Development of a New Model System to Study Long-Distance Interactions Supported by Architectural Proteins.

Chromatin architecture is critical for the temporal and tissue-specific activation of genes that determine eukaryotic development. The functional interaction between enhancers and promoters is controlled by insulators and tethering elements that support specific long-distance interactions. However, the mechanisms of the formation and maintenance of long-range interactions between genome regulatory elements remain poorly understood, primarily due to the lack of convenient model systems. Drosophila became the first model organism in which architectural proteins that determine the activity of insulators were described. In Drosophila, one of the best-studied DNA-binding architectural proteins, Su(Hw), forms a complex with Mod(mdg4)-67.2 and CP190 proteins. Using a combination of CRISPR/Cas9 genome editing and attP-dependent integration technologies, we created a model system in which the promoters and enhancers of two reporter genes are separated by 28 kb. In this case, enhancers effectively stimulate reporter gene promoters in cis and trans only in the presence of artificial Su(Hw) binding sites (SBS), in both constructs. The expression of the mutant Su(Hw) protein, which cannot interact with CP190, and the mutation inactivating Mod(mdg4)-67.2, lead to the complete loss or significant weakening of enhancer-promoter interactions, respectively. The results indicate that the new model system effectively identifies the role of individual subunits of architectural protein complexes in forming and maintaining specific long-distance interactions in the D. melanogaster model.

Genetic, Environmental, and Stochastic Components of Lifespan Variability: The Drosophila Paradigm.

Lifespan is a complex quantitative trait involving genetic and non-genetic factors as well as the peculiarities of ontogenesis. As with all quantitative traits, lifespan shows considerable variation within populations and between individuals. Drosophila, a favourite object of geneticists, has greatly advanced our understanding of how different forms of variability affect lifespan. This review considers the role of heritable genetic variability, phenotypic plasticity and stochastic variability in controlling lifespan in Drosophila melanogaster. We discuss the major historical milestones in the development of the genetic approach to study lifespan, the breeding of long-lived lines, advances in lifespan QTL mapping, the environmental factors that have the greatest influence on lifespan in laboratory maintained flies, and the mechanisms, by which individual development affects longevity. The interplay between approaches to study ageing and lifespan limitation will also be discussed. Particular attention will be paid to the interaction of different types of variability in the control of lifespan.

The N-terminal dimerization domains of human and Drosophila CTCF have similar functionality.

BACKGROUND: CTCF is highly likely to be the ancestor of proteins that contain large clusters of C2H2 zinc finger domains, and its conservation is observed across most bilaterian organisms. In mammals, CTCF is the primary architectural protein involved in organizing chromosome topology and mediating enhancer-promoter interactions over long distances. In Drosophila, CTCF (dCTCF) cooperates with other architectural proteins to establish long-range interactions and chromatin boundaries. CTCFs of various organisms contain an unstructured N-terminal dimerization domain (DD) and clusters comprising eleven zinc-finger domains of the C2H2 type. The Drosophila (dCTCF) and human (hCTCF) CTCFs share sequence homology in only five C2H2 domains that specifically bind to a conserved 15 bp motif. RESULTS: Previously, we demonstrated that CTCFs from different organisms carry unstructured N-terminal dimerization domains (DDs) that lack sequence homology. Here we used the CTCFattP(mCh) platform to introduce desired changes in the Drosophila CTCF gene and generated a series of transgenic lines expressing dCTCF with different variants of the N-terminal domain. Our findings revealed that the functionality of dCTCF is significantly affected by the deletion of the N-terminal DD. Additionally, we observed a strong impact on the binding of the dCTCF mutant to chromatin upon deletion of the DD. However, chromatin binding was restored in transgenic flies expressing a chimeric CTCF protein with the DD of hCTCF. Although the chimeric protein exhibited lower expression levels than those of the dCTCF variants, it efficiently bound to chromatin similarly to the wild type (wt) protein. CONCLUSIONS: Our findings suggest that one of the evolutionarily conserved functions of the unstructured N-terminal dimerization domain is to recruit dCTCF to its genomic sites in vivo.

Interaction of MLE with CLAMP zinc finger is involved in proper MSL proteins binding to chromosomes in Drosophila.

The Drosophila male-specific lethal (MSL) complex binds to the male X chromosome to activate transcription. It comprises five proteins (MSL1, MSL2, MSL3, male absent on the first (MOF), and maleless (MLE)) and two long noncoding RNAs (lncRNAs; roX1 and roX2). The MLE helicase remodels the roX lncRNAs, enabling the lncRNA-mediated assembly of the Drosophila dosage compensation complex. MSL2 is expressed only in males and interacts with the N-terminal zinc finger of the transcription factor chromatin-linked adapter for MSL proteins (CLAMP), which is important for the specific recruitment of the MSL complex to the male X chromosome. Here, we found that MLE's unstructured C-terminal region interacts with the sixth zinc-finger domain of CLAMP. In vitro, 4-5 zinc fingers are critical for the specific DNA-binding of CLAMP with GA repeats, which constitute the core motif at the high affinity binding sites for MSL proteins. Deleting the CLAMP binding region in MLE decreases the association of MSL proteins with the male X chromosome and increases male lethality. These results suggest that interactions of unstructured regions in MSL2 and MLE with CLAMP zinc finger domains are important for the specific recruitment of the MSL complex to the male X chromosome.

The N-Terminal Part of Drosophila CP190 Is a Platform for Interaction with Multiple Architectural Proteins.

CP190 is a co-factor in many Drosophila architectural proteins, being involved in the formation of active promoters and insulators. CP190 contains the N-terminal BTB/POZ (Broad-Complex, Tramtrack and Bric a brac/POxvirus and Zinc finger) domain and adjacent conserved regions involved in protein interactions. Here, we examined the functional roles of these domains of CP190 in vivo. The best-characterized architectural proteins with insulator functions, Pita, Su(Hw), and dCTCF, interacted predominantly with the BTB domain of CP190. Due to the difficulty of mutating the BTB domain, we obtained a transgenic line expressing a chimeric CP190 with the BTB domain of the human protein Kaiso. Another group of architectural proteins, M1BP, Opbp, and ZIPIC, interacted with one or both of the highly conserved regions in the N-terminal part of CP190. Transgenic lines of D. melanogaster expressing CP190 mutants with a deletion of each of these domains were obtained. The results showed that these mutant proteins only partially compensated for the functions of CP190, weakly binding to selective chromatin sites. Further analysis confirmed the essential role of these domains in recruitment to regulatory regions associated with architectural proteins. We also found that the N-terminal of CP190 was sufficient for recruiting Z4 and Chromator proteins and successfully achieving chromatin opening. Taken together, our results and the results of previous studies showed that the N-terminal region of CP190 is a platform for simultaneous interaction with various DNA-binding architectural proteins and transcription complexes.

The MADF-BESS Protein CP60 Is Recruited to Insulators via CP190 and Has Redundant Functions in Drosophila.

Drosophila CP190 and CP60 are transcription factors that are associated with centrosomes during mitosis. CP190 is an essential transcription factor and preferentially binds to housekeeping gene promoters and insulators through interactions with architectural proteins, including Su(Hw) and dCTCF. CP60 belongs to a family of transcription factors that contain the N-terminal MADF domain and the C-terminal BESS domain, which is characterized by the ability to homodimerize. In this study, we show that the conserved CP60 region adjacent to MADF is responsible for interacting with CP190. In contrast to the well-characterized MADF-BESS transcriptional activator Adf-1, CP60 is recruited to most chromatin sites through its interaction with CP190, and the MADF domain is likely involved in protein-protein interactions but not in DNA binding. The deletion of the Map60 gene showed that CP60 is not an essential protein, despite the strong and ubiquitous expression of CP60 at all stages of Drosophila development. Although CP60 is a stable component of the Su(Hw) insulator complex, the inactivation of CP60 does not affect the enhancer-blocking activity of the Su(Hw)-dependent gypsy insulator. Overall, our results indicate that CP60 has an important but redundant function in transcriptional regulation as a partner of the CP190 protein.

Boundary bypass activity in the abdominal-B region of the Drosophila bithorax complex is position dependent and regulated.

Expression of Abdominal-B (Abd-B) in abdominal segments A5-A8 is controlled by four regulatory domains, iab-5-iab-8. Each domain has an initiator element (which sets the activity state), elements that maintain this state and tissue-specific enhancers. To ensure their functional autonomy, each domain is bracketed by boundary elements (Mcp, Fab-7, Fab-7 and Fab-8). In addition to blocking crosstalk between adjacent regulatory domains, the Fab boundaries must also have bypass activity so the relevant regulatory domains can 'jump over' intervening boundaries and activate the Abd-B promoter. In the studies reported here we have investigated the parameters governing bypass activity. We find that the bypass elements in the Fab-7 and Fab-8 boundaries must be located in the regulatory domain that is responsible for driving Abd-B expression. We suggest that bypass activity may also be subject to regulation.

lncRNA read-through regulates the BX-C insulator Fub-1.

Though long non-coding RNAs (lncRNAs) represent a substantial fraction of the Pol II transcripts in multicellular animals, only a few have known functions. Here we report that the blocking activity of the Bithorax complex (BX-C) Fub-1 boundary is segmentally regulated by its own lncRNA. The Fub-1 boundary is located between the Ultrabithorax (Ubx) gene and the bxd/pbx regulatory domain, which is responsible for regulating Ubx expression in parasegment PS6/segment A1. Fub-1 consists of two hypersensitive sites, HS1 and HS2. HS1 is an insulator while HS2 functions primarily as an lncRNA promoter. To activate Ubx expression in PS6/A1, enhancers in the bxd/pbx domain must be able to bypass Fub-1 blocking activity. We show that the expression of the Fub-1 lncRNAs in PS6/A1 from the HS2 promoter inactivates Fub-1 insulating activity. Inactivation is due to read-through as the HS2 promoter must be directed toward HS1 to disrupt blocking.

Transcriptional Readthrough Interrupts Boundary Function in Drosophila.

In higher eukaryotes, distance enhancer-promoter interactions are organized by topologically associated domains, tethering elements, and chromatin insulators/boundaries. While insulators/boundaries play a central role in chromosome organization, the mechanisms regulating their functions are largely unknown. In the studies reported here, we have taken advantage of the well-characterized Drosophila bithorax complex (BX-C) to study one potential mechanism for controlling boundary function. The regulatory domains of BX-C are flanked by boundaries, which block crosstalk with their neighboring domains and also support long-distance interactions between the regulatory domains and their target gene. As many lncRNAs have been found in BX-C, we asked whether readthrough transcription (RT) can impact boundary function. For this purpose, we took advantage of two BX-C boundary replacement platforms, Fab-7attP50 and F2attP, in which the Fab-7 and Fub boundaries, respectively, are deleted and replaced with an attP site. We introduced boundary elements, promoters, and polyadenylation signals arranged in different combinations and then assayed for boundary function. Our results show that RT can interfere with boundary activity. Since lncRNAs represent a significant fraction of Pol II transcripts in multicellular eukaryotes, it is therefore possible that RT may be a widely used mechanism to alter boundary function and regulation of gene expression.

Corticosteroid-Associated Avascular Necrosis of the Femoral Head in Patients with Severe COVID-19: A Single-Center Study.

BACKGROUND Avascular necrosis (AVN) of the femoral head can result from high-dose corticosteroid therapy. Given that severe COVID-19 pneumonia patients respond positively to corticosteroids, this study aimed to explore the incidence of femoral head AVN associated with corticosteroid therapy in 24 patients diagnosed with severe COVID-19 at a single center. MATERIAL AND METHODS The study included 24 patients who were diagnosed with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection through real-time reverse transcription polymerase chain reaction test (rRT-PCR) and with COVID-19 pneumonia via high-resolution computed tomography (HRCT). Moderate cases received 2×4 mg Dexamethasone while severe cases were also administered with 3×40 mg Methylprednisolone. Diagnosis of femoral head AVN was confirmed with magnetic resonance imaging (MRI) and radiographs, which was subsequently treated by a total hip arthroplasty (THA) or a core decompression surgery (CDS) in line with the Ficat and Arlet classifications RESULTS Among the patients, 8 had a moderate infection course, while 16 were severe. The mean corticosteroid duration was 15±5 days for Dexamethasone and 30 days for Methylprednisolone. Severe patients presented with higher grade femoral head AVN and greater pain levels compared to moderate cases (p<0.05). Four patients developed bilateral AVN. The treatment resulted in 23 THAs and 5 CDSs CONCLUSIONS The data from this study corroborate earlier studies and case reports, suggesting an increased occurrence of AVN of the femoral head during the COVID-19 pandemic due to the high-dose corticosteroid therapy employed for patients hospitalized with severe COVID-19 pneumonia.

Boundary Bypass Activity in the Abdominal-B Region of the Drosophila Bithorax Complex is Position Dependent and Regulated.

Expression of Abdominal-B ( Abd-B ) in abdominal segments A5 - A8 is controlled by four regulatory domains, iab-5 - iab-8 . Each domain has an initiator element (which sets the activity state), elements that maintain this state and tissue-specific enhancers. To ensure their functional autonomy, each domain is bracketed by boundary elements ( Mcp , Fab-7 , Fab-7 and Fab-8 ). In addition to blocking crosstalk between adjacent regulatory domains, the Fab boundaries must also have bypass activity so the relevant regulatory domains can "jump over" intervening boundaries and activate the Abd-B promoter. In the studies reported here we have investigated the parameters governing bypass activity. We find that the bypass elements in the Fab-7 and Fab-8 boundaries must be located in the regulatory domain that is responsible for driving Abd-B expression. We suggest that bypass activity may also be subject to regulation. Summary Statement: Boundaries separating Abd-B regulatory domains block crosstalk between domains and mediate their interactions with Abd-B . The latter function is location but not orientation dependent.

Crol contributes to PRE-mediated repression and Polycomb group proteins recruitment in Drosophila.

The Polycomb group (PcG) proteins are fundamental epigenetic regulators that control the repressive state of target genes in multicellular organisms. One of the open questions is defining the mechanisms of PcG recruitment to chromatin. In Drosophila, the crucial role in PcG recruitment is thought to belong to DNA-binding proteins associated with Polycomb response elements (PREs). However, current data suggests that not all PRE-binding factors have been identified. Here, we report the identification of the transcription factor Crooked legs (Crol) as a novel PcG recruiter. Crol is a C2H2-type Zinc Finger protein that directly binds to poly(G)-rich DNA sequences. Mutation of Crol binding sites as well as crol CRISPR/Cas9 knockout diminish the repressive activity of PREs in transgenes. Like other PRE-DNA binding proteins, Crol co-localizes with PcG proteins inside and outside of H3K27me3 domains. Crol knockout impairs the recruitment of the PRC1 subunit Polyhomeotic and the PRE-binding protein Combgap at a subset of sites. The decreased binding of PcG proteins is accompanied by dysregulated transcription of target genes. Overall, our study identified Crol as a new important player in PcG recruitment and epigenetic regulation.

Mechanisms of Interaction between Enhancers and Promoters in Three Drosophila Model Systems.

In higher eukaryotes, the regulation of developmental gene expression is determined by enhancers, which are often located at a large distance from the promoters they regulate. Therefore, the architecture of chromosomes and the mechanisms that determine the functional interaction between enhancers and promoters are of decisive importance in the development of organisms. Mammals and the model animal Drosophila have homologous key architectural proteins and similar mechanisms in the organization of chromosome architecture. This review describes the current progress in understanding the mechanisms of the formation and regulation of long-range interactions between enhancers and promoters at three well-studied key regulatory loci in Drosophila.

Transcriptional read through interrupts boundary function in Drosophila.

In higher eukaryotes enhancer-promoter interactions are known to be restricted by the chromatin insulators/boundaries that delimit topologically associated domains (TADs); however, there are instances in which enhancer-promoter interactions span one or more boundary elements/TADs. At present, the mechanisms that enable cross-TAD regulatory interaction are not known. In the studies reported here we have taken advantage of the well characterized Drosophila Bithorax complex (BX-C) to study one potential mechanism for controlling boundary function and TAD organization. The regulatory domains of BX-C are flanked by boundaries which function to block crosstalk with their neighboring domains and also to support long distance interactions between the regulatory domains and their target gene. As many lncRNAs have been found in BX-C, we asked whether transcriptional readthrough can impact boundary function. For this purpose, we took advantage of two BX-C boundary replacement platforms, Fab-7 attP50 and F2 attP , in which the Fab-7 and Fub boundaries, respectively, are deleted and replaced with an attP site. We introduced boundary elements, promoters and polyadenylation signals arranged in different combinations and then assayed for boundary function. Our results show that transcriptional readthrough can interfere with boundary activity. Since lncRNAs represent a significant fraction of Pol II transcripts in multicellular eukaryotes, it is possible that many of them may function in the regulation of TAD organization. Author Summary: Recent studies have shown that much genome in higher eukaryotes is transcribed into non-protein coding lncRNAs. It is though that lncRNAs may preform important regulatory functions, including the formation of protein complexes, organization of functional interactions between enhancers and promoters and the maintenance of open chromatin. Here we examined how transcription from promoters inserted into the Drosophila Bithorax complex can impact the boundaries that are responsible for establishing independent regulatory domains. Surprisingly, we found that even a relatively low level of transcriptional readthrough can impair boundary function. Transcription also affects the activity of enhancers located in BX-C regulatory domains. Taken together, our results raise the possibility that transcriptional readthrough may be a widely used mechanism to alter chromosome structure and regulate gene expression.

BTB domains: A structural view of evolution, multimerization, and protein-protein interactions.

Broad-complex, Tramtrack, and Bric-à-brac/poxvirus and zinc finger (BTB/POZ) is a conserved domain found in many eukaryotic proteins with diverse cellular functions. Recent studies revealed its importance in multiple developmental processes as well as in the onset and progression of oncological diseases. Most BTB domains can form multimers and selectively interact with non-BTB proteins. Structural studies of BTB domains delineated the presence of different interfaces involved in various interactions mediated by BTBs and provided a basis for the specific inhibition of distinct protein-interaction interfaces. BTB domains originated early in eukaryotic evolution and progressively adapted their structural elements to perform distinct functions. In this review, we summarize and discuss the structural principles of protein-protein interactions mediated by BTB domains based on the recently published structural data and advances in protein modeling. We propose an update to the structure-based classification of BTB domain families and discuss their evolutionary interconnections.

The Essential Role of Prolines and Their Conformation in Allosteric Regulation of Kaiso Zinc Finger DNA-Binding Activity by the Adjacent C-Terminal Loop.

Kaiso is a methyl-DNA-binding protein containing three C2H2 zinc fingers with a C-terminal extension that participates in DNA binding. The linker between the last zinc finger and the DNA-binding portion of the extension contains two prolines that are highly conserved in vertebrates and in cognate ZBTB4 and ZBTB38 proteins. Prolines provide chain rigidity and can exist in cis and trans conformations that can be switched by proline isomerases, affecting protein function. We found that substitution of the conserved proline P588, but not of P577, to alanine, negatively affected KaisoDNA-binding according to molecular dynamics simulation and in vitro DNA-binding assays. Molecular dynamics simulations of the Kaiso DNA-binding domain with P588 either substituted to alanine or switched to the cis-conformation revealed similar alterations in the H-bonding network and uncovered allosteric effects leading to structural rearrangements in the entire domain that resulted in the weakening of DNA-binding affinity. The substitution of proline with a large hydrophobic residue led to the same negative effects despite its ability to partially rescue the intrinsic DNA-binding activity of the C-terminal loop. Thus, the presence of the C-terminal extension and cis-conformation of proline residues are essential for efficient Kaiso-DNA binding, which likely involves intramolecular tension squeezing the DNA chain.

Mechanisms of enhancer-promoter communication and chromosomal architecture in mammals and Drosophila.

The spatial organization of chromosomes is involved in regulating the majority of intranuclear processes in higher eukaryotes, including gene expression. Drosophila was used as a model to discover many transcription factors whose homologs play a key role in regulation of gene expression in mammals. According to modern views, a cohesin complex mostly determines the architecture of mammalian chromosomes by forming chromatin loops on anchors created by the CTCF DNA-binding architectural protein. The role of the cohesin complex in chromosome architecture is poorly understood in Drosophila, and CTCF is merely one of many Drosophila architectural proteins with a proven potential to organize specific long-range interactions between regulatory elements in the genome. The review compares the mechanisms responsible for long-range interactions and chromosome architecture between mammals and Drosophila.

Drosophila as a Model Organism to Study Basic Mechanisms of Longevity.

The spatio-temporal regulation of gene expression determines the fate and function of various cells and tissues and, as a consequence, the correct development and functioning of complex organisms. Certain mechanisms of gene activity regulation provide adequate cell responses to changes in environmental factors. Aside from gene expression disorders that lead to various pathologies, alterations of expression of particular genes were shown to significantly decrease or increase the lifespan in a wide range of organisms from yeast to human. Drosophila fruit fly is an ideal model system to explore mechanisms of longevity and aging due to low cost, easy handling and maintenance, large number of progeny per adult, short life cycle and lifespan, relatively low number of paralogous genes, high evolutionary conservation of epigenetic mechanisms and signalling pathways, and availability of a wide range of tools to modulate gene expression in vivo. Here, we focus on the organization of the evolutionarily conserved signaling pathways whose components significantly influence the aging process and on the interconnections of these pathways with gene expression regulation.

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.

Comparative interactome analysis of the PRE DNA-binding factors: purification of the Combgap-, Zeste-, Psq-, and Adf1-associated proteins.

The Polycomb group (PcG) and Trithorax group (TrxG) proteins are key epigenetic regulators controlling the silenced and active states of genes in multicellular organisms, respectively. In Drosophila, PcG/TrxG proteins are recruited to the chromatin via binding to specific DNA sequences termed polycomb response elements (PREs). While precise mechanisms of the PcG/TrxG protein recruitment remain unknown, the important role is suggested to belong to sequence-specific DNA-binding factors. At the same time, it was demonstrated that the PRE DNA-binding proteins are not exclusively localized to PREs but can bind other DNA regulatory elements, including enhancers, promoters, and boundaries. To gain an insight into the PRE DNA-binding protein regulatory network, here, using ChIP-seq and immuno-affinity purification coupled to the high-throughput mass spectrometry, we searched for differences in abundance of the Combgap, Zeste, Psq, and Adf1 PRE DNA-binding proteins. While there were no conspicuous differences in co-localization of these proteins with other functional transcription factors, we show that Combgap and Zeste are more tightly associated with the Polycomb repressive complex 1 (PRC1), while Psq interacts strongly with the TrxG proteins, including the BAP SWI/SNF complex. The Adf1 interactome contained Mediator subunits as the top interactors. In addition, Combgap efficiently interacted with AGO2, NELF, and TFIID. Combgap, Psq, and Adf1 have architectural proteins in their networks. We further investigated the existence of direct interactions between different PRE DNA-binding proteins and demonstrated that Combgap-Adf1, Psq-Dsp1, and Pho-Spps can interact in the yeast two-hybrid assay. Overall, our data suggest that Combgap, Psq, Zeste, and Adf1 are associated with the protein complexes implicated in different regulatory activities and indicate their potential multifunctional role in the regulation of transcription.