Biology subjects

Maksimenko, O.

Publications and source records attributed to Maksimenko, O..

9 recordsLinked to original sources

bioRxiv2025

Beyond DNA Binding: single C2H2 zinc fingers with adjacent β-strands mediate dimerization in Drosophila transcription factors

C2H2 proteins, characterized by DNA-binding C2H2-type zinc finger domains, constitute the largest group of transcription factors. In addition to binding DNA, C2H2 domains can mediate protein-protein interactions, facilitating oligomerization of C2H2 proteins. In this study, we identified eight C2H2 proteins in the Drosophila genome that feature a unique single C2H2 domain containing a conservedCGEx motif. Yeast two-hybrid assays, SEC-MALS, and chemical cross-linking experiments revealed a strong propensity of these domains to form dimers. Using NMR spectroscopy, we determined the solution structure of the dimeric C2H2 domain from IMZF (Immune-mediated Zinc Finger) protein, providing structural evidence towards the dimerization of C2H2 domains. The structure revealed that dimerization is mediated by the interface between the core C2H2 fold and the adjacent {beta}-strand containingCGEx motif, which was further validated by structure-guided mutagenesis. A bioinformatic survey showed thatCGEx-type C2H2 domains are specific to Diptera. Finally, we demonstrate that putatively dimerizing C2H2 domains containing additional structural elements beyond the canonical fold are widespread among diverse eukaryotic taxa, with the highest prevalence observed in insects. These findings establish that single C2H2 domains can mediate self-association and identify theCGEx-type C2H2 domain as a distinct structural subclass specific to Dipteran insects.

molecular biology↗

bioRxiv2022

Structural basis for interaction between CLAMP and MSL2 proteins involved in the specific recruitment of the dosage compensation complex in Drosophila

Transcriptional regulators select their targets from a large pool of similar genomic sites. The binding of the Drosophila dosage compensation complex (DCC) exclusively to the male X chromosome provides insight into binding site selectivity rules. Previous studies showed that the male-specific organizer of the complex, MSL2, and ubiquitous DNA-binding protein CLAMP directly interact and play an important role in the specificity of X chromosome binding. Here we studied the highly specific interaction between the intrinsically disordered region of MSL2 and the N-terminal zinc-finger C2H2-type (C2H2) domain of CLAMP. We obtained the NMR structure of the CLAMP N-terminal C2H2 zinc finger, which has a classic C2H2 zinc-finger fold with a rather unusual distribution of residues typically used in DNA recognition. Substitutions of residues in this C2H2 domain had the same effect on the viability of males and females, suggesting that it plays a general role in CLAMP activity. The N-terminal C2H2 domain of CLAMP is highly conserved in insects. However, the MSL2 region involved in the interaction is conserved only within the Drosophila genus, suggesting that this interaction emerged during the evolution of a mechanism for the specific recruitment of the DCC on the male X chromosome in Drosophilidae.

molecular biology↗

bioRxiv2021

Drosophila architectural protein CTCF is not essential for fly survival and is able to function independently of CP190

CTCF is the most likely ancestor of proteins that contain large clusters of C2H2 zinc finger domains (C2H2) and is conserved among most bilateral organisms. In mammals, CTCF functions as the main architectural protein involved in the organization of topology-associated domains (TADs). In vertebrates and Drosophila, CTCF is involved in the regulation of homeotic genes. Previously, null mutations in the dCTCF gene were found to result in death during the stage of pharate adults, which failed to eclose from their pupal case. Here, we obtained several new null dCTCF mutations and found that the complete inactivation of dCTCF appears to be limited to phenotypic manifestations of the Abd-B gene and fertility of adult flies. Many modifiers that are not associated with an independent phenotypic manifestation can significantly enhance the expressivity of the null dCTCF mutations, indicating that other architectural proteins are able to functionally compensate for dCTCF inactivation in Drosophila. We also mapped the 715-735 aa region of dCTCF as being essential for the interaction with the BTB (Broad-Complex, Tramtrack, and Bric a brac) and microtubule-targeting (M) domains of the CP190 protein, which binds to many architectural proteins. However, the mutational analysis showed that the interaction with CP190 was not essential for the functional activity of dCTCF in vivo. HighlightsO_LIThe dCTCF 715-735 aa region interacts with the BTB and M domains of CP190 C_LIO_LIInteraction with CP190 is not essential for dCTCF function in vivo C_LIO_LINull dCTCF mutants are viable but show severely reduced fertility C_LI

molecular biology↗

bioRxiv2020

N-terminus of Drosophila MSL1 interacts with DNA-binding proteins and is critical for the recruitment of the dosage compensation complex to the X chromosome

The male-specific lethal complex (MSL), which consists of five proteins and two non-coding roX RNAs, is involved in the transcriptional enhancement of X-linked genes to compensate for the sex chromosome monosomy in Drosophila XY males compared with XX females. The MSL1 and MSL2 proteins form the heterotetrameric core of MSL complex and are critical for the specific recruitment of the complex to the high-affinity "entry" sites (HAS) on the X chromosome. In this study, we demonstrated that the N-terminal region of MSL1 is critical for stability and functions of MSL1. Amino acid deletions and substitutions in the N-terminal region of MSL1 strongly affect both the interaction with roX2 RNA and the MSL complex binding to HAS on the X chromosome. In particular, substitution of the conserved N-terminal amino-acids 3-7 in MSL1 (MSL1GS) affects male viability similar to the inactivation of genes encoding roX RNAs. In addition, MSL1GS binds to promoters such as MSL1WT but does not co-bind with MSL2 and MSL3 to X chromosomal HAS. However, overexpression of MSL2 partially restores the dosage compensation. Thus, the interaction of MSL1 with roX RNA is critical for the efficient assembly of the MSL complex on HAS of the male X chromosome.

molecular biology↗

bioRxiv2020

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

The architectural protein Pita is critical for Drosophila embryogenesis and predominantly binds to gene promoters and insulators. In particular, Pita is involved in the organization of boundaries between regulatory domains that controlled the expression of three hox genes in the Bithorax complex (BX-C). The best-characterized partner for Pita is the BTB/POZ-domain containing protein CP190. 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. The expression of the mutant protein completely complemented the null pita mutation. ChIP-seq experiments with wild-type and mutant embryos showed that the deletion of the CP190-interacting regions did not significantly affect the binding of the mutant Pita protein to most chromatin sites. However, the mutant Pita protein does not support the ability of multimerized Pita sites to prevent cross-talk between the iab-6 and iab-7 regulatory domains that activate the expression of Abdominal-B (Abd-B), one of the genes in the BX-C. The recruitment of a chimeric protein consisting of the DNA-binding domain of GAL4 and CP190-interacting region of the Pita to the GAL4 binding sites on the polytene chromosomes of larvae induces the formation of a new interband, which is a consequence of the formation of open chromatin in this region. These results suggested that the interaction with CP190 is required for the primary Pita activities, but other architectural proteins may also recruit CP190 in flies expressing only the mutant Pita protein. Author SummaryPita 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. The CP190 protein is recruited to chromatin through interaction with the architectural proteins. Here we mapped two regions in Pita that are required for interaction with the CP190 protein. 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. 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. These results support a model in which the regulatory elements are composed of combinations of binding sites that interact with several architectural proteins with similar functions.

molecular biology↗

bioRxiv2020

A Non-stop identity complex (NIC) supervises enterocyte identity and protects from pre-mature aging

A hallmark of aging is the inability of differentiated cells to maintain their identity. In the aged Drosophila midgut differentiated enterocytes (ECs) lose their identity, and the integrity of the midgut tissue and its homeostasis are impaired. To discover regulators of EC identity relevant to aging we performed an RNAi screen targeting 453 ubiquitin-related genes in fully differentiated ECs. Seventeen genes were identified, including the de-ubiquitinase Non-stop (Not/dUSP22; CG4166). Acute loss of Non-stop in young ECs phenotypically resembled aged ECs. Lineage tracing experiments established that Non-stop-deficient young ECs as well as wild-type aged ECs are no longer differentiated. Aging or acute loss of Non-stop also resulted in progenitor cell hyperproliferation and mis-differentiation, loss of gut integrity, and reduced organismal survival. Proteomic analysis unveiled that Non-stop maintains identity as part of a Non-stop identity complex (NIC) that contains E(y)2, Sgf11, Cp190, (Mod) mdg4, and Nup98. Transcriptionally, Non-stop ensured chromatin accessibility at EC genes, maintained an EC-specific gene expression signature, and silenced non-EC-relevant transcriptional programs. Within the NIC, Non-stop was required for stabilizing of NIC subunits. Upon aging, the levels of Non-stop and NIC subunits declined, and the large-scale organization of the nucleus was distorted. Maintaining youthful levels of Non-stop in wildtype aged ECs safeguarded the protein level of NIC subunits, restored the large-scale organization of the differentiated nucleus, and suppressed aging phenotypes and tissue integrity. Thus, the isopeptidase Non-stop, and NIC, supervise EC identity and protects from premature aging.

cell biology↗

bioRxiv2018

CLAMP directly interacts with MSL2 to facilitate Drosophila dosage compensation

The binding of Drosophila male-specific lethal (MSL) dosage compensation complex exclusively to 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 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 MSL complex recruitment. Here we demonstrated that MSL2 directly interacts with the N-terminal zinc-finger domain of CLAMP. CLAMP-MSL2 and CXC-DNA interactions are cooperatively involved in recruitment of MSL complex to the X chromosome.

molecular biology↗

bioRxiv2018

Factor cooperation for chromosome discrimination in Drosophila

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.\n\nThe process of dosage compensation in Drosophila allows exploring the rules underlying binding site selectivity. The male-specific-lethal (MSL) Dosage Compensation Complex 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.

molecular biology↗

bioRxiv2018

The bithorax complex iab-7 Polycomb Response Element has a novel role in the functioning of the Fab-7 chromatin boundary

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 in between the iab-6 and iab-7 regulatory domains. Transgene 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 presented 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.\n\nAuthor SummaryPolycomb group proteins (PcG) are important epigenetic regulators of developmental genes in all higher eukaryotes. In Drosophila, these proteins are bound to specific regulatory DNA elements called Polycomb group Response Elements (PREs). PcG support proper patterns of homeotic gene expression throughout development. Drosophila PREs are made up of binding sites for a complex array of DNA binding proteins, including GAF and Pho. In the regulatory region of the bithorax complex (BX-C), the boundary/insulator elements organize the autonomous regulatory domains, and their active or repressed states are regulated by PREs. Here, we studied the domain organization of the Fab-7 boundary and the neighboring PRE, which separate the iab-6 and iab-7 domains involved in transcription of the Abd-B gene. It was previously thought that PRE recruits PcG proteins that inhibit activation of the iab-7 enhancers in the inappropriate domains. However, here we found that PRE contributes to boundary activity and in combination with a key 242 bp Fab-7 region (dHS1) can form a completely functional boundary. Late Boundary Complex (LBC) binds not only to dHS1 but also to PRE and is required for the boundary activity of both elements. At the same time, mutations of Pho binding sites strongly diminish recruiting of PcG but do not considerably affect boundary function, suggesting that these activities can be separated in PRE.

genetics↗