Publications by authors named "Oksana Maksimenko"

32 Publications

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

A Non-stop identity complex (NIC) supervises enterocyte identity and protects from premature aging.

Elife 2021 Feb 25;10. Epub 2021 Feb 25.

Rappaport Research Institute and Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.

A hallmark of aging is loss of differentiated cell identity. Aged midgut differentiated enterocytes (ECs) lose their identity, impairing tissue homeostasis. To discover identity regulators, we performed an RNAi screen targeting ubiquitin-related genes in ECs. Seventeen genes were identified, including the deubiquitinase Non-stop (CG4166). Lineage tracing established that acute loss of Non-stop in young ECs phenocopies aged ECs at cellular and tissue levels. Proteomic analysis unveiled that Non-stop maintains identity as part of a Non-stop identity complex (NIC) containing E(y)2, Sgf11, Cp190, (Mod) mdg4, and Nup98. Non-stop ensured chromatin accessibility, maintaining the EC-gene signature, and protected NIC subunit stability. Upon aging, the levels of Non-stop and NIC subunits declined, distorting the unique organization of the EC nucleus. Maintaining youthful levels of Non-stop in wildtype aged ECs safeguards NIC subunits, nuclear organization, and suppressed aging phenotypes. Thus, Non-stop and NIC, supervise EC identity and protects from premature aging.
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http://dx.doi.org/10.7554/eLife.62312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7936876PMC
February 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

N-terminal domain of the architectural protein CTCF has similar structural organization and ability to self-association in bilaterian organisms.

Sci Rep 2020 02 14;10(1):2677. Epub 2020 Feb 14.

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

CTCF is the main architectural protein found in most of the examined bilaterian organisms. The cluster of the C2H2 zinc-finger domains involved in recognition of long DNA-binding motif is only part of the protein that is evolutionarily conserved, while the N-terminal domain (NTD) has different sequences. Here, we performed biophysical characterization of CTCF NTDs from various species representing all major phylogenetic clades of higher metazoans. With the exception of Drosophilides, the N-terminal domains of CTCFs show an unstructured organization and absence of folded regions in vitro. In contrast, NTDs of Drosophila melanogaster and virilis CTCFs contain unstructured folded regions that form tetramers and dimers correspondingly in vitro. Unexpectedly, most NTDs are able to self-associate in the yeast two-hybrid and co-immunoprecipitation assays. These results suggest that NTDs of CTCFs might contribute to the organization of CTCF-mediated long-distance interactions and chromosomal architecture.
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http://dx.doi.org/10.1038/s41598-020-59459-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021899PMC
February 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

The simultaneous interaction of MSL2 with CLAMP and DNA provides redundancy in the initiation of dosage compensation in males.

Development 2019 08 23;146(19). Epub 2019 Aug 23.

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

The binding of the male-specific lethal dosage compensation complex (DCC) exclusively to the male X chromosome provides an excellent model system to understand mechanisms of selective recruitment of protein complexes to chromatin. Previous studies showed that the male-specific organizer of the complex, MSL2, and the ubiquitous DNA-binding protein CLAMP are key players in the specificity of X chromosome binding. The CXC domain of MSL2 binds to genomic sites of DCC recruitment Another conserved domain of MSL2, named Clamp-binding domain (CBD) directly interacts with the N-terminal zinc-finger domain of CLAMP. Here, we found that inactivation of CBD or CXC individually only modestly affected recruitment of the DCC to the X chromosome in males. However, combination of these two genetic lesions within the same MSL2 mutant resulted in an increased loss of DCC recruitment to the X chromosome. Thus, proper MSL2 positioning requires an interaction with either CLAMP or DNA to initiate dosage compensation in males.
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http://dx.doi.org/10.1242/dev.179663DOI Listing
August 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

Removal of extra sequences with I-SceI in combination with CRISPR/Cas9 technique for precise gene editing in Drosophila.

Biotechniques 2019 04;66(4):198-201

Group of Molecular Organization of Genome, Russian Academy of Sciences, 34/5 Vavilov St, Moscow 119334, Russia.

The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies and has been adopted for many organisms, including Drosophila. Previously, an efficient two-step strategy was developed to engineer the fly genome by combining CRISPR/Cas9 with recombinase-mediated cassette exchange (RMCE). This strategy allows the introduction of designed mutations into a gene of interest in vivo. However, the loxP or frt site remains in the edited locus. Here, we propose a modification of this approach for rapid and efficient seamless genome editing with CRISPR/Cas9 and site-specific recombinase-mediated integration (SSRMI) combined with recombination between homologous sequences induced by the rare-cutting endonuclease I-SceI. The induced homological recombination leads to the removal of the remaining extraneous sequences from the target locus.
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http://dx.doi.org/10.2144/btn-2018-0147DOI Listing
April 2019

Transcription termination sequences support the expression of transgene product secreted with milk.

Transgenic Res 2019 08 28;28(3-4):401-410. Epub 2019 Mar 28.

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

Expression of the reporter gene in transgenic animals depends on the surrounding chromatin environment. Recent genome-wide studies have shown that, in mammals, the entire genome is transcribed. Transcription through a transgene often has a negative effect on the expression of a reporter gene. Here, we compared the ability of well-studied chicken chromatin insulator HS4 and bidirectional transcription terminators from the human genome to support high-level expression of the firefly luciferase gene (Fluc) under control of the previously characterized goat β-casein gene promoter. The insertion of HS4 or either of the two transcription terminators upstream of the promoter resulted in tenfold enhancement of Fluc expression in the mammary glands of transgenic mice. These results suggest that transcriptional terminators, similar to the HS4 insulator, can be used to improve the reporter gene expression in transgenic animals.
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http://dx.doi.org/10.1007/s11248-019-00122-9DOI Listing
August 2019

Factor cooperation for chromosome discrimination in Drosophila.

Nucleic Acids Res 2019 02;47(4):1706-1724

Molecular Biology Division, Biomedical Center, Faculty of Medicine and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität Munich, 82151 Martinsried, Germany.

Transcription regulators select their genomic binding sites from a large pool of similar, non-functional sequences. Although general principles that allow such discrimination are known, the complexity of DNA elements often precludes a prediction of functional sites. The process of dosage compensation in Drosophila allows exploring the rules underlying binding site selectivity. The male-specific-lethal (MSL) Dosage Compensation Complex (DCC) selectively binds to some 300 X chromosomal 'High Affinity Sites' (HAS) containing GA-rich 'MSL recognition elements' (MREs), but disregards thousands of other MRE sequences in the genome. The DNA-binding subunit MSL2 alone identifies a subset of MREs, but fails to recognize most MREs within HAS. The 'Chromatin-linked adaptor for MSL proteins' (CLAMP) also interacts with many MREs genome-wide and promotes DCC binding to HAS. Using genome-wide DNA-immunoprecipitation we describe extensive cooperativity between both factors, depending on the nature of the binding sites. These are explained by physical interaction between MSL2 and CLAMP. In vivo, both factors cooperate to compete with nucleosome formation at HAS. The male-specific MSL2 thus synergises with a ubiquitous GA-repeat binding protein for refined X/autosome discrimination.
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http://dx.doi.org/10.1093/nar/gky1238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393291PMC
February 2019

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

Conserved sequences in the Drosophila mod(mdg4) intron promote poly(A)-independent transcription termination and trans-splicing.

Nucleic Acids Res 2018 11;46(20):10608-10618

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

Alternative splicing (AS) is a regulatory mechanism of gene expression that greatly expands the coding capacities of genomes by allowing the generation of multiple mRNAs from a single gene. In Drosophila, the mod(mdg4) locus is an extreme example of AS that produces more than 30 different mRNAs via trans-splicing that joins together the common exons and the 3' variable exons generated from alternative promoters. To map the regions required for trans-splicing, we have developed an assay for measuring trans-splicing events and identified a 73-bp region in the last common intron that is critical for trans-splicing of three pre-mRNAs synthesized from different DNA strands. We have also found that conserved sequences in the distal part of the last common intron induce polyadenylation-independent transcription termination and are enriched by paused RNA polymerase II (RNAP II). These results suggest that all mod(mdg4) mRNAs are formed by joining in trans the 5' splice site in the last common exon with the 3' splice site in one of the alternative exons.
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http://dx.doi.org/10.1093/nar/gky716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237743PMC
November 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

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

The Differences Between Cis- and Trans-Gene Inactivation Caused by Heterochromatin in Drosophila.

Genetics 2016 Jan 23;202(1):93-106. Epub 2015 Oct 23.

Department of Molecular Genetics of the Cell, Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia

Position-effect variegation (PEV) is the epigenetic disruption of gene expression near the de novo-formed euchromatin-heterochromatin border. Heterochromatic cis-inactivation may be accompanied by the trans-inactivation of genes on a normal homologous chromosome in trans-heterozygous combination with a PEV-inducing rearrangement. We characterize a new genetic system, inversion In(2)A4, demonstrating cis-acting PEV as well as trans-inactivation of the reporter transgenes on the homologous nonrearranged chromosome. The cis-effect of heterochromatin in the inversion results not only in repression but also in activation of genes, and it varies at different developmental stages. While cis-actions affect only a few juxtaposed genes, trans-inactivation is observed in a 500-kb region and demonstrates а nonuniform pattern of repression with intermingled regions where no transgene repression occurs. There is no repression around the histone gene cluster and in some other euchromatic sites. trans-Inactivation is accompanied by dragging of euchromatic regions into the heterochromatic compartment, but the histone gene cluster, located in the middle of the trans-inactivated region, was shown to be evicted from the heterochromatin. We demonstrate that trans-inactivation is followed by de novo HP1a accumulation in the affected transgene; trans-inactivation is specifically favored by the chromatin remodeler SAYP and prevented by Argonaute AGO2.
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http://dx.doi.org/10.1534/genetics.115.181693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4701106PMC
January 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

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

Mechanisms and proteins involved in long-distance interactions.

Front Genet 2014 18;5:28. Epub 2014 Feb 18.

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

Due to advances in genome-wide technologies, consistent distant interactions within chromosomes of higher eukaryotes have been revealed. In particular, it has been shown that enhancers can specifically and directly interact with promoters by looping out intervening sequences, which can be up to several hundred kilobases long. This review is focused on transcription factors that are supposed to be involved in long-range interactions. Available data are in agreement with the model that several known transcription factors and insulator proteins belong to an abundant but poorly studied class of proteins that are responsible for chromosomal architecture.
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http://dx.doi.org/10.3389/fgene.2014.00028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3927085PMC
June 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

Drosophila BTB/POZ domains of "ttk group" can form multimers and selectively interact with each other.

J Mol Biol 2011 Sep 29;412(3):423-36. Epub 2011 Jul 29.

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

The BTB (bric-a-brac, tramtrack and broad complex)/POZ (poxvirus and zinc finger) domain is a conserved protein-protein interaction motif contained in a variety of transcription factors involved in development, chromatin remodeling, insulator activity, and carcinogenesis. All well-studied mammalian BTB domains form obligate homodimers and, rarely, tetramers. Only the BTB domain of the Drosophila GAGA factor (GAF) has been shown to exist as higher-order multimers. The BTB domain of GAF belongs to the "ttk group" that contains several highly conserved sequences not found in other BTB domains. Here, we have shown by size-exclusion chromatography, chemical cross-linking, and nondenaturing PAGE that four additional BTB domains of the ttk group-Batman, Mod(mdg4), Pipsqueak, and Tramtrack-can form multimers, like GAF. Interestingly, the BTB domains of GAF and Batman have formed a wide range of complexes and interacted in the yeast two-hybrid assay with other BTB domains tested. In contrast, the BTB domains of Mod(mdg4), Pipsqueak, and Tramtrack have formed stable high-order multimer complexes and failed to interact with each other. The BTB domain of Drosophila CP190 protein does not belong to the ttk group. This BTB domain has formed stable dimers and has not interacted with domains of the ttk group. Previously, it was suggested that GAF oligomerization into higher-order complexes facilitates long-range activation by providing a protein bridge between an enhancer and a promoter. Unexpectedly, experiments in the Drosophila model system have not supported the role of GAF in organization of long-distance interaction between the yeast GAL4 activator and the white promoter.
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http://dx.doi.org/10.1016/j.jmb.2011.07.052DOI 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

Expression of humanized anti-Her2/neu single-chain IgG1-like antibody in mammary glands of transgenic mice.

Biochimie 2011 Mar 10;93(3):628-30. Epub 2010 Dec 10.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya street 16/10, Moscow, Russia.

A system for production of single-chain antibody in mammary glands of mice was developed on the basis of a hybrid gene constructed from the coding sequence of anti-Her2/neu single-chain antibody inserted into the first exon of the sheep beta-lactoglobulin gene. Lines of transgenic mice were obtained that expressed humanized single-chain anti-Her2/neu IgG1-like antibody in their milk. These antibodies interact with Her2/neu antigen with high affinity (K(d) = 0.4 nM). The expression level of the transgene depended on its integration site in the genome but not on the copy number. The transgene had no toxic effect on the mice and was stably inherited, at least for two generations. The results reveal new opportunities of producing single-chain antibodies in the milk of animals.
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http://dx.doi.org/10.1016/j.biochi.2010.12.001DOI Listing
March 2011

Drosophila mini-white model system: new insights into positive position effects and the role of transcriptional terminators and gypsy insulator in transgene shielding.

Nucleic Acids Res 2010 Jan 23;38(1):39-47. Epub 2009 Oct 23.

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

The white gene, which is responsible for eye pigmentation, is widely used to study position effects in Drosophila. As a result of insertion of P-element vectors containing mini-white without enhancers into random chromosomal sites, flies with different eye color phenotypes appear, which is usually explained by the influence of positive/negative regulatory elements located around the insertion site. We found that, in more than 70% of cases when mini-white expression was subject to positive position effects, deletion of the white promoter had no effect on eye pigmentation; in these cases, the transposon was inserted into the transcribed regions of genes. Therefore, transcription through the mini-white gene could be responsible for high levels of its expression in most of chromosomal sites. Consistently with this conclusion, transcriptional terminators proved to be efficient in protecting mini-white expression from positive position effects. On the other hand, the best characterized Drosophila gypsy insulator was poorly effective in terminating transcription and, as a consequence, only partially protected mini-white expression from these effects. Thus, to ensure maximum protection of a transgene from position effects, a perfect boundary/insulator element should combine three activities: to block enhancers, to provide a barrier between active and repressed chromatin, and to terminate transcription.
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http://dx.doi.org/10.1093/nar/gkp877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2800232PMC
January 2010

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

Enhancer-promoter communication is regulated by insulator pairing in a Drosophila model bigenic locus.

Mol Cell Biol 2008 Sep 23;28(17):5469-77. Epub 2008 Jun 23.

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

The complexity of regulatory systems in higher eukaryotes, featuring many distantly located enhancers that nonetheless properly activate the target promoters, has prompted the hypothesis that the action of enhancers should be restricted by insulators. Continuing our research on the functional role of insulators and the consequences of their interaction in Drosophila, we studied the interplay of different Su(Hw)-dependent Drosophila insulators. The set of transgenic constructs comprised two consecutive genes (yellow and white) with their enhancers and insulator elements differently arranged in between and/or around the gene(s). All insulators were found to interact in twin or mixed tandems, demonstrating the bypass phenomenon. However, insulator pairing around a gene did not always improve its isolation from an outside enhancer. On the other hand, merely two insulator elements (identical or different) in appropriate positions can permit the expression of one gene but not the gene next to it or, conversely, largely block the transcription of the first gene, while allowing full enhancement of the second, or make them behave similarly. Thus, the results of this study support the model that loop formation by insulators is an essential component of insulator action on a positive and negative regulation of an enhancer-promoter communication.
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http://dx.doi.org/10.1128/MCB.00461-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2519739PMC
September 2008

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

Nucleic Acids Res 2008 Feb 17;36(3):929-37. Epub 2007 Dec 17.

Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, University of Oslo, Centre for Medical Studies in Russia, Moscow 199334, Russia.

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.
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http://dx.doi.org/10.1093/nar/gkm992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2241909PMC
February 2008