Publications by authors named "Pavel Georgiev"

88 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

Structural basis of diversity and homodimerization specificity of zinc-finger-associated domains in Drosophila.

Nucleic Acids Res 2021 02;49(4):2375-2389

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

In arthropods, zinc finger-associated domains (ZADs) are found at the N-termini of many DNA-binding proteins with tandem arrays of Cys2-His2 zinc fingers (ZAD-C2H2 proteins). ZAD-C2H2 proteins undergo fast evolutionary lineage-specific expansion and functional diversification. Here, we show that all ZADs from Drosophila melanogaster form homodimers, but only certain ZADs with high homology can also heterodimerize. CG2712, for example, is unable to heterodimerize with its paralog, the previously characterized insulator protein Zw5, with which it shares 46% homology. We obtained a crystal structure of CG2712 protein's ZAD domain that, in spite of a low sequence homology, has similar spatial organization with the only known ZAD structure (from Grauzone protein). Steric clashes prevented the formation of heterodimers between Grauzone and CG2712 ZADs. Using detailed structural analysis, site-directed mutagenesis, and molecular dynamics simulations, we demonstrated that rapid evolutionary acquisition of interaction specificity was mediated by the more energy-favorable formation of homodimers in comparison to heterodimers, and that this specificity was achieved by multiple amino acid substitutions resulting in the formation or breaking of stabilizing interactions. We speculate that specific homodimerization of ZAD-C2H2 proteins is important for their architectural role in genome organization.
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http://dx.doi.org/10.1093/nar/gkab061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7913770PMC
February 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

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

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

HIPP1 stabilizes the interaction between CP190 and Su(Hw) in the Drosophila insulator complex.

Sci Rep 2019 12 13;9(1):19102. Epub 2019 Dec 13.

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

Suppressor of Hairy-wing [Su(Hw)] is one of the best characterized architectural proteins in Drosophila and recruits the CP190 and Mod(mdg4)-67.2 proteins to chromatin, where they form a well-known insulator complex. Recently, HP1 and insulator partner protein 1 (HIPP1), a homolog of the human co-repressor Chromodomain Y-Like (CDYL), was identified as a new partner for Su(Hw). Here, we performed a detailed analysis of the domains involved in the HIPP1 interactions with Su(Hw)-dependent complexes. HIPP1 was found to directly interact with the Su(Hw) C-terminal region (aa 720-892) and with CP190, but not with Mod(mdg4)-67.2. We have generated Hipp1 null mutants (Hipp) and found that the loss of Hipp1 does not affect the enhancer-blocking or repression activities of the Su(Hw)-dependent complex. However, the simultaneous inactivation of both HIPP1 and Mod(mdg4)-67.2 proteins resulted in reduced CP190 binding with Su(Hw) sites and significantly altered gypsy insulator activity. Taken together, these results suggested that the HIPP1 protein stabilized the interaction between CP190 and the Su(Hw)-dependent complex.
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http://dx.doi.org/10.1038/s41598-019-55617-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6911044PMC
December 2019

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

The same domain of Su(Hw) is required for enhancer blocking and direct promoter repression.

Sci Rep 2019 03 29;9(1):5314. Epub 2019 Mar 29.

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

Suppressor of Hairy-wing [Su(Hw)] is a DNA-binding architectural protein that participates in the organization of insulators and repression of promoters in Drosophila. This protein contains acidic regions at both ends and a central cluster of 12 zinc finger domains, some of which are involved in the specific recognition of the binding site. One of the well-described in vivo function of Su(Hw) is the repression of transcription of neuronal genes in oocytes. Here, we have found that the same Su(Hw) C-terminal region (aa 720-892) is required for insulation as well as for promoter repression. The best characterized partners of Su(Hw), CP190 and Mod(mdg4)-67.2, are not involved in the repression of neuronal genes. Taken together, these results suggest that an unknown protein or protein complex binds to the C-terminal region of Su(Hw) and is responsible for the direct repression activity of Su(Hw).
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http://dx.doi.org/10.1038/s41598-019-41761-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441048PMC
March 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

Functional dissection of the developmentally restricted BEN domain chromatin boundary factor Insensitive.

Epigenetics Chromatin 2019 01 3;12(1). Epub 2019 Jan 3.

Department of Molecular Biology, Princeton University, Princeton, NJ, USA.

Background: Boundaries in the Drosophila bithorax complex delimit autonomous regulatory domains that activate the parasegment (PS)-specific expression of homeotic genes. The Fab-7 boundary separates the iab-6 and iab-7 regulatory domains that control Abd-B expression in PS11 and PS12. This boundary is composed of multiple functionally redundant elements and has two key activities: it blocks crosstalk between iab-6 and iab-7 and facilitates boundary bypass.

Results: Here, we have used a structure-function approach to elucidate the biochemical properties and the in vivo activities of a conserved BEN domain protein, Insensitive, that is associated with Fab-7. Our biochemical studies indicate that in addition to the C-terminal BEN DNA-binding domain, Insv has two domains that mediate multimerization: one is a coiled-coil domain in the N-terminus, and the other is next to the BEN domain. These multimerization domains enable Insv to bind simultaneously to two canonical 8-bp recognition motifs, as well as to a ~ 100-bp non-canonical recognition sequence. They also mediate the assembly of higher-order multimers in the presence of DNA. Transgenic proteins lacking the N-terminal coiled-coil domain are compromised for boundary function in vivo. We also show that Insv interacts directly with CP190, a protein previously implicated in the boundary functions of several DNA-binding proteins, including Su(Hw) and dCTCF. While CP190 interaction is required for Insv binding to a subset of sites on polytene chromosomes, it has only a minor role in the boundary activity of Insv in the context of Fab-7.

Conclusions: The subdivision of eukaryotic chromosomes into discrete topological domains depends upon the pairing of boundary elements. In flies, pairing interactions are specific and typically orientation dependent. They occur in cis between neighboring heterologous boundaries, and in trans between homologous boundaries. One potential mechanism for ensuring pairing-interaction specificity is the use of sequence-specific DNA-binding proteins that can bind simultaneously with two or more recognition sequences. Our studies indicate that Insv can assemble into a multivalent DNA-binding complex and that the N-terminal Insv multimerization domain is critical for boundary function.
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http://dx.doi.org/10.1186/s13072-018-0249-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317261PMC
January 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

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

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

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

PLoS One 2018 23;13(2):e0193497. Epub 2018 Feb 23.

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

Su(Hw) belongs to the class of proteins that organize chromosome architecture and boundaries/insulators between regulatory domains. This protein contains a cluster of 12 zinc finger domains most of which are responsible for binding to three different modules in the consensus site. Su(Hw) forms a complex with CP190 and Mod(mdg4)-67.2 proteins that binds to well-known Drosophila insulators. To understand how Su(Hw) performs its activities and binds to specific sites in chromatin, we have examined the previously described su(Hw)f mutation that disrupts the 10th zinc finger (ZF10) responsible for Su(Hw) binding to the upstream module. The results have shown that Su(Hw)f loses the ability to interact with CP190 in the absence of DNA. In contrast, complete deletion of ZF10 does not prevent the interaction between Su(Hw)Δ10 and CP190. Having studied insulator complex formation in different mutant backgrounds, we conclude that both association with CP190 and Mod(mdg4)-67.2 partners and proper organization of DNA binding site are essential for the efficient recruitment of the Su(Hw) complex to chromatin insulators.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193497PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5825117PMC
June 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

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

Open Biol 2017 10;7(10)

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

The best-studied insulator complex consists of two BTB-containing proteins, the Mod(mdg4)-67.2 isoform and CP190, which are recruited to the chromatin through interactions with the DNA-binding Su(Hw) protein. It was shown previously that Mod(mdg4)-67.2 is critical for the enhancer-blocking activity of the Su(Hw) insulators and it differs from more than 30 other Mod(mdg4) isoforms by the C-terminal domain required for a specific interaction with Su(Hw) only. The mechanism of the highly specific association between Mod(mdg4)-67.2 and Su(Hw) is not well understood. Therefore, we have performed a detailed analysis of domains involved in the interaction of Mod(mdg4)-67.2 with Su(Hw) and CP190. We found that the N-terminal region of Su(Hw) interacts with the glutamine-rich domain common to all the Mod(mdg4) isoforms. The unique C-terminal part of Mod(mdg4)-67.2 contains the Su(Hw)-interacting domain and the FLYWCH domain that facilitates a specific association between Mod(mdg4)-67.2 and the CP190/Su(Hw) complex. Finally, interaction between the BTB domain of Mod(mdg4)-67.2 and the M domain of CP190 has been demonstrated. By using transgenic lines expressing different protein variants, we have shown that all the newly identified interactions are to a greater or lesser extent redundant, which increases the reliability in the formation of the protein complexes.
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http://dx.doi.org/10.1098/rsob.170150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666082PMC
October 2017

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

Chromosoma 2018 03 22;127(1):59-71. Epub 2017 Sep 22.

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

The best-studied Drosophila insulator complex consists of two BTB-containing proteins, the Mod(mdg4)-67.2 isoform and CP190, which are recruited cooperatively to chromatin through interactions with the DNA-binding architectural protein Su(Hw). While Mod(mdg4)-67.2 interacts only with Su(Hw), CP190 interacts with many other architectural proteins. In spite of the fact that CP190 is critical for the activity of Su(Hw) insulators, interaction between these proteins has not been studied yet. Therefore, we have performed a detailed analysis of domains involved in the interaction between the Su(Hw) and CP190. The results show that the BTB domain of CP190 interacts with two adjacent regions at the N-terminus of Su(Hw). Deletion of either region in Su(Hw) only weakly affected recruiting of CP190 to the Su(Hw) sites in the presence of Mod(mdg4)-67.2. Deletion of both regions in Su(Hw) prevents its interaction with CP190. Using mutations in vivo, we found that interactions with Su(Hw) and Mod(mdg4)-67.2 are essential for recruiting of CP190 to the Su(Hw) genomic sites.
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http://dx.doi.org/10.1007/s00412-017-0645-6DOI Listing
March 2018

Presenilin-1 Delta E9 Mutant Induces STIM1-Driven Store-Operated Calcium Channel Hyperactivation in Hippocampal Neurons.

Mol Neurobiol 2018 Jun 13;55(6):4667-4680. Epub 2017 Jul 13.

Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Ave, St. Petersburg, 194064, Russia.

Presenilins regulate calcium homeostasis in the endoplasmic reticulum, and dysregulation of intracellular calcium has been implicated in the pathogenesis of Alzheimer disease. Elevated presenilin-1 (PS1) holoprotein levels have been detected in postmortem brains of patients carrying familial Alzheimer disease (FAD) PS1 mutations. This study examines the effect of the FAD presenilin mutant that lacks the ninth exon (PS1 ∆E9) and does not undergo endoproteolysis on store-operated calcium (SOC) entry. Significant enhancement of SOC channel activation was detected by electrophysiological measurements in hippocampal neurons with PS1 ∆E9 mutant expression. Here, we show that (i) the hyperactivation of SOC channels is mediated by the STIM1 sensor and can be attenuated by STIM1 knockdown or 2-aminoethoxydiphenyl borate application, (ii) the STIM2 is not involved in pathological changes of SOC entry, (iii) the pathological SOC entry demonstrates properties of both TRPC and Orai subunit composition, and (iiii) transgenic Drosophila flies with PS1 ∆E9 expression in the cholinergic neuron system show short-term memory loss, which can be abolished by 2-aminoethoxydiphenyl borate feeding.
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http://dx.doi.org/10.1007/s12035-017-0674-4DOI Listing
June 2018

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

The GAGA factor regulatory network: Identification of GAGA factor associated proteins.

PLoS One 2017 15;12(3):e0173602. Epub 2017 Mar 15.

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

The Drosophila GAGA factor (GAF) has an extraordinarily diverse set of functions that include the activation and silencing of gene expression, nucleosome organization and remodeling, higher order chromosome architecture and mitosis. One hypothesis that could account for these diverse activities is that GAF is able to interact with partners that have specific and dedicated functions. To test this possibility we used affinity purification coupled with high throughput mass spectrometry to identify GAF associated partners. Consistent with this hypothesis the GAF interacting network includes a large collection of factors and complexes that have been implicated in many different aspects of gene activity, chromosome structure and function. Moreover, we show that GAF interactions with a small subset of partners is direct; however for many others the interactions could be indirect, and depend upon intermediates that serve to diversify the functional capabilities of the GAF protein.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0173602PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5351981PMC
September 2017

Boundaries of loop domains (insulators): Determinants of chromosome form and function in multicellular eukaryotes.

Bioessays 2017 03 30;39(3). Epub 2017 Jan 30.

Department of Molecular Biology, Princeton University, Princeton, NJ, USA.

Chromosomes in multicellular animals are subdivided into a series of looped domains. In addition to being the underlying principle for organizing the chromatin fiber, looping is critical for processes ranging from gene regulation to recombination and repair. The subdivision of chromosomes into looped domains depends upon a special class of architectural elements called boundaries or insulators. These elements are distributed throughout the genome and are ubiquitous building blocks of chromosomes. In this review, we focus on features of boundaries that are critical in determining the topology of the looped domains and their genetic properties. We highlight the properties of fly boundaries that are likely to have an important bearing on the organization of looped domains in vertebrates, and discuss the functional consequences of the observed similarities and differences.
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http://dx.doi.org/10.1002/bies.201600233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5536339PMC
March 2017

Large-scale ATP-independent nucleosome unfolding by a histone chaperone.

Nat Struct Mol Biol 2016 Dec 7;23(12):1111-1116. Epub 2016 Nov 7.

Biology Faculty, Lomonosov, Moscow State University, Moscow, Russia.

DNA accessibility to regulatory proteins is substantially influenced by nucleosome structure and dynamics. The facilitates chromatin transcription (FACT) complex increases the accessibility of nucleosomal DNA, but the mechanism and extent of its nucleosome reorganization activity are unknown. Here we determined the effects of FACT from the yeast Saccharomyces cerevisiae on single nucleosomes by using single-particle Förster resonance energy transfer (spFRET) microscopy. FACT binding results in dramatic ATP-independent, symmetrical and reversible DNA uncoiling that affects at least 70% of the DNA within a nucleosome, occurs without apparent loss of histones and proceeds via an 'all-or-none' mechanism. A mutated version of FACT is defective in uncoiling, and a histone mutation that suppresses phenotypes caused by this FACT mutation in vivo restores the uncoiling activity in vitro. Thus, FACT-dependent nucleosome unfolding modulates the accessibility of nucleosomal DNA, and this activity is an important function of FACT in vivo.
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http://dx.doi.org/10.1038/nsmb.3321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518926PMC
December 2016

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