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Long-range transcriptional activation of gene promoters by abundant enhancers in animal genomes calls for mechanisms to limit inappropriate regulation. DNA elements called insulators serve this purpose by shielding promoters from an enhancer when interposed. Unlike promoters and enhancers, insulators have not been systematically characterized due to lacking high-throughput screening assays, and questions regarding how insulators are distributed and encoded in the genome remain. Here, we establish "insulator-seq" as a plasmid-based massively parallel reporter assay in Drosophila cultured cells to perform a systematic insulator screen of selected genomic loci. Screening developmental gene loci showed that not all insulator protein binding sites effectively block enhancer-promoter communication. Deep insulator mutagenesis identified sequences flexibly positioned around the CTCF insulator protein binding motif that are critical for functionality. The ability to screen millions of DNA sequences without positional effect has enabled functional mapping of insulators and provided further insights into the determinants of insulators., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This study analyzes the optimization of Extra High Voltage (EHV) transmission line design parameters by Uttar Pradesh Power Corporation Limited (UPPCL) in India to improve power grid efficiency and reliability. UPPCL reduced insulation specifications for 400 kV lines from 27 to 21 disc insulators and decreased live metal clearance from 2600 to 2400 mm, resulting in 7-9% cost savings per line and reduced right-of-way requirements. While initial test lines performed satisfactorily for over 5 years, longer lines experienced operational challenges related to insulator quality, bird activity, and design margins. Tripping issues emerged during adverse weather conditions, particularly in foggy winter periods. Root cause analysis revealed the need for stringent insulator quality control, regular cleaning to mitigate bird dropping contamination, and potential corona ring design improvements. This case study demonstrates how targeted EHV transmission line optimization can yield significant cost and environmental benefits, while highlighting the importance of balancing economic gains with reliability and safety considerations. The findings provide valuable insights for future EHV transmission line designs and upgrades across India's power grid., (© 2024. The Author(s).)

Topologically associating domain (TAD) boundaries are the flanking edges of TADs, also known as insulated neighborhoods, within the 3D structure of genomes. A prominent feature of TAD boundaries in mammalian genomes is the enrichment of clustered CTCF sites often with mixed orientations, which can either block or facilitate enhancer-promoter (E-P) interactions within or across distinct TADs, respectively. We will discuss recent progress in the understanding of fundamental organizing principles of the clustered CTCF insulator codes at TAD boundaries. Specifically, both inward- and outward-oriented CTCF sites function as topological chromatin insulators by asymmetrically blocking improper TAD-boundary-crossing cohesin loop extrusion. In addition, boundary stacking and enhancer clustering facilitate long-distance E-P interactions across multiple TADs. Finally, we provide a unified mechanism for RNA-mediated TAD boundary function via R-loop formation for both insulation and facilitation. This mechanism of TAD boundary formation and insulation has interesting implications not only on how the 3D genome folds in the Euclidean nuclear space but also on how the specificity of E-P interactions is developmentally regulated., (© 2024 The Author(s). BioEssays published by Wiley Periodicals LLC.)

Aiming at the problems of a large volume, slow processing speed, and difficult deployment in the edge terminal, this paper proposes a lightweight insulator detection algorithm based on an improved SSD. Firstly, the original feature extraction network VGG-16 is replaced by a lightweight Ghost Module network to initially achieve the lightweight model. A Feature Pyramid structure and Feature Pyramid Network (FPN+PAN) are integrated into the Neck part and a Simplified Spatial Pyramid Pooling Fast (SimSPPF) module is introduced to realize the integration of local features and global features. Secondly, multiple Spatial and Channel Squeeze-and-Excitation (scSE) attention mechanisms are introduced in the Neck part to make the model pay more attention to the channels containing important feature information. The original six detection heads are reduced to four to improve the inference speed of the network. In order to improve the recognition performance of occluded and overlapping targets, DIoU-NMS was used to replace the original non-maximum suppression (NMS). Furthermore, the channel pruning strategy is used to reduce the unimportant weight matrix of the model, and the knowledge distillation strategy is used to fine-adjust the network model after pruning, so as to ensure the detection accuracy. The experimental results show that the parameter number of the proposed model is reduced from 26.15 M to 0.61 M, the computational load is reduced from 118.95 G to 1.49 G, and the mAP is increased from 96.8% to 98%. Compared with other models, the proposed model not only guarantees the detection accuracy of the algorithm, but also greatly reduces the model volume, which provides support for the realization of visible light insulator target detection based on edge intelligence.

Resilience of the power system against natural disasters is a vital need for sustainable energy supply. As a result of global warming, lakes and rivers have dried out, resulting in dust hubs that threaten the normal operation of outdoor power system equipment. Unlike other events like hurricanes and blizzards, the impact of extreme salt dust on power system insulator failures and network resilience in affected areas remains unexamined. In this paper, to avoid power curtailment caused by insulators breakdown in electricity distribution networks, the resiliency assessment and enhancement of these networks against salt dust is investigated. Failure mechanism analysis of insulators and fragility curves extraction of them in face of salt pollution and relative humidity are done using mathematical modelling and experimental tests to extract the breakdown probability; Experimental tests are conducted in the High Voltage Laboratory, University of Tehran (HVLUT) and a novel method is proposed to extract 3-dimensional fragility curves of insulators. A Monte Carlo-based resiliency assessment method is then employed to obtain resiliency curve against the salt dust. Some suitable indicators are introduced for this purpose. In addition, several resiliency enhancement measures are proposed and ranked using a benefit to cost ratio (BCR) index. Numerical simulations are conducted on two real distribution feeders in a distribution grid around Urmia Salt Lake, Iran. Numerical results confirm the effectiveness and applicability of the proposed method., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Transvection is a phenomenon of interallelic communication in which enhancers can activate a specific promoter located on a homologous chromosome. Insulators play a significant role in ensuring functional interactions between enhancers and promoters. In the presented work, we created a model where two or three copies of the insulator are located next to enhancers and promoters localized on homologous chromosomes. Using the Su(Hw) insulator as a model, we showed that the functional interaction between a pair of insulators promotes enhancer-promoter trans-interactions. The interaction between the three insulators, on the contrary, can lead to the formation of chromatin loops that sterically hinder the full enhancer-promoter interaction. The results of the work suggest the participation of insulators in the regulation of homologous chromosome pairing and in communication between distant genomic loci., (© 2024. Pleiades Publishing, Ltd.)

Using density functional theory, we have studied the intrinsic properties of styrene. First, we determine the optimized structures, structural parameters and thermodynamic properties to make our simulations more realistic to experimental results and check the stability. Second, we investigate optoelectronic, electronic and global descriptors, transport properties of holes and electrons, natural bond orbital analysis, absorption and fluorescence properties. Finally, we study nonlinear optical (NLO) properties: first- and second-order hyperpolarizability, second and third-order optical susceptibilities, hyper-Rayleigh scattering hyperpolarizability, electro-optical Pockel effect, direct current Kerr effects and quadratic refractive index. The bandgap energy E = 5.146 eV and dielectric constant ε r = 3.062 show that styrene is a good insulator with an average electric field value of 4.43 × 10 g = 5.146 eV and dielectric constant ε r = 3.062 show that styrene is a good insulator with an average electric field value of 4.43 × 10 8 Vm -1 . Thermodynamic findings show that our molecule is thermodynamically and chemically stable. Electron and hole reorganization energies of 0.393 and 0.295 eV, respectively, show that styrene is more favourable to hole transport than electron transport. Styrene is transparent with linear refractive index n = 1.750 and quadratic n 2 = 1.748 × 10 -20 m 2  W -1 . At the NLO, styrene has a non-zero value of β H R S , which confirms the existence of first-order NLO activity. Globally the study shows that the styrene monomer is suitable for the architecture design of new polymer materials for NLO applications and optoelectronic by functionalization., Competing Interests: We declare we have no competing interests., (© 2024 The Author(s).)

β 0 /β 0 thalassemia is the most severe type of transfusion-dependent β-thalassemia (TDT) and is still a challenge facing lentiviral gene therapy. Here, we report the interim analysis of a single-center, single-arm pilot trial (NCT05015920) evaluating the safety and efficacy of a β-globin expression-optimized and insulator-engineered lentivirus-modified cell product (BD211) in β 0 /β 0 TDT. Two female children were enrolled, infused with BD211, and followed up for an average of 25.5 months. Engraftment of genetically modified hematopoietic stem and progenitor cells was successful and sustained in both patients. No unexpected safety issues occurred during conditioning or after infusion. Both patients achieved transfusion independence for over 22 months. The treatment extended the lifespan of red blood cells by over 42 days. Single-cell DNA/RNA-sequencing analysis of the dynamic changes of gene-modified cells, transgene expression, and oncogene activation showed no notable adverse effects. Optimized lentiviral gene therapy may safely and effectively treat all β-thalassemia., Competing Interests: Declaration of interests Y.C. is a co-founder and advisor of BDgene Therapeutics. S. Ling and X.W. filed two patents related to this work (“Lentiviral vector applicable to gene therapy of thalassemia and sickle anemia,” ZL201910824134.8, September 2, 2019, China; and “Lentiviral vector applicable to gene therapy of thalassemia and sickle anemia,” ZL202210013838.9, January 6, 2022, China)., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Due to the ever-increasing growth of electric energy consumption, the production of high-quality, reliable and high-reliability electricity is very important. Therefore, it is essential to have distribution and sub-transmission networks with a good reliability factor. In power distribution and sub-transmission lines, it is necessary to somehow isolate the conductors under voltage from the towers, and insulators are used for this purpose. These insulators have two main tasks. One of the main tasks of insulators is to isolate (insulate) the line conductor from the body of the tower. The insulators must be able to isolate the high voltages of the lines from the body of the tower without having a leakage current, and on the other hand, the insulators must be able to withstand the mechanical forces resulting from the weight of the conductors and the applied forces caused by wind and ice. Also, leakage current is one of the important parameters for condition monitoring of insulators in power grid lines. Failure to inspect the insulation for contamination and health conditions will lead to insulator failure and will cause faults in the electrical system. Therefore, it is very important to monitor the condition of the insulator. Based on this, in this paper, according to the data related to leakage current and also according to the introduced wear out function, a procedure for measuring the condition of insulators has been obtained. Finally, the condition of each insulator will be determined according to the defined indicators. Also, the failure level of each monitored data will be obtained using sensitivity analysis., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 Published by Elsevier Ltd.)

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.

Insulators play a pivotal role in the reliability of power distribution networks, necessitating precise defect detection. However, compared with aerial insulator images of transmission network, insulator images of power distribution network contain more complex backgrounds and subtle insulator defects, it leads to high false detection rates and omission rates in current mainstream detection algorithms. In response, this study presents ID-YOLOv7, a tailored convolutional neural network. First, we design a novel Edge Detailed Shape Data Augmentation (EDSDA) method to enhance the model's sensitivity to insulator's edge shapes. Meanwhile, a Cross-Channel and Spatial Multi-Scale Attention (CCSMA) module is proposed, which can interactively model across different channels and spatial domains, to augment the network's attention to high-level insulator defect features. Second, we design a Re-BiC module to fuse multi-scale contextual features and reconstruct the Neck component, alleviating the issue of critical feature loss during inter-feature layer interaction in traditional FPN structures. Finally, we utilize the MPDIoU function to calculate the model's localization loss, effectively reducing redundant computational costs. We perform comprehensive experiments using the Su22kV_broken and PASCAL VOC 2007 datasets to validate our algorithm's effectiveness. On the Su22kV_broken dataset, our approach attains an 85.7% mAP on a single NVIDIA RTX 2080ti graphics card, marking a 7.2% increase over the original YOLOv7. On the PASCAL VOC 2007 dataset, we achieve an impressive 90.3% mAP at a processing speed of 53 FPS, showing a 2.9% improvement compared to the original YOLOv7., Competing Interests: BC, WZ, WW, and ZC were employed by State Grid Fujian Electric Power Research Institute. YL was employed by State Grid Fujian Electric Power Co., Ltd. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Chen, Zhang, Wu, Li, Chen and Li.)

Using Density Functional Theory (DFT) and time-dependent DFT (TD-DFT), we studied a compound widely used in daily life, namely: Methyl Methacrylate (MMA) monomer in its two stable isomers, cis-MMA and trans-MMA. The impact of conformation on the optical and electronic properties of MMA was highlighted. Structural parameters and thermodynamic properties were also assessed, and allowed to discuss the stability and reactivity of this compound. In addition, analysis of optoelectronic and electronic properties, global reactivity descriptors, natural bond orbital (NBO), and hole and electron reorganization energies was performed. The absorption and fluorescence properties, as well as the linear and nonlinear optical (NLO) properties of MMA were also investigated. The findings show that MMA is an insulator due to its wide band gap of 6.20 eV. It also exhibits good optoelectronic properties with high average electric field values of about 5.90 × 10 9 Vm -1 for cis-MMA and 5.42 × 10 9 Vm -1 for trans-MMA and high electric displacement values of at least 14.65 × 10 -2 cm -2 for cis-MMA and 14.33 × 10 -2 cm -2 for trans-MMA, suggesting a potential use for the design of piezoelectric and pyroelectric materials. In addition, trans-MMA has a dielectric constant close to that of good insulators; while cis-MMA exhibits a dielectric behaviour close to that of polymeric substances. Further, these materials are thermodynamically stable in its two conformations, with a good reactivity which can lead to good ability of polymerization. The analysis of the UV-vis spectra revealed that both forms of MMA absorb and emit mainly in the UV and that the Stokes shift of MMA is low, reducing its potential use in devices such as solar cells. At NLO level, MMA isomers exhibit weak optical properties such as second- and third-order optical susceptibility and cannot suitable for second and third harmonic generation; however, the conformation enhances second-order NLO response by at least 2 times when moving from cis- to trans-MMA, whereas the third-order response is almost unaffected. In addition, the conformation also affects the electronic and optoelectronic properties of MMA., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The transcriptome data of skin cells from domestic cats with brown, orange, and white coats were analyzed using a public database to investigate the possible relationship between coat color-related gene expression and squamous cell carcinoma risk, as well as the mechanism of deafness in white cats. We found that the ratio of the expression level of genes suppressing squamous cell carcinoma to that of genes promoting squamous cell carcinoma might be considerably lower than the theoretical estimation in skin cells with orange and white coats in white-spotted cat. We also found the possibility of the frequent production of KIT lacking the first exon (d1KIT) in skin cells with white coats, and d1KIT production exhibited a substantial negative correlation with the expression of SOX10, which is essential for melanocyte formation and adjustment of hearing function. Additionally, the production of d1KIT was expected to be due to the insulating activity of the feline endogenous retrovirus 1 (FERV1) LTR in the first intron of KIT by its CTCF binding sequence repeat. These results contribute to basic veterinary research to understand the relationship between cat skin coat and disease risk, as well as the underlying mechanism., (© 2023 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype that exhibits a high incidence of distant metastases and lacks targeted therapeutic options. Here we explored how the epigenome contributes to matrix metalloprotease (MMP) dysregulation impacting tumor invasion, which is the first step of the metastatic process., Methods: We combined RNA expression and chromatin interaction data to identify insulator elements potentially associated with MMP gene expression and invasion. We employed CRISPR/Cas9 to disrupt the CCCTC-Binding Factor (CTCF) binding site on an insulator element downstream of the MMP8 gene (IE8) in two TNBC cellular models. We characterized these models by combining Hi-C, ATAC-seq, and RNA-seq with functional experiments to determine invasive ability. The potential of our findings to predict the progression of ductal carcinoma in situ (DCIS), was tested in data from clinical specimens., Results: We explored the clinical relevance of an insulator element located within the Chr11q22.2 locus, downstream of the MMP8 gene (IE8). This regulatory element resulted in a topologically associating domain (TAD) boundary that isolated nine MMP genes into two anti-correlated expression clusters. This expression pattern was associated with worse relapse-free (HR = 1.57 [1.06 - 2.33]; p = 0.023) and overall (HR = 2.65 [1.31 - 5.37], p = 0.005) survival of TNBC patients. After CRISPR/Cas9-mediated disruption of IE8, cancer cells showed a switch in the MMP expression signature, specifically downregulating the pro-invasive MMP1 gene and upregulating the antitumorigenic MMP8 gene, resulting in reduced invasive ability and collagen degradation. We observed that the MMP expression pattern predicts DCIS that eventually progresses into invasive ductal carcinomas (AUC = 0.77, p < 0.01)., Conclusion: Our study demonstrates how the activation of an IE near the MMP8 gene determines the regional transcriptional regulation of MMP genes with opposing functional activity, ultimately influencing the invasive properties of aggressive forms of breast cancer., (© 2023. The Author(s).)

Introduction: Gene expression is often controlled via cis-regulatory elements (CREs) that modulate the production of transcripts. For multi-gene genetic engineering and synthetic biology, precise control of transcription is crucial, both to insulate the transgenes from unwanted native regulation and to prevent readthrough or cross-regulation of transgenes within a multi-gene cassette. To prevent this activity, insulator-like elements, more properly referred to as transcriptional blockers, could be inserted to separate the transgenes so that they are independently regulated. However, only a few validated insulator-like elements are available for plants, and they tend to be larger than ideal., Methods: To identify additional potential insulator-like sequences, we conducted a genome-wide analysis of Utricularia gibba (humped bladderwort), one of the smallest known plant genomes, with genes that are naturally close together. The 10 best insulator-like candidates were evaluated in vivo for insulator-like activity., Results: We identified a total of 4,656 intergenic regions with expression profiles suggesting insulator-like activity. Comparisons of these regions across 45 other plant species (representing Monocots, Asterids, and Rosids) show low levels of syntenic conservation of these regions. Genome-wide analysis of unmethylated regions (UMRs) indicates ~87% of the targeted regions are unmethylated; however, interpretation of this is complicated because U. gibba has remarkably low levels of methylation across the genome, so that large UMRs frequently extend over multiple genes and intergenic spaces. We also could not identify any conserved motifs among our selected intergenic regions or shared with existing insulator-like elements for plants. Despite this lack of conservation, however, testing of 10 selected intergenic regions for insulator-like activity found two elements on par with a previously published element (EXOB) while being significantly smaller., Discussion: Given the small number of insulator-like elements currently available for plants, our results make a significant addition to available tools. The high hit rate (2 out of 10) also implies that more useful sequences are likely present in our selected intergenic regions; additional validation work will be required to identify which will be most useful for plant genetic engineering., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Laspisa, llla-Berenguer, Bang, Schmitz, Parrott and Wallace.)

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.

An unexpected interaction between a long non-coding RNA locus and a genetic insulator called Fub-1 has an important role in gene regulation during development in Drosophila ., Competing Interests: QD No competing interests declared, (© 2023, Dai.)

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., Competing Interests: AI, YS, ML, PG, PS No competing interests declared, XB is affiliated with BlueRock Therapeutics. The author has no other competing interests to declare, (© 2023, Ibragimov et al.)

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.

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-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 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.

Insulators are architectural elements implicated in the organization of higher-order chromatin structures and transcriptional regulation. However, it is still unknown how insulators contribute to Drosophila telomere maintenance. Although the Drosophila telomeric retrotransposons HeT-A and TART occupy a common genomic niche, they are regulated independently. TART elements are believed to provide reverse transcriptase activity, whereas HeT-A transcripts serve as a template for telomere elongation. Here, we report that insulator complexes associate with TART and contribute to its transcriptional regulation in the Drosophila germline. Chromatin immunoprecipitation revealed that the insulator complex containing BEAF32, Chriz, and DREF proteins occupy the TART promoter. BEAF32 depletion causes derepression and chromatin changes at TART in ovaries. Moreover, an expansion of TART copy number was observed in the genome of the BEAF32 mutant strain. BEAF32 localizes between the TART enhancer and promoter, suggesting that it blocks enhancer-promoter interactions. Our study found that TART repression is released in the germ cysts as a result of the normal reduction of BEAF32 expression at this developmental stage. We suggest that coordinated expression of telomeric repeats during development underlies telomere elongation control., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The cohesin complex has a range of crucial functions in the cell. Cohesin is essential for mediating chromatid cohesion during mitosis, for repair of double-strand DNA breaks, and for control of gene transcription. This last function has been the subject of intense research ever since the discovery of cohesin's role in the long-range regulation of the cut gene in Drosophila. Subsequent research showed that the expression of some genes is exquisitely sensitive to cohesin depletion, while others remain relatively unperturbed. Sensitivity to cohesin depletion is also remarkably cell type- and/or condition-specific. The relatively recent discovery that cohesin is integral to forming chromatin loops via loop extrusion should explain much of cohesin's gene regulatory properties, but surprisingly, loop extrusion has failed to identify a 'one size fits all' mechanism for how cohesin controls gene expression. This review will illustrate how early examples of cohesin-dependent gene expression integrate with later work on cohesin's role in genome organization to explain mechanisms by which cohesin regulates gene expression., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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.

Uncovering the cis-regulatory code that governs when and how much each gene is transcribed in a given genome and cellular state remains a central goal of biology. Here, we discuss major layers of regulation that influence how transcriptional outputs are encoded by DNA sequence and cellular context. We first discuss how transcription factors bind specific DNA sequences in a dosage-dependent and cooperative manner and then proceed to the cofactors that facilitate transcription factor function and mediate the activity of modular cis-regulatory elements such as enhancers, silencers, and promoters. We then consider the complex and poorly understood interplay of these diverse elements within regulatory landscapes and its relationships with chromatin states and nuclear organization. We propose that a mechanistically informed, quantitative model of transcriptional regulation that integrates these multiple regulatory layers will be the key to ultimately cracking the cis-regulatory code., Competing Interests: Declaration of interests J.W. is paid scientific advisory board member at Camp4 and Paratus Sciences. J.W. is an advisory board member at Cell Press journals, including Cell, Molecular Cell, and Developmental Cell., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The accuracy of insulators and their defect identification by UAVs (unmanned aerial vehicles) in transmission-line inspection needs to be further improved, and the model size of the detection algorithm is significantly reduced to make it more suitable for edge-end deployment. In this paper, the algorithm uses a lightweight GhostNet module to reconstruct the backbone feature extraction network of the YOLOv4 model and employs depthwise separable convolution in the feature fusion layer. The model is lighter on the premise of ensuring the effect of image information extraction. Meanwhile, the ECA-Net channel attention mechanism is embedded into the feature extraction layer and PANet (Path Aggregation Network) to improve the recognition accuracy of the model for small targets. The experimental results show that the size of the improved model is reduced from 244 MB to 42 MB, which is only 17.3% of the original model. At the same time, the mAp of the improved model is 0.77% higher than that of the original model, reaching 95.4%. Moreover, the mAP compared with YOLOv5-s and YOLOX-s, respectively, is improved by 1.98% and 1.29%. Finally, the improved model is deployed into Jetson Xavier NX and run at a speed of 8.8 FPS, which is 4.3 FPS faster than the original model.

The failure of insulators may seriously threaten the safe operation of the power system, where the state detection of high-voltage insulators is a must for the normal and safe operation of the power system. Based on the data of insulators in aerial images, this work explored an enhanced particle swarm algorithm to optimize the parameters of the support vector machine. A support vector machine model was therefore established for the identification of the normal and defective states of the insulators. This methodology works with the structure minimization principle of SVM and the characteristics of particle swarm fast optimization. First, the aerial insulator image was segmented as a target by way of the seed region growth based on double-layer cascade morphological improvements, and then, HOG features plus GLCM features were extracted as sample data. Finally, an ameliorated PSO-SVM classifier was designed to realize insulator state identification. Comparisons were made between PSO-SVM and conventional machine learning algorithms, SVM and Random Forest, and an optimization algorithm, Gray Wolf Optimization Support Vector Machine (GWO-SVM), and advanced neural network CNN. The experimental results showed that the performance of the algorithm proposed in this paper touched the top level, where the recognition accuracy rate was 92.11%, the precision rate 90%, the recall rate 94.74%, and the F1-score 92.31%.

Amongst the most important outputs of the biopharmaceutical industry are recombinant proteins, many of which are produced by integrating transgenes into the genomes of mammalian cells. However, expression is highly variable and can be unstable during prolonged culture. This is often due to epigenetic mechanisms silencing the transgenes. To combat this problem, vectors have been engineered to include ubiquitous chromatin opening elements (UCOEs) that protect against silencing. Here, we recount the evidence that UCOEs can modify chromatin environments and benefit biomanufacturing., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early versus late in different cell types remain incompletely understood. Intercalary heterochromatin domains replicate very late in both diploid chromosomes of dividing cells and in endoreplicating polytene chromosomes where they are also underreplicated. Drosophila SNF2-related factor SUUR imparts locus-specific underreplication of polytene chromosomes. SUUR negatively regulates DNA replication fork progression; however, its mechanism of action remains obscure. Here, we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates SUUR ATPase activity and is required for a normal spatiotemporal distribution of SUUR in vivo. SUUR and Mod(Mdg4)-67.2 together mediate the activities of gypsy insulator that prevent certain enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, SuUR or mod(mdg4 ) mutations reverse underreplication of intercalary heterochromatin. Thus, SUMM4 can impart late replication of intercalary heterochromatin by attenuating the progression of replication forks through euchromatin/heterochromatin boundaries. Our findings implicate a SNF2 family ATP-dependent motor protein SUUR in the insulator function, reveal that DNA replication can be delayed by a chromatin barrier, and uncover a critical role for architectural proteins in replication control. They suggest a mechanism for the establishment of late replication that does not depend on an asynchronous firing of late replication origins., Competing Interests: EA, AE, MN, EV, CH, RD, AS, DF No competing interests declared, LS Lu Sun is employed by Epicypher, Inc, a commercial developer and supplier of the EpiDyne nucleosomes and associated remodeling assay platforms used in this study, MK Michael C Keogh is employed by Epicypher, Inc, a commercial developer and supplier of the EpiDyne nucleosomes and associated remodeling assay platforms used in this study, (© 2022, Andreyeva, Emelyanov et al.)

Insulators in vertebrates play a role in genome architecture and orchestrate temporo-spatial enhancer-promoter interactions. In plants, insulators and their associated binding factors have not been documented as of yet, largely as a result of a lack of characterized insulators. In this study, we took a comprehensive strategy to identify and validate the enhancer-blocking insulator CW198. We show that a 1.08-kb CW198 fragment from Arabidopsis can, when interposed between an enhancer and a promoter, efficiently abrogate the activation function of both constitutive and floral organ-specific enhancers in transgenic Arabidopsis and tobacco plants. In plants, both transcriptional crosstalk and spreading of histone modifications were rarely detectable across CW198, which resembles the insulation property observed across the CTCF insulator in the mammalian genome. Taken together, our findings support that CW198 acts as an enhancer-blocking insulator in both Arabidopsis and tobacco. The significance of the present findings and their relevance to the mitigation of mutual interference between enhancers and promoters, as well as multiple promoters in transgenes, is discussed., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Performing ultrasonic nondestructive testing experiments on insulators and then using machine learning algorithms to classify and identify the signals is an important way to achieve an intelligent diagnosis of insulators. However, in most cases, we can obtain only a limited number of data from the experiments, which is insufficient to meet the requirements for training an effective classification and recognition model. In this paper, we start with an existing data augmentation method called DBA (for dynamic time warping barycenter averaging) and propose a new data enhancement method called AWDBA (adaptive weighting DBA). We first validated the proposed method by synthesizing new data from insulator sample datasets. The results show that the AWDBA proposed in this study has significant advantages relative to DBA in terms of data enhancement. Then, we used AWDBA and two other data augmentation methods to synthetically generate new data on the original dataset of insulators. Moreover, we compared the performance of different machine learning algorithms for insulator health diagnosis on the dataset with and without data augmentation. In the SVM algorithm especially, we propose a new parameter optimization method based on GA (genetic algorithm). The final results show that the use of the data augmentation method can significantly improve the accuracy of insulator defect identification.

Transformers play an essential role in power networks, ensuring that generated power gets to consumers at the safest voltage level. However, they are prone to insulation failure from ageing, which has fatal and economic consequences if left undetected or unattended. Traditional detection methods are based on scheduled maintenance practices that often involve taking samples from in situ transformers and analysing them in laboratories using several techniques. This conventional method exposes the engineer performing the test to hazards, requires specialised training, and does not guarantee reliable results because samples can be contaminated during collection and transportation. This paper reviews the transformer oil types and some traditional ageing detection methods, including breakdown voltage (BDV), spectroscopy, dissolved gas analysis, total acid number, interfacial tension, and corresponding regulating standards. In addition, a review of sensors, technologies to improve the reliability of online ageing detection, and related online transformer ageing systems is covered in this work. A non-destructive online ageing detection method for in situ transformer oil is a better alternative to the traditional offline detection method. Moreover, when combined with the Internet of Things (IoT) and artificial intelligence, a prescriptive maintenance solution emerges, offering more advantages and robustness than offline preventive maintenance approaches.

CCCTC-binding factor (CTCF) is an eleven zinc finger (ZF), multivalent transcriptional regulator, that recognizes numerous motifs thanks to the deployment of distinct combinations of its ZFs. The great majority of the ~50,000 genomic locations bound by the CTCF protein in a given cell type is intergenic, and a fraction of these sites overlaps with transcriptional enhancers. Furthermore, a proportion of the regions bound by CTCF intersect genes and promoters. This suggests multiple ways in which CTCF may impact gene expression. At promoters, CTCF can directly affect transcription. At more distal sites, CTCF may orchestrate interactions between regulatory elements and help separate eu- and heterochromatic areas in the genome, exerting a chromatin barrier function. In this review, we outline how CTCF contributes to the regulation of the three-dimensional structure of chromatin and the formation of chromatin domains. We discuss how CTCF binding and architectural functions are regulated. We examine the literature implicating CTCF in controlling gene expression in development and disease both by acting as an insulator and a factor facilitating regulatory elements to efficiently interact with each other in the nuclear space., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Chromatin insulators are DNA-protein complexes localized throughout the genome capable of establishing independent transcriptional domains. It was previously reported that the Drosophila su(Hw) mRNA physically associates with the gypsy chromatin insulator protein complex within the nucleus and may serve a noncoding function to affect insulator activity. However, how this mRNA is recruited to the gypsy complex is not known. Here, we utilized RNA-affinity pulldown coupled with mass spectrometry to identify a novel RNA-binding protein, Isha (CG4266), that associates with su(Hw) mRNA in vitro and in vivo. Isha harbors a conserved RNA recognition motif and RNA Polymerase II C-terminal domain-interacting domain (CID). We found that Isha physically interacts with total and elongating Polymerase II and associates with chromatin at the 5' end of genes in an RNA-dependent manner. Furthermore, ChIP-seq analysis reveals Isha overlaps particularly with the core gypsy insulator component CP190 on chromatin. Depletion of Isha reduces enhancer-blocking and barrier activities of the gypsy insulator and disrupts the nuclear localization of insulator bodies. Our results reveal a novel factor Isha that promotes gypsy insulator activity that may act as a nuclear RNA-binding protein adapter for su(Hw) noncoding mRNA., (Published by Oxford University Press on behalf of Genetics Society of America 2022.)

Eukaryotic chromosomes are divided into domains with distinct structural and functional properties, such as differing levels of chromatin compaction and gene transcription. Domains of relatively compact chromatin and minimal transcription are termed heterochromatic, whereas euchromatin is more open and actively transcribed. Insulators separate these domains and maintain their distinct features. Disruption of insulators can cause diseases such as cancer. Many insulators contain tRNA genes (tDNAs), examples of which have been shown to block the spread of activating or silencing activities. This characteristic of specific tDNAs is conserved through evolution, such that human tDNAs can serve as barriers to the spread of silencing in fission yeast. Here we demonstrate that tDNAs from the methylotrophic fungus Pichia pastoris can function effectively as insulators in distantly-related budding yeast. Key to the function of tDNAs as insulators is TFIIIC, a transcription factor that is also required for their expression. TFIIIC binds additional loci besides tDNAs, some of which have insulator activity. Although the mechanistic basis of TFIIIC-based insulation has been studied extensively in yeast, it is largely uncharacterized in metazoa. Utilising publicly-available genome-wide ChIP-seq data, we consider the extent to which mechanisms conserved from yeast to man may suffice to allow efficient insulation by TFIIIC in the more challenging chromatin environments of metazoa and suggest features that may have been acquired during evolution to cope with new challenges. We demonstrate the widespread presence at human tDNAs of USF1, a transcription factor with well-established barrier activity in vertebrates. We predict that tDNA-based insulators in higher organisms have evolved through incorporation of modules, such as binding sites for factors like USF1 and CTCF that are absent from yeasts, thereby strengthening function and providing opportunities for regulation between cell types., (Copyright © 2022. Published by Elsevier B.V.)

The organization of the genome into transcriptionally active and inactive chromatin domains requires well-delineated chromatin boundaries and insulator functions in order to maintain the identity of adjacent genomic loci with antagonistic chromatin marks and functionality. In plants that lack known chromatin insulators, the mechanisms that prevent heterochromatin spreading into euchromatin remain to be identified. Here, we show that DNA Topoisomerase VI participates in a chromatin boundary function that safeguards the expression of genes in euchromatin islands within silenced heterochromatin regions. While some transposable elements are reactivated in mutants of the Topoisomerase VI complex, genes insulated in euchromatin islands within heterochromatic regions of the Arabidopsis thaliana genome are specifically down-regulated. H3K9me2 levels consistently increase at euchromatin island loci and decrease at some transposable element loci. We further show that Topoisomerase VI physically interacts with S-adenosylmethionine synthase methionine adenosyl transferase 3 (MAT3), which is required for H3K9me2. A Topoisomerase VI defect affects MAT3 occupancy on heterochromatic elements and its exclusion from euchromatic islands, thereby providing a possible mechanistic explanation to the essential role of Topoisomerase VI in the delimitation of chromatin domains.

Rising energy and raw material prices, dwindling resources, increased recycling, and the need for sustainable management have led to growth in the smart materials sector. In recent years, the importance and diversity of bio-based adhesives for industrial applications has grown steadily. This article focuses on the production and characterization of insulation panels consisting of peat moss and two bio-based adhesives. The panels were pressed with tannin and animal-based resins and compared to panels bonded with urea formaldehyde. The physical-mechanical properties, namely, thermal conductivity (TC), water vapor diffusion resistance, modulus of rupture (MOR), modulus of elasticity (MOE), internal bond (IB), compression resistance (CR), water absorption (WA) and thickness swelling (TS) were measured and analyzed. The results show that the insulation effectiveness and mechanical stability of moss panels bound with tannin and animal glue are comparable to standard adhesives used in the composite industry.

Background: The increasing need for therapeutic monoclonal antibodies (mAbs) entails the development of innovative and improved expression strategies. Chromatin insulators have been utilized for the enhancement of the heterologous proteins in mammalian cells., Methods and Results: In the current study the Ccnb1ip1 gene insulator element was utilized to construct a novel vector system for the expression of an anti-CD52 mAb in Chinese hamster ovary (CHO) cells. The insulator containing (pIns-mAb) and control (pmAb) vectors were generated and stable cell pools were established using these constructs. The expression level in the cells created with pIns-mAb vector was calculated to be 233 ng/mL, and the expression rate in the control vector was 210 ng/mL, which indicated a 10.9% increase in mAb expression in pIns-mAb pool. In addition, analysis of mAb expression in clonal cells established from each pool showed a 10% increase in antibody productivity in the highest mAb producing clone derived from the pIns-mAb pool compared to the clone isolated from pmAb pool., Conclusions: More studies are needed to fully elucidate the effects of Ccnb1ip1 gene insulator on recombinant therapeutic protein expression in mammalian cells. The combination of this element with other chromatin-modifying elements might improve its augmentation effect which could pave the way for efficient and cost-effective production of therapeutic drugs., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

HSV-1 is a human pathogen that establishes a lifelong infection in the host. HSV-1 is transported by retrograde axonal transport to sensory neurons in the peripheral nervous system where latent viral genomes can reactivate. The resulting virus travels via anterograde axonal transport to the periphery and can cause clinical disease. CTCF insulators flank the LAT and IE regions of HSV-1 and during latency and maintain the integrity of transcriptional domains through a myriad of functions, including enhancer-blocking or barrier-insulator functions. Importantly, during reactivation, CTCF protein is evicted from the HSV-1 genome, especially from the CTRL2 insulator. CTRL2 is a functional insulator downstream of the 5'exon region of the LAT, so these results suggest that the disruption of this insulator may be required for efficient HSV-1 reactivation. To further explore this, we used a recombinant virus containing a deletion of the CTRL2 insulator (ΔCTRL2) in a rabbit ocular model of HSV-1 infection and induced reactivation. We show that, in the absence of the CTRL2 insulator, HSV-1 established an equivalent latent infection in rabbits, but those rabbits failed to efficiently reactivate from latency. Furthermore, we found a significant decrease in the expression of the gene Us9-, a gene that codes for a type II membrane protein that has been shown to be required for anterograde transport in neurons. Taken together, these results suggest that the functions of the CTRL2 insulator and Us9 activation in reactivating neurons are intrinsically linked through the regulation of a gene responsible for the axonal transport of HSV-1 to the periphery.

Metabolic engineering of microbial cell factories through integrating the heterologous synthetic pathway into the chromosome is most commonly used for industrial applications. However, the position of the foreign gene in the chromosome can affect its transcriptional level. As a microorganism that is generally regarded as safe (GRAS) and commonly applied in industrial manufacture with large-scale operations, Saccharomyces cerevisiae is also confronted with this position effect. In this study, we characterized 12 different chromosome sites by inserting the lycopene biosynthetic pathway as a reporter cassette. Due to the different integration loci, the gene transcription and lycopene yield exhibited more than 58-fold and 3.8-fold differences, respectively. Furthermore, changing the gene order also revealed a remarkable influence (30-fold and 14-fold) on gene transcription and lycopene yield. Besides, the upstream activation sequence of a strong promoter (defined as an insulator) in S. cerevisiae could reduce the impact by gene order, and increased the gene transcription (tenfold) and lycopene yield (sevenfold). Taken together, our results demonstrated that gene order and insulator affected gene transcription and heterogeneous biosynthesis, opening the opportunity to regulate gene transcription by insulator against position effect in S. cerevisiae., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Insulators are one of the many components responsible for the reliability of electricity supply as part of transmission and distribution lines. Failure of the insulator can cause considerable economic problems that are much greater than the insulator cost. When the failure occurs on the transmission line, a large area can be without electricity supply or other transmission lines will be overloaded. Because of the consequences of the insulator's failure, diagnostics of the insulator plays a significant role in the reliability of the power supply. Basic diagnostic methods require experienced personnel, and inspection requires moving in the field. New diagnostic methods require online measurement if it is possible. Diagnostic by measuring the leakage current flowing on the surface of the insulator is well known. However, many other quantities can be used as a good tool for diagnostics of insulators. We present in this article results obtained on the investigated porcelain insulators that are one of the most used insulation materials for housing the insulator's core. Leakage current, dielectric loss factor, capacity, and electric charge are used as diagnostic quantities to investigate porcelain insulators in different pollution conditions and different ambient relative humidity. Pollution and humidity are the main factors that decrease the insulator´s electric strength and reliability.

The transcription of each gene is tightly regulated by elements like promoters, enhancers, silencers and insulators. These elements determine the temporal and tissue-specific expression in development and disease. Drug resistance is the major obstacle in successfully treating cancer patients. In the recent years, it became evident that epigenetic changes represent one of the mechanisms that contribute to the onset and progression of cancer but also to the development of therapy resistance. The assay for transposase-accessible chromatin coupled with next generation sequencing (ATAC-seq) is a fast and easy technique to track epigenetic changes that result in different opening of the chromatin in regulatory regions genome-wide. The transposase cuts DNA in regions that are open and therefore accessible for transcription factors, regulatory RNAs and proteins that alter the architectural structure of the DNA and drive or inhibit transcription through the RNA polymerase. Here we describe a detailed protocol to perform an ATAC-seq of cells from culture or tissue., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mutations in enhancers have been shown to often underlie natural variation but the evolved differences in enhancer activity can be difficult to identify in vivo. Threespine sticklebacks (Gasterosteus aculeatus) are a robust system for studying enhancer evolution due to abundant natural genetic variation, a diversity of evolved phenotypes between ancestral marine and derived freshwater forms, and the tractability of transgenic techniques. Previous work identified a series of polymorphisms within an intronic enhancer of the Bone morphogenetic protein 6 (Bmp6) gene that are associated with evolved tooth gain, a derived increase in freshwater tooth number that arises late in development. Here, we use a bicistronic reporter construct containing a genetic insulator and a pair of reciprocal two-color transgenic reporter lines to compare enhancer activity of marine and freshwater alleles of this enhancer. In older fish, the two alleles drive partially overlapping expression in both mesenchyme and epithelium of developing teeth, but the freshwater enhancer drives a reduced mesenchymal domain and a larger epithelial domain relative to the marine enhancer. In younger fish, these spatial shifts in enhancer activity are less pronounced. Comparing Bmp6 expression by in situ hybridization in developing teeth of marine and freshwater fish reveals similar evolved spatial shifts in gene expression. Together, these data support a model in which the polymorphisms within this enhancer underlie evolved tooth gain by shifting the spatial expression of Bmp6 during tooth development, and provide a general strategy to identify spatial differences in enhancer activity in vivo., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Heparanase is an endo-β-glucuronidase that is best known for its pro-cancerous effects but is also implicated in the pathogenesis of various viruses. Activation of heparanase is a common strategy to increase viral spread and trigger the subsequent inflammatory cascade. Using a Single Nucleotide Polymorphisms (SNP)-associated approach we identified enhancer and insulator regions that regulate HPSE expression. Although a role for heparanase in viral infection has been noticed, the impact of HPSE functional SNPs has not been determined. We investigated the effect of cytomegalovirus (CMV) serostatus on the involvement of HPSE enhancer and insulator functional SNPs in the risk of acute graft versus host disease (GVHD) and granulocyte-colony stimulating factor related CD34 + mobilization. A significant correlation between the C alleles of insulator rs4364254 and rs4426765 and CMV seropositivity was found in healthy donors and patients with hematological malignancies. The risk of developing acute GVHD after hematopoietic stem cell transplantation was identified only in CMV-seropositive patients. A significant correlation between the enhancer rs4693608 and insulator rs28649799 and CD34 + cell mobilization was demonstrated in the CMV-seropositive donors. It is thus conceivable that latent CMV infection modulates heparanase regulatory regions and enhances the effect of functional SNPs on heparanase function in normal and pathological processes.

The HPSE gene encodes heparanase (HPSE), a key player in cancer, inflammation, and autoimmunity. We have previously identified a strong HPSE gene enhancer involved in self-regulation of heparanase by negative feedback exerted in a functional rs4693608 single-nucleotide polymorphism (SNP) dependent manner. In the present study, we analyzed the HPSE gene insulator region, located in intron 9 and containing rs4426765, rs28649799, and rs4364254 SNPs. Our results indicate that this region exhibits HPSE regulatory activity. SNP substitutions lead to modulation of a unique DNA-protein complex that affects insulator activity. Analysis of interactions between enhancer and insulator SNPs revealed that rs4693608 has a major effect on HPSE expression and the risk of post-transplantation acute graft versus host disease (GVHD). The C alleles of insulator SNPs rs4364254 and rs4426765 modify the activity of the HPSE enhancer, resulting in altered HPSE expression and increased risk of acute GVHD. Moreover, rs4426765 correlated with HPSE expression in activated mononuclear cells, as well as with CD3 levels and lymphocyte counts following G-CSF mobilization. rs4363084 and rs28649799 were found to be associated with CD34 + levels. Our study provides new insight into the mechanism of HPSE gene regulation and its impact on normal and pathological processes in the hematopoietic system.

In interphase eukaryotic nuclei, chromatin is folded to form a higher-order topological structure. The spatial organization of such chromatin domain has an important impact on the regulation of gene expression. As a key architectural structural protein, CTCF (CCCTC-binding factor) plays an important role in the formation of chromatin three-dimensional chromatin structure. CTCF can also bind to many insulator elements in the genome and insulate enhancers from activating target genes via modulating remote chromatin interactions. A recent study by Dr. Chunliang Li and his team at St. Jude Children's Research Hospital in the United States showed that when CTCF was acutely degraded, significant changes were found in the three-dimensional structure of chromatin. The mechanism by which CTCF binding sites function as insulator elements was investigated by Prof. Qiang Wu's team at Institute of Systems Biomedicine and Shanghai Jiao Tong University in China and Prof. Bing Ren's team at Ludwig Institute for Cancer Research in the United States. Here we mainly review and discuss some of these latest progresses.

The genomes of higher eukaryotes are partitioned into topologically associated domains or TADs, and insulators (also known as boundary elements) are the key elements responsible for their formation and maintenance. Insulators were first identified and extensively studied in Drosophila as well as mammalian genomes, and have also been described in yeast and plants. In addition, many insulator proteins are known in Drosophila, and some have been investigated in mammals. However, much less is known about this important class of non-coding DNA elements in plant genomes. In this review, we take a detailed look at known plant insulators across different species and provide an overview of potential determinants of plant insulator functions, including cis-elements and boundary proteins. We also discuss methods previously used in attempts to identify plant insulators, provide a perspective on their importance for research and biotechnology, and discuss areas of potential future research.

Immunoglobulin class switch recombination (CSR) plays a crucial role in adaptive immune responses through a change of the effector functions of antibodies and is triggered by T-cell-dependent as well as T-cell-independent antigens. Signals generated following encounter with each type of antigen direct CSR to different isotypes. At the genomic level, CSR occurs between highly repetitive switch sequences located upstream of the constant gene exons of the immunoglobulin heavy chain locus. Transcription of switch sequences is mandatory for CSR and is induced in a stimulation-dependent manner. Switch transcription takes place within dynamic chromatin domains and is regulated by long-range regulatory elements which promote alignment of partner switch regions in CSR centers. Here, we review recent work and models that account for the function of long-range transcriptional regulatory elements and the chromatin-based mechanisms involved in the control of CSR., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dauba and Khamlichi.)

CTCF (CCCTC-binding factor) is a zinc-finger protein which plays a vital role in the three-dimensional (3D) genome architecture. A pair of forward-reverse convergent CTCF binding sites (CBS elements) mediates long-distance DNA interactions to form chromatin loops with the assistance of the cohesin complex, while CBS elements at the chromatin domain boundaries show reverse-forward divergent patterns and function as insulators to discriminate against DNA interaction between chromatin domains. However, there are still many unresolved problems regarding CTCF-mediated insulation function. In order to study the connections between chromatin loops and the insulation function of CBS elements, we combinatorically inverted CBS elements at the HOXD locus by using CRISPR/Cas9 DNA-fragment editing methods in the HEK293T cell line and obtained five different kinds of single-cell CRISPR clones. By performing quantitative high-resolution chromosome conformation capture copy (QHR-4C) experiments, we found that boundary CBS inversions abolish original chromatin loops and establish new loops from the opposite direction, thus shifting the insulator boundary to the new divergent CTCF sites. Furthermore, tandem CBS elements block cohesin permeated from the opposite orientation to function as insulators. RNA-seq experiments showed that alterations of local three-dimensional genome architecture would further influence gene expression of the HOXD cluster. In conclusion, a pair of divergent CBS elements function as insulators by forming chromatin loops within chromatin domains to block cohesin sliding.

The genome architectural protein CTCF (CCCTC-binding factor) not only mediates long-distance chromatin interactions between distal enhancers and target promoters, but also functions as an important insulator-binding factor to block improper enhancer activation of non-target promoters, and is thus of great significance to transcriptional regulation of developmental genes. The Hox (Homeobox) gene family plays an important role in the development of the brain, bones, and limbs. The spatiotemporal colinear expression of the HOXD cluster along the proximal-distal axis of limbs is regulated by two clusters of enhancers known as super-enhancers located in the flanking regulatory regions. We focused on the HOXD cluster to explore the architectural role of CTCF in transcriptional regulation of developmental genes. The HOXD cluster contains 9 paralogous genes intermixed with a series of CBS (CTCF-binding site) elements. Using the CRISPR DNA-fragment editing system, we generated a series of single-cell HEK293T clones with deletion of increasing numbers of reverse CBS elements. RNA-seq experiments revealed decreased levels of HOXD gene expression. In addition, chromosome conformation capture experiments revealed increased long-distance chromatin interactions between HOXD and the upstream enhancer cluster and corresponding decreased interactions between HOXD and the downstream enhancer cluster. Thus, tandem reverse CTCF sites function as insulators to maintain HOXD regulatory balance between the upstream and downstream enhancer clusters. This study has interesting implications on the precise gene expression control of the Hox family during animal development.