1016/j.mod.2015.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4890074PMC
November 2015

Two new insulator proteins, Pita and ZIPIC, target CP190 to chromatin.

Genome Res 2015 Jan 23;25(1):89-99. Epub 2014 Oct 23.

Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia

Insulators are multiprotein-DNA complexes that regulate the nuclear architecture. The Drosophila CP190 protein is a cofactor for the DNA-binding insulator proteins Su(Hw), CTCF, and BEAF-32. The fact that CP190 has been found at genomic sites devoid of either of the known insulator factors has until now been unexplained. We have identified two DNA-binding zinc-finger proteins, Pita, and a new factor named ZIPIC, that interact with CP190 in vivo and in vitro at specific interaction domains. Genomic binding sites for these proteins are clustered with CP190 as well as with CTCF and BEAF-32. Model binding sites for Pita or ZIPIC demonstrate a partial enhancer-blocking activity and protect gene expression from PRE-mediated silencing. The function of the CTCF-bound MCP insulator sequence requires binding of Pita. These results identify two new insulator proteins and emphasize the unifying function of CP190, which can be recruited by many DNA-binding insulator proteins.
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http://dx.doi.org/10.1101/gr.174169.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4317163PMC
January 2015

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

Epigenetics 2014 Sep 1;9(9):1261-70. Epub 2014 Aug 1.

Department of the Control of Genetic Processes; Institute of Gene Biology; Russian Academy of Sciences; Moscow, Russia.

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.
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http://dx.doi.org/10.4161/epi.32086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4169018PMC
September 2014

Chromatin insulators and long-distance interactions in Drosophila.

FEBS Lett 2014 Jan 5;588(1):8-14. Epub 2013 Nov 5.

Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St., Moscow 119334, Russia. Electronic address:

Data on long-distance enhancer-mediated activation of gene promoters and complex regulation of gene expression by multiple enhancers have prompted the hypothesis that the action of enhancers is restricted by insulators. Studies with transgenic lines have shown that insulators are responsible for establishing proper local interactions between regulatory elements, but not for defining independent transcriptional domains that restrict the activity of enhancers. It has also become apparent that enhancer blocking is only one of several functional activities of known insulator proteins, which also contribute to the organization of chromosome architecture and the integrity of regulatory elements.
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http://dx.doi.org/10.1016/j.febslet.2013.10.039DOI Listing
January 2014

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

PLoS Genet 2013 4;9(7):e1003606. Epub 2013 Jul 4.

Group of Transcriptional Regulation, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia.

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.
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http://dx.doi.org/10.1371/journal.pgen.1003606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3701704PMC
January 2014

New properties of Drosophila scs and scs' insulators.

PLoS One 2013 24;8(4):e62690. Epub 2013 Apr 24.

Group of Transcriptional Regulation, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0062690PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3634774PMC
December 2013

Insulators form gene loops by interacting with promoters in Drosophila.

Development 2011 Sep;138(18):4097-106

Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St, Moscow, 119334 Russia.

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.
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http://dx.doi.org/10.1242/dev.062836DOI Listing
September 2011

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

Nucleic Acids Res 2011 Apr 10;39(8):3042-52. Epub 2010 Dec 10.

Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St, Moscow 119334, Russia.

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.
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http://dx.doi.org/10.1093/nar/gkq1248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3082887PMC
April 2011

Insulators, not Polycomb response elements, are required for long-range interactions between Polycomb targets in Drosophila melanogaster.

Mol Cell Biol 2011 Feb 6;31(4):616-25. Epub 2010 Dec 6.

Rutgers University, Department of Molecular Biology and Biochemistry, Piscataway, NJ 08854, USA.

The genomic binding sites of Polycomb group (PcG) complexes have been found to cluster, forming Polycomb "bodies" or foci in mammalian or fly nuclei. These associations are thought to be driven by interactions between PcG complexes and result in enhanced repression. Here, we show that a Polycomb response element (PRE) with strong PcG binding and repressive activity cannot mediate trans interactions. In the case of the two best-studied interacting PcG targets in Drosophila, the Mcp and the Fab-7 regulatory elements, we find that these associations are not dependent on or caused by the Polycomb response elements they contain. Using functional assays and physical colocalization by in vivo fluorescence imaging or chromosome conformation capture (3C) methods, we show that the interactions between remote copies of Mcp or Fab-7 elements are dependent on the insulator activities present in these elements and not on their PREs. We conclude that insulator binding proteins rather than PcG complexes are likely to be the major determinants of the long-range higher-order organization of PcG targets in the nucleus.
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http://dx.doi.org/10.1128/MCB.00849-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3028641PMC
February 2011

Orientation-dependent interaction between Drosophila insulators is a property of this class of regulatory elements.

Nucleic Acids Res 2008 Dec 5;36(22):7019-28. Epub 2008 Nov 5.

Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia.

Insulators are defined as a class of regulatory elements that delimit independent transcriptional domains within eukaryotic genomes. According to previous data, an interaction (pairing) between some Drosophila insulators can support distant activation of a promoter by an enhancer. Here, we have demonstrated that pairs of well-studied insulators such as scs-scs, scs'-scs', 1A2-1A2 and Wari-Wari support distant activation of the white promoter by the yeast GAL4 activator in an orientation-dependent manner. The same is true for the efficiency of the enhancer that stimulates white expression in the eyes. In all insulator pairs tested, stimulation of the white gene was stronger when insulators were inserted between the eye enhancer or GAL4 and the white promoter in opposite orientations relative to each other. As shown previously, Zw5, Su(Hw) and dCTCF proteins are required for the functioning of different insulators that do not interact with each other. Here, strong functional interactions have been revealed between DNA fragments containing binding sites for either Zw5 or Su(Hw) or dCTCF protein but not between heterologous binding sites [Zw5-Su(Hw), dCTCF-Su(Hw), or dCTCF-Zw5]. These results suggest that insulator proteins can support selective interactions between distant regulatory elements.
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http://dx.doi.org/10.1093/nar/gkn781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2602758PMC
December 2008

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

Mol Cell Biol 2008 Jun 21;28(12):4188-95. Epub 2008 Apr 21.

Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St., Moscow 119334, Russia.

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.
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http://dx.doi.org/10.1128/MCB.00229-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2423118PMC
June 2008

New properties of Drosophila fab-7 insulator.

Genetics 2007 Sep;177(1):113-21

Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia.

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.
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http://dx.doi.org/10.1534/genetics.107.075887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2013716PMC
September 2007

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

Mol Cell Biol 2007 Apr 5;27(8):3035-43. Epub 2007 Feb 5.

Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St., Moscow 119334, Russia.

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.
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http://dx.doi.org/10.1128/MCB.02203-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1899939PMC
April 2007

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

Mol Cell Biol 2005 May;25(9):3682-9

Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St., 119334 Moscow, Russia.

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.
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http://dx.doi.org/10.1128/MCB.25.9.3682-3689.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1084309PMC
May 2005