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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.)

A key question in regulatory genomics is whether cis-regulatory elements (CREs) are modular elements that can function anywhere in the genome, or whether they are adapted to certain genomic locations. To distinguish between these possibilities we develop MPIRE (Massively Parallel Integrated Regulatory Elements), a technology for recurrently assaying CREs at thousands of defined locations across the genome in parallel. MPIRE allows us to separate the intrinsic activity of CREs from the effects of their genomic environments. We apply MPIRE to assay three insulator sequences at thousands of genomic locations and find that each insulator functions in locations with distinguishable properties. All three insulators can block enhancers, but each insulator blocks specific enhancers at specific locations. However, only ALOXE3 appears to block heterochromatin silencing. We conclude that insulator function is highly context dependent and that MPIRE is a robust method for revealing the context dependencies of CREs., (© 2024. The Author(s).)

The article presents an innovative approach for detecting defects in insulators used in high-voltage power transmission lines, employing an enhanced Detection Transformer (DETR) model, termed IF-DETR. The study addresses the significant challenges in traditional insulator defect detection methods, such as the loss of small defect features and confusion with background features. Firstly, we propose a multi-scale backbone network to better extract features of small objects. Secondly, as the contextual information surrounding objects plays a critical role in detecting small objects, we introduce a fusion module composed of ECA-Net and SAU to replace the original attention module for improved contextual information extraction. Lastly, we introduce the insulator defect (IDIoU) loss to optimize the instability in the matching process caused by small defects. Extensive experiments demonstrate the model's effectiveness, particularly in detecting small defects, marking a notable advancement in insulator defect detection technology. The IF-DETR achieved a 2.3% increase in AP compared to existing advanced methods. This method not only enhances the accuracy of defect detection, crucial for maintaining the reliability and safety of power transmission systems but also has broader implications for the maintenance and inspection of high-voltage power infrastructure., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

While the elements encoding enhancers and promoters have been relatively well studied, the full spectrum of insulator elements which bind the CCCTC binding factor (CTCF), is relatively poorly characterized. This is partly due to the genomic context of CTCF sites greatly influencing their roles and activity. Here we have developed an experimental system to determine the ability of minimal, consistently sized, individual CTCF elements to interpose between enhancers and promoters and thereby reduce gene expression during differentiation. Importantly, each element is tested in the identical location thereby minimising the effect of genomic context. We found no correlation between the ability of CTCF elements to block enhancer-promoter activity with the degree of evolutionary conservation; their resemblance to the consensus core sequences; or the number of CTCF core motifs harboured in the element. Nevertheless, we have shown that the strongest enhancer-promoter blockers include a previously described bound element lying upstream of the CTCF core motif. In addition, we found other uncharacterised DNaseI footprints located close to the core motif that may affect function. We have developed an assay of CTCF sequences which will enable researchers to sub-classify individual CTCF elements in a uniform and unbiased way., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The specificity of gene expression during development requires the insulation of regulatory domains to avoid inappropriate enhancer-gene interactions. In vertebrates, this insulator function is mostly attributed to clusters of CTCF sites located at topologically associating domain (TAD) boundaries. However, TAD boundaries allow some physical crosstalk across regulatory domains, which is at odds with the specific and precise expression of developmental genes. Here we show that developmental genes and nearby clusters of CTCF sites cooperatively foster the robust insulation of regulatory domains. By genetically dissecting a couple of representative loci in mouse embryonic stem cells, we show that CTCF sites prevent undesirable enhancer-gene contacts (i.e. physical insulation), while developmental genes preferentially contribute to regulatory insulation through non-structural mechanisms involving promoter competition rather than enhancer blocking. Overall, our work provides important insights into the insulation of regulatory domains, which in turn might help interpreting the pathological consequences of certain structural variants., (© 2024. The Author(s).)

Chromatin insulators are DNA-protein complexes that promote specificity of enhancer-promoter interactions and maintain distinct transcriptional states through control of 3D genome organization. In this review, we highlight recent work visualizing how mammalian CCCTC-binding factor acts as a boundary to dynamic DNA loop extrusion mediated by cohesin. We also discuss new studies in both mammals and Drosophila that elucidate biological redundancy of chromatin insulator function and interplay with transcription with respect to topologically associating domain formation. Finally, we present novel concepts in spatiotemporal regulation of chromatin insulator function during differentiation and development and possible consequences of disrupted insulator activity on cellular proliferation., 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., (Published by Elsevier Ltd.)

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.)

In various eukaryotic kingdoms, long terminal repeat (LTR) retrotransposons repress transcription by infiltrating heterochromatin generated within their elements. In contrast, the budding yeast LTR retrotransposon Ty1 does not itself undergo transcriptional repression, although it is capable of repressing the transcription of the inserted genes within it. In this study, we identified a DNA region within Ty1 that exerts its silencing effect via sequence orientation. We identified a DNA region within the Ty1 group-specific antigen (GAG) gene that causes gene silencing, termed GAG silencing (GAGsi), in which the silent chromatin in the GAGsi region is created by euchromatin-specific histone modifications. A characteristic inverted repeat (IR) sequence is present at the 5' end of this region, forming a chromatin boundary between promoter-specific chromatin upstream of the IR sequence and silent chromatin downstream of the IR sequence. In addition, Esc2 and Rad57, which are involved in DNA repair, were required for GAGsi silencing. Finally, the chromatin boundary was required for the transcription of Ty1 itself. Thus, the GAGsi sequence contributes to the creation of a chromatin environment that promotes Ty1 transcription., (© 2024. The Author(s).)

In Drosophila, a group of zinc finger architectural proteins recruits the CP190 protein to the chromatin, an interaction that is essential for the functional activity of promoters and insulators. In this study, we describe a new architectural C2H2 protein called Madf and Zinc-Finger Protein 1 (Mzfp1) that interacts with CP190. Mzfp1 has an unusual structure that includes six C2H2 domains organized in a C-terminal cluster and two tandem MADF domains. Mzfp1 predominantly binds to housekeeping gene promoters located in both euchromatin and heterochromatin genome regions. In vivo mutagenesis studies showed that Mzfp1 is an essential protein, and both MADF domains and the CP190 interaction region are required for its functional activity. The C2H2 cluster is sufficient for the specific binding of Mzfp1 to regulatory elements, while the second MADF domain is required for Mzfp1 recruitment to heterochromatin. Mzfp1 binds to the proximal part of the Fub boundary that separates regulatory domains of the Ubx and abd-A genes in the Bithorax complex. Mzfp1 participates in Fub functions in cooperation with the architectural proteins Pita and Su(Hw). Thus, Mzfp1 is a new architectural C2H2 protein involved in the organization of active promoters and insulators in Drosophila., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

Drosophila insulators were the first DNA elements found to regulate gene expression by delimiting chromatin contacts. We still do not know how many of them exist and what impact they have on the Drosophila genome folding. Contrary to vertebrates, there is no evidence that fly insulators block cohesin-mediated chromatin loop extrusion. Therefore, their mechanism of action remains uncertain. To bridge these gaps, we mapped chromatin contacts in Drosophila cells lacking the key insulator proteins CTCF and Cp190. With this approach, we found hundreds of insulator elements. Their study indicates that Drosophila insulators play a minor role in the overall genome folding but affect chromatin contacts locally at many loci. Our observations argue that Cp190 promotes cobinding of other insulator proteins and that the model, where Drosophila insulators block chromatin contacts by forming loops, needs revision. Our insulator catalog provides an important resource to study mechanisms of genome folding.

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.)

Chromatin insulators are responsible for orchestrating long-range interactions between enhancers and promoters throughout the genome and align with the boundaries of Topologically Associating Domains (TADs). Here, we demonstrate an association between gypsy insulator proteins and the phosphorylated histone variant H2Av (γH2Av), normally a marker of DNA double strand breaks. Gypsy insulator components colocalize with γH2Av throughout the genome, in polytene chromosomes and in diploid cells in which Chromatin IP data shows it is enriched at TAD boundaries. Mutation of insulator components su(Hw) and Cp190 results in a significant reduction in γH2Av levels in chromatin and phosphatase inhibition strengthens the association between insulator components and γH2Av and rescues γH2Av localization in insulator mutants. We also show that γH2Av, but not H2Av, is a component of insulator bodies, which are protein condensates that form during osmotic stress. Phosphatase activity is required for insulator body dissolution after stress recovery. Together, our results implicate the H2A variant with a novel mechanism of insulator function and boundary formation., Competing Interests: The authors have declared that no competing interests exist.

Chromatin insulators are DNA-protein complexes that can prevent the spread of repressive chromatin and block communication between enhancers and promoters to regulate gene expression. In Drosophila, the gypsy chromatin insulator complex consists of three core proteins: CP190, Su(Hw), and Mod(mdg4)67.2. These factors concentrate at nuclear foci termed insulator bodies, and changes in insulator body localization have been observed in mutants defective for insulator function. Here, we identified NURF301/E(bx), a nucleosome remodeling factor, as a novel regulator of gypsy insulator body localization through a high-throughput RNAi imaging screen. NURF301 promotes gypsy-dependent insulator barrier activity and physically interacts with gypsy insulator proteins. Using ChIP-seq, we found that NURF301 co-localizes with insulator proteins genome-wide, and NURF301 promotes chromatin association of Su(Hw) and CP190 at gypsy insulator binding sites. These effects correlate with NURF301-dependent nucleosome repositioning. At the same time, CP190 and Su(Hw) both facilitate recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy insulator DNA binding sites, and NURF301 is required for proper nuclear positioning of gypsy binding sites. Our data provide new insights into how a nucleosome remodeling factor and insulator proteins cooperatively contribute to nuclear organization., (Published by Oxford University Press on behalf of Nucleic Acids Research 2022.)

CTCF is the most thoroughly studied chromatin architectural protein and it is found in both Drosophila and mammals. CTCF preferentially binds to promoters and insulators and is thought to facilitate formation of chromatin loops. In a subset of sites, CTCF binding depends on the epigenetic status of the surrounding chromatin. One such variable CTCF site (vCTCF) was found in the intron of the Ubx gene, in close proximity to the BRE and abx enhancers. CTCF binds to the variable site in tissues where Ubx gene is active, suggesting that the vCTCF site plays a role in facilitating contacts between the Ubx promoter and its enhancers. Using CRISPR/Cas9 and attP/attB site-specific integration methods, we investigated the functional role of vCTCF and showed that it is not required for normal Drosophila development. Furthermore, a 2161-bp fragment containing vCTCF does not function as an effective insulator when substituted for the Fab-7 boundary in the Bithorax complex. Our results suggest that vCTCF function is redundant in the regulation of Ubx., (© 2022. Pleiades Publishing, Ltd.)

Germline stem cells (GSCs) are the progenitor cells of the germline for the lifetime of an animal. In Drosophila, these cells reside in a cellular niche that is required for both their maintenance (self-renewal) and differentiation (asymmetric division resulting in a daughter cell that differs from the GSC). The stem cell-daughter cell transition is tightly regulated by a number of processes, including an array of proteins required for genome stability. The germline stem-cell maintenance factor Stonewall (Stwl) associates with heterochromatin, but its molecular function is poorly understood. We performed RNA-Seq on stwl mutant ovaries and found significant derepression of many transposon families but not heterochromatic genes. We also discovered inappropriate expression of multiple classes of genes. Most prominent are testis-enriched genes, including the male germline sex-determination switch Phf7, the differentiation factor bgcn, and a large testis-specific gene cluster on chromosome 2, all of which are upregulated or ectopically expressed in stwl mutant ovaries. Surprisingly, we also found that RNAi knockdown of stwl in somatic S2 cells results in ectopic expression of these testis genes. Using parallel ChIP-Seq and RNA-Seq experiments in S2 cells, we discovered that Stwl localizes upstream of transcription start sites and at heterochromatic sequences including repetitive sequences associated with telomeres. Stwl is also enriched at bgcn, suggesting that it directly regulates this essential differentiation factor. Finally, we identify Stwl binding motifs that are shared with known insulator binding proteins. We propose that Stwl affects gene regulation, including repression of male transcripts in the female germline, by binding insulators and establishing chromatin boundaries., Competing Interests: The authors have declared that no competing interests exist.

Transcriptional terminators signal where transcribing RNA polymerases (RNAPs) should halt and disassociate from DNA. However, because termination is stochastic, two different forms of transcript could be produced: one ending at the terminator and the other reading through. An ability to control the abundance of these transcript isoforms would offer bioengineers a mechanism to regulate multi-gene constructs at the level of transcription. Here, we explore this possibility by repurposing terminators as 'transcriptional valves' that can tune the proportion of RNAP read-through. Using one-pot combinatorial DNA assembly, we iteratively construct 1780 transcriptional valves for T7 RNAP and show how nanopore-based direct RNA sequencing (dRNA-seq) can be used to characterize entire libraries of valves simultaneously at a nucleotide resolution in vitro and unravel genetic design principles to tune and insulate termination. Finally, we engineer valves for multiplexed regulation of CRISPR guide RNAs. This work provides new avenues for controlling transcription and demonstrates the benefits of long-read sequencing for exploring complex sequence-function landscapes., (© 2022. The Author(s).)

The genome-wide promoter interactome is primarily maintained and regulated by architectural proteins such as CTCF and cohesin. However, some studies suggest a role for non-coding RNAs (ncRNAs) in this process. We aimed to characterise the regulatory role of RNA-mediated promoter interactions in the control of gene expression. We integrated genome-wide datasets of RNA-chromatin and promoter-genome interactions in human embryonic stem cells (hESCs) to identify putative RNA-mediated promoter interactions. We discovered that CTCF sites were enriched in RNA-PIRs (promoter interacting regions co-localising with RNA-chromatin interaction sites) and genes interacting with RNA-PIRs containing CTCF sites showed higher expression levels. One of the long noncoding RNAs (lncRNAs) expressed in hESCs, Syntaxin 18-Antisense 1 (STX18-AS1), appeared to be involved in an insulating promoter interaction with the neighbouring gene, MSX1. By knocking down STX18-AS1, the MSX1 promoter-PIR interaction was intensified and the target gene (MSX1) expression was down-regulated. Conversely, reduced MSX1 promoter-PIR interactions, resulting from CRISPR-Cas9 deletion of the PIR, increased the expression of MSX1. We conclude that STX18-AS1 RNA antagonised local CTCF-mediated insulating promoter interactions to augment gene expression. Such down-regulation of the insulating promoter interactions by this novel mechanism may explain the higher expression of genes interacting with RNA-PIRs linked to CTCF sites., (© 2021. The Author(s).)

Despite the unequivocal success of hematopoietic stem and progenitor cell gene therapy, limitations still exist including genotoxicity and variegation/silencing of transgene expression. A class of DNA regulatory elements known as chromatin insulators (CIs) can mitigate both vector transcriptional silencing (barrier CIs) and vector-induced genotoxicity (enhancer-blocking CIs) and have been proposed as genetic modulators to minimize unwanted vector/genome interactions. Recently, a number of human, small-sized, and compact CIs bearing strong enhancer-blocking activity were identified. To ultimately uncover an ideal CI with a dual, enhancer-blocking and barrier activity, we interrogated these elements in vitro and in vivo . After initial screening of a series of these enhancer-blocking insulators for potential barrier activity, we identified three distinct categories with no, partial, or full protection against transgene silencing. Subsequently, the two CIs with full barrier activity (B4 and C1) were tested for their ability to protect against position effects in primary cells, after incorporation into lentiviral vectors (LVs) and transduction of human CD34 + cells. B4 and C1 did not adversely affect vector titers due to their small size, while they performed as strong barrier insulators in CD34 + cells, both in vitro and in vivo , shielding transgene's long-term expression, more robustly when placed in the forward orientation. Overall, the incorporation of these dual-functioning elements into therapeutic viral vectors will potentially provide a new generation of safer and more efficient LVs for all hematopoietic stem cell gene therapy applications.

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.

Glioblastoma (GBM) is the most aggressive primary brain tumor, having a poor prognosis and a median overall survival of less than two years. Over the last decade, numerous findings regarding the distinct molecular and genetic profiles of GBM have led to the emergence of several therapeutic approaches. Unfortunately, none of them has proven to be effective against GBM progression and recurrence. Epigenetic mechanisms underlying GBM tumor biology, including histone modifications, DNA methylation, and chromatin architecture, have become an attractive target for novel drug discovery strategies. Alterations on chromatin insulator elements (IEs) might lead to aberrant chromatin remodeling via DNA loop formation, causing oncogene reactivation in several types of cancer, including GBM. Importantly, it is shown that mutations affecting the isocitrate dehydrogenase (IDH) 1 and 2 genes, one of the most frequent genetic alterations in gliomas, lead to genome-wide DNA hypermethylation and the consequent IE dysfunction. The relevance of IEs has also been observed in a small population of cancer stem cells known as glioma stem cells (GSCs), which are thought to participate in GBM tumor initiation and drug resistance. Recent studies revealed that epigenomic alterations, specifically chromatin insulation and DNA loop formation, play a crucial role in establishing and maintaining the GSC transcriptional program. This review focuses on the relevance of IEs in GBM biology and their implementation as a potential theranostic target to stratify GBM patients and develop novel therapeutic approaches. We will also discuss the state-of-the-art emerging technologies using big data analysis and how they will settle the bases on future diagnosis and treatment strategies in GBM patients., (© 2021. The Author(s).)

The reliability of the insulator has directly affected the stable operation of electric power system. The detection of defective insulators has always been an important issue in smart grid systems. However, the traditional transmission line detection method has low accuracy and poor real-time performance. We present an insulator defect detection method based on CenterNet. In order to improve detection efficiency, we simplified the backbone network. In addition, an attention mechanism is utilized to suppress useless information and improve the accuracy of network detection. In image preprocessing, the blurring of some detected images results in the samples being discarded, so we use super-resolution reconstruction algorithm to reconstruct the blurred images to enhance the dataset. The results show that the AP of the proposed method reaches 96.16% and the reasoning speed reaches 30FPS under the test condition of NVIDIA GTX 1080 test conditions. Compared with Faster R-CNN, YOLOV3, RetinaNet and FSAF, the detection accuracy of proposed method is greatly improved, which fully proves the effectiveness of the proposed method., Competing Interests: The authors have declared that no competing interests exist.

Genome organization is driven by forces affecting transcriptional state, but the relationship between transcription and genome architecture remains unclear. Here, we identified the Drosophila transcription factor Motif 1 Binding Protein (M1BP) in physical association with the gypsy chromatin insulator core complex, including the universal insulator protein CP190. M1BP is required for enhancer-blocking and barrier activities of the gypsy insulator as well as its proper nuclear localization. Genome-wide, M1BP specifically colocalizes with CP190 at Motif 1-containing promoters, which are enriched at topologically associating domain (TAD) borders. M1BP facilitates CP190 chromatin binding at many shared sites and vice versa. Both factors promote Motif 1-dependent gene expression and transcription near TAD borders genome-wide. Finally, loss of M1BP reduces chromatin accessibility and increases both inter- and intra-TAD local genome compaction. Our results reveal physical and functional interaction between CP190 and M1BP to activate transcription at TAD borders and mediate chromatin insulator-dependent genome organization.

Several distinct activities and functions have been described for chromatin insulators, which separate genes along chromosomes into functional units. Here, we describe a novel mechanism of functional separation whereby an insulator prevents gene repression. When the homie insulator is deleted from the end of a Drosophila even skipped (eve) locus, a flanking P-element promoter is activated in a partial eve pattern, causing expression driven by enhancers in the 3' region to be repressed. The mechanism involves transcriptional read-through from the flanking promoter. This conclusion is based on the following. Read-through driven by a heterologous enhancer is sufficient to repress, even when homie is in place. Furthermore, when the flanking promoter is turned around, repression is minimal. Transcriptional read-through that does not produce anti-sense RNA can still repress expression, ruling out RNAi as the mechanism in this case. Thus, transcriptional interference, caused by enhancer capture and read-through when the insulator is removed, represses eve promoter-driven expression. We also show that enhancer-promoter specificity and processivity of transcription can have decisive effects on the consequences of insulator removal. First, a core heat shock 70 promoter that is not activated well by eve enhancers did not cause read-through sufficient to repress the eve promoter. Second, these transcripts are less processive than those initiated at the P-promoter, measured by how far they extend through the eve locus, and so are less disruptive. These results highlight the importance of considering transcriptional read-through when assessing the effects of insulators on gene expression., Competing Interests: The authors have declared that no competing interests exist.

The autonomy of segment-specific regulatory domains in the Bithorax complex is conferred by boundary elements and associated Polycomb response elements (PREs). The Fab-6 boundary is located at the junction of the iab-5 and iab-6 domains. Previous studies mapped it to a nuclease hypersensitive region 1 (HS1), while the iab-6 PRE was mapped to a second hypersensitive region HS2 nearly 3 kb away. To analyze the role of HS1 and HS2 in boundary we generated deletions of HS1 or HS1 + HS2 that have attP site for boundary replacement experiments. The 1389 bp HS1 deletion can be rescued by a 529 bp core Fab-6 sequence that includes two CTCF sites. However, Fab-6 HS1 cannot rescue the HS1 + HS2 deletion or substitute for another BX-C boundary - Fab-7. For this it must be combined with a PRE, either Fab-7 HS3, or Fab-6 HS2. These findings suggest that the boundary function of Fab-6 HS1 must be bolstered by a second element that has PRE activity.

Key Message: Genetic analysis identifies multiple, potential protein binding sites important for insulator function in Arabidopsis thaliana: Rap1 site in UASrpg, Su(Hw) site in UASrpg, and CTCF site in BEAD1c. Three non-plant insulators UASrpg, BEAD1c, and gypsy isolated from Ashbya gossypii, Homo sapiens and Drosophila melanogaster gypsy retrotransposon, respectively, demonstrate insulator function in transgenic Arabidopsis thaliana. Here, the hypothesis that DNA sequences functional in A. thaliana are the same as those in the original host as previously assumed, was tested. Genetic analyses of the cloned fragments in an enhancer blocking assay system was performed through deletions and mutations to identify more precisely which sequences within the cloned fragments function as insulators. Significant loss of insulator activity was observed when the UASrpg Rap1 binding site R2 was mutated but not R1. Cloned fragments containing BEAD1c are effective insulators in our assay system and the previously investigated gypsy insulator is non-functional. Further analyses identified potential Su(Hw) and CTCF sites within UASrpg, of which only the Su(Hw) site was functional. Thus, the activity of non-plant insulators in A. thaliana is context dependent. These results support the hypothesis that insulator function is conserved across kingdoms.

Latest advancements in genomics involving individuals from different races and geographical locations has led to the identification of thousands of common as well as rare genetic variants and copy number variations (CNVs). These studies have surprisingly revealed that the majority of genetic variation is not present within the coding region but rather in the non-coding region of the genome, which is also termed as "Medical Genome". This short review describes how mutations/variations within; regulatory sequences, architectural proteins and transcriptional regulators give rise to the aberrant gene expression profiles that drives cellular transformations and malignancies.

Synthetic biology circuits are often constructed with multiple gene expression units assembled in close proximity, and they can be used to perform complex functions in embryonic stem cells (ESCs). However, mutual interference between transcriptional units has not been well studied in mouse ESCs. To assess the efficiency of insulators at suppressing promoter interference in mouse ESCs, we used an evaluation scheme in which a tunable tetracycline response element promoter is connected to a constant Nanog promoter. The chicken hypersensitive site 4 (cHS4) insulator, widely used both for enhancer blocking and for barrier insulation in vitro and in vivo, was positioned between the two expression units for assessment. By inserting the cassette into various loci of the mouse ESC genome with PiggyBac transposon, we were able to quantitatively examine the protective effect of cHS4 by gradually increasing the transcriptional activity of the tetracycline response element promoter with doxycycline and then measuring the transcriptional activity of the Nanog promoter. Our results indicate that the cHS4 insulator has minimal insulating effects on promoter interference in mouse ESCs. Further studies show that the cHS4 insulation effect may be promoter specific and related to interaction with CCCTC-binding factor-mediated loop formation. In addition, we also compared DNA transposition and transgene expression with or without the cHS4 insulator using well-established ESC reporters. The results indicate that cHS4 has no apparent effects on DNA transposition and transgene expression levels, but exerts modest protective effects on long-term transgene silencing., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Drosophila Myb (Dm-Myb) encodes a protein that plays a key role in regulation of mitotic phase genes. Here, we further refine its role in the context of a developing tissue as a potentiator of gene expression required for proper RNA polymerase II (RNA Pol II) function and efficient H3K4 methylation at promoters. In contrast to its role in gene activation, Myb is also required for repression of many genes, although no specific mechanism for this role has been proposed. We now reveal a critical role for Myb in contributing to insulator function, in part by promoting binding of insulator proteins BEAF-32 and CP190 and stabilizing H3K27me3 Polycomb-group (PcG) domains. In the absence of Myb, H3K27me3 is markedly reduced throughout the genome, leading to H3K4me3 spreading and gene derepression. Finally, Myb is enriched at boundaries that demarcate chromatin environments, including chromatin loop anchors. These results reveal functions of Myb that extend beyond transcriptional regulation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Recent evidence shows that the disruption of constitutive insulated neighbourhoods might lead to oncogene dysregulation. We present here a systematic pan-cancer characterisation of the associations between constitutive boundaries and genome alterations in cancer. Specifically, we investigate the enrichment of somatic mutation, abnormal methylation, and copy number alteration events in the proximity of CTCF bindings overlapping with topological boundaries (junctions) in 26 cancer types. Focusing on CTCF motifs that are both in-boundary (overlapping with junctions) and active (overlapping with peaks of CTCF expression), we find a significant enrichment of somatic mutations in several cancer types. Furthermore, mutated junctions are significantly conserved across cancer types, and we also observe a positive selection of transversions rather than transitions in many cancer types. We also analyzed the mutational signature found on the different classes of CTCF motifs, finding some signatures (such as SBS26) to have a higher weight within in-boundary than off-bounday motifs. Regarding methylation, we find a significant number of over-methylated active in-boundary CTCF motifs in several cancer types; similarly to somatic-mutated junctions, they also have a significant conservation across cancer types. Finally, in several cancer types we observe that copy number alterations tend to overlap with active junctions more often than in matched normal samples. While several articles have recently reported a mutational enrichment at CTCF binding sites for specific cancer types, our analysis is pan-cancer and investigates abnormal methylation and copy number alterations in addition to somatic mutations. Our method is fully replicable and suggests several follow-up tumour-specific analyses., Competing Interests: The authors have declared that no competing interests exist.

Homologous chromosomes colocalize to regulate gene expression in processes including genomic imprinting, X-inactivation, and transvection. In Drosophila, homologous chromosomes pair throughout development, promoting transvection. The "button" model of pairing proposes that specific regions along chromosomes pair with high affinity. Here, we identify buttons interspersed across the fly genome that pair with their homologous sequences, even when relocated to multiple positions in the genome. A majority of transgenes that span a full topologically associating domain (TAD) function as buttons, but not all buttons contain TADs. Additionally, buttons are enriched for insulator protein clusters. Fragments of buttons do not pair, suggesting that combinations of elements within a button are required for pairing. Pairing is necessary but not sufficient for transvection. Additionally, pairing and transvection are stronger in some cell types than in others, suggesting that pairing strength regulates transvection efficiency between cell types. Thus, buttons pair homologous chromosomes to facilitate cell-type-specific interchromosomal gene regulation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Boundaries in the bithorax complex (BX-C) delimit autonomous regulatory domains that drive parasegment-specific expression of the Hox genes Ubx , abd-A, and Abd-B The Fab-7 boundary is located between the iab-6 and iab-7 domains and has two key functions: blocking cross-talk between these domains and at the same time promoting communication (boundary bypass) between iab-6 and the Abd-B promoter. Using a replacement strategy, we found that multimerized binding sites for the architectural proteins Pita, Su(Hw), and dCTCF function as conventional insulators and block cross-talk between the iab-6 and iab-7 domains; however, they lack bypass activity, and iab-6 is unable to regulate Abd-B Here we show that an ∼200-bp sequence of dHS1 from the Fab-7 boundary rescues the bypass defects of these multimerized binding sites. The dHS1 sequence is bound in embryos by a large multiprotein complex, Late Boundary Complex (LBC), that contains the zinc finger proteins CLAMP and GAF. Using deletions and mutations in critical GAGAG motifs, we show that bypass activity correlates with the efficiency of recruitment of LBC components CLAMP and GAF to the artificial boundary. These results indicate that LBC orchestrates long-distance communication between the iab-6 regulatory domain and the Abd-B gene, while the Pita, Su(Hw), and dCTCF proteins function to block local cross-talk between the neighboring regulatory domains iab-6 and iab-7 ., Competing Interests: The authors declare no conflict of interest.

For more than 20 years, plant biologists have tried to achieve complete control of transgene expression. Until the techniques to target transgenes to safe harbor sites in the genome become routine, flanking transgenes with genetic insulators, DNA sequences that create independent domains of gene expression, can help avoid positional effects and stabilize their expression. We have, for the first time, compared the effect of three insulator sequences previously described in the literature and one never tested before. Our results indicate that their use increases transgene expression, but only the last one reduces variability between lines and between individuals. We have analyzed the integration of insulator-flanked T-DNAs using whole genome re-sequencing (to our knowledge, also for the first time) and found data suggesting that chiMARs can shelter transgene insertions from neighboring repressive epigenetic states. Finally, we could also observe a loss of accuracy of the RB insertion in the lines harboring insulators, evidenced by a high frequency of truncation of T-DNAs and of insertion of vector backbone that, however, did not affect transgene expression. Our data supports that the effect of each genetic insulator is different and their use in transgenic constructs should depend on the needs of each specific experiment.

Transvection is the phenomenon where a transcriptional enhancer activates a promoter located on the homologous chromosome. It has been amply documented in Drosophila where homologs are closely paired in most, if not all, somatic nuclei, but it has been known to rarely occur in mammals as well. We have taken advantage of site-directed transgenesis to insert reporter constructs into the same genetic locus in Drosophila and have evaluated their ability to engage in transvection by testing many heterozygous combinations. We find that transvection requires the presence of an insulator element on both homologs. Homotypic trans -interactions between four different insulators can support transvection: the gypsy insulator ( GI ), Wari , Fab-8 and 1A2 ; GI and Fab-8 are more effective than Wari or 1A2 We show that, in the presence of insulators, transvection displays the characteristics that have been previously described: it requires homolog pairing, but can happen at any of several loci in the genome; a solitary enhancer confronted with an enhancerless reporter is sufficient to drive transcription; it is weaker than the action of the same enhancer-promoter pair in cis , and it is further suppressed by cis -promoter competition. Though necessary, the presence of homotypic insulators is not sufficient for transvection; their position, number and orientation matters. A single GI adjacent to both enhancer and promoter is the optimal configuration. The identity of enhancers and promoters in the vicinity of a trans -interacting insulator pair is also important, indicative of complex insulator-enhancer-promoter interactions., (Copyright © 2019 by the Genetics Society of America.)

Coordinated changes of DNA (de)methylation, nucleosome positioning, and chromatin binding of the architectural protein CTCF play an important role for establishing cell-type-specific chromatin states during differentiation. To elucidate molecular mechanisms that link these processes, we studied the perturbed DNA modification landscape in mouse embryonic stem cells (ESCs) carrying a double knockout (DKO) of the Tet1 and Tet2 dioxygenases. These enzymes are responsible for the conversion of 5-methylcytosine (5mC) into its hydroxymethylated (5hmC), formylated (5fC), or carboxylated (5caC) forms. We determined changes in nucleosome positioning, CTCF binding, DNA methylation, and gene expression in DKO ESCs and developed biophysical models to predict differential CTCF binding. Methylation-sensitive nucleosome repositioning accounted for a significant portion of CTCF binding loss in DKO ESCs, whereas unmethylated and nucleosome-depleted CpG islands were enriched for CTCF sites that remained occupied. A number of CTCF sites also displayed direct correlations with the CpG modification state: CTCF was preferentially lost from sites that were marked with 5hmC in wild-type (WT) cells but not from 5fC-enriched sites. In addition, we found that some CTCF sites can act as bifurcation points defining the differential methylation landscape. CTCF loss from such sites, for example, at promoters, boundaries of chromatin loops, and topologically associated domains (TADs), was correlated with DNA methylation/demethylation spreading and can be linked to down-regulation of neighboring genes. Our results reveal a hierarchical interplay between cytosine modifications, nucleosome positions, and DNA sequence that determines differential CTCF binding and regulates gene expression., (© 2019 Wiehle et al.; Published by Cold Spring Harbor Laboratory Press.)

Contact domains are closely linked to gene regulation and lineage commitment, while current understanding of contact domains and their boundaries is still limited. Here, we present a novel method HiCDB, which is constructively based on local relative insulation metric and multi-scale aggregation approach to detect contact domain boundaries (CDBs) on Hi-C maps. Compared with other 'state-of-art' methods, HiCDB shows improved sensitivity and specificity in determining CDBs at various Hi-C resolutions. The superiority of HiCDB enabled us to study the epigenetic features of detected CDBs and showed enrichment of architectural proteins and cell-type-specific transcription factor binding sites at CDBs. The further comparison of GM12878 and IMR90 Hi-C datasets suggested that cell-type-specific CDBs are marked by active regulatory signals and correlate with activation of nearby cell identity genes.

Cell line development is one of the most critical steps in the production of complex recombinant therapeutic proteins such as monoclonal antibodies in mammalian cells. Generation of industrial cell lines is mainly based on the time-consuming and laborious process of selection and screening of a large number of clones. With the increasing demand for therapeutic proteins during the past years, more effort is invested to improve the efficiency of cell line development. In line with this premise, several studies employed expression vector engineering strategies based on incorporation of epigenetic regulatory elements, which can enhance the expression level and stability of the transgenes. Main examples of such elements include ubiquitous chromatin opening elements, scaffold or matrix attachment regions, stabilizing antirepressor elements, and insulators. This work evaluates the utility of the tDNA insulator element for stable expression of an IgG1 monoclonal antibody as well as the enhanced green fluorescent protein (EGFP) reporter gene in Chinese hamster ovary (CHO) cells. Initial analysis of EGFP transfected cells showed improved mean fluorescent intensity in cell pools and single cell clones when tDNA element was included in the expression vector. Our results also indicated up to nine- and sixfold enhancements in antibody titer and specific productivity of clones derived from tDNA containing vectors, respectively. Moreover, improved single cell cloning efficiency was observed for transfectants generated using tDNA harboring expression constructs. Our study clearly shows the beneficial effects of the tDNA insulator on monoclonal antibody expression in CHO cells.

Chromatin organization is crucial for nuclear functions such as gene regulation, DNA replication and DNA repair. Insulator binding proteins, such as the Drosophila Boundary Element-Associated Factor (BEAF), are involved in chromatin organization. To further understand the role of BEAF, we detected cis- and trans-interaction partners of four BEAF binding regions (viewpoints) using 4C (circular chromosome conformation capture) and analyzed their association with different genomic features. Previous genome-wide mapping found that BEAF usually binds near transcription start sites, often of housekeeping genes, so our viewpoints were selected to reflect this. Our 4C data show the interaction partners of our viewpoints are highly variable and generally enriched for active chromatin marks. The most consistent association was with housekeeping genes, a feature in common with our viewpoints. Fluorescence in situ hybridization indicated that the long-distance interactions occur even in the absence of BEAF. These data are most consistent with a model in which BEAF is redundant with other factors found at active promoters. Our results point to principles of long-distance interactions made by active chromatin, supporting a previously proposed model in which condensed chromatin is sticky and associates into topologically associating domains (TADs) separated by active chromatin. We propose that the highly variable long-distance interactions we detect are driven by redundant factors that open chromatin to promote transcription, combined with active chromatin filling spaces between TADs while packing of TADs relative to each other varies from cell to cell., Competing Interests: The authors have declared that no competing interests exist.

Chromatin insulators or boundary elements protect genes from regulatory activities from neighboring genes or chromatin domains. In the Drosophila Abdominal-B (Abd-B) locus, the deletion of such elements, such as Frontabdominal-7 (Fab-7) or Fab-8 led to dominant gain of function phenotypes, presumably due to the loss of chromatin barriers. Homologous chromosomes are paired in Drosophila, creating a number of pairing dependent phenomena including transvection, and whether transvection may affect the function of Polycomb response elements (PREs) and thus contribute to the phenotypes are not known. Here, we studied the chromatin barrier activity of Fab-8 and how it is affected by the zygosity of the transgene, and found that Fab-8 is able to block the silencing effect of the Ubx PRE on the DsRed reporter gene in a CTCF binding sites dependent manner. However, the blocking also depends on the zygosity of the transgene in that the barrier activity is present when the transgene is homozygous, but absent when the transgene is heterozygous. To analyze this effect, we performed chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) experiments on homozygous transgenic embryos, and found that H3K27me3 and H3K9me3 marks are restricted by Fab-8, but they spread beyond Fab-8 into the DsRed gene when the two CTCF binding sites within Fab-8 were mutated. Consistent with this, the mutation reduced H3K4me3 and RNA Pol II binding to the DsRed gene, and consequently, DsRed expression. Importantly, in heterozygous embryos, Fab-8 is unable to prevent the spread of H3K27me3 and H3K9me3 marks from crossing Fab-8 into DsRed, suggesting an insulator bypass. These results suggest that in the Abd-B locus, deletion of the insulator in one copy of the chromosome could lead to the loss of insulator activity on the homologous chromosome, and in other loci where chromosomal deletion created hemizygous regions of the genome, the chromatin barrier could be compromised. This study highlights a role of homologous chromosome pairing in the regulation of gene expression in the Drosophila genome., Competing Interests: The authors have declared that no competing interests exist.

Aim: Identification and functional characterization of cis-regulatory elements in human PPARD gene., Methods: We used various bioinformatic tools on the publicly available human genome and Encyclopedia of DNA Elements databases to explore potential cis-regulatory elements in PPARD gene region., Results: We predicted an insulator and an enhancer element in intron 2 of PPARD gene. Functional characterization using transient transfection, reporter assay and CTCF binding confirmed the insulator status. However, the predicted enhancer element showed repressor/silencer activity. Finally, we observed a potential interaction between these two cis-regulatory elements which is in agreement with 5C-Encyclopedia of DNA Elements data., Conclusion: We report two functionally validated cis-regulatory elements in PPARD gene which will aid in understanding its regulation and role in metabolic functions.

The three-dimensional (3D) organization of the genome is intimately related to numerous key biological functions including gene expression and DNA replication regulations. The mechanisms by which molecular drivers functionally organize the 3D genome, such as topologically associating domains (TADs), remain to be explored. Current approaches consist in assessing the enrichments or influences of proteins at TAD borders. Here, we propose a TAD-free model to directly estimate the blocking effects of architectural proteins, insulators and DNA motifs on long-range contacts, making the model intuitive and biologically meaningful. In addition, the model allows analyzing the whole Hi-C information content (2D information) instead of only focusing on TAD borders (1D information). The model outperforms multiple logistic regression at TAD borders in terms of parameter estimation accuracy and is validated by enhancer-blocking assays. In Drosophila, the results support the insulating role of simple sequence repeats and suggest that the blocking effects depend on the number of repeats. Motif analysis uncovered the roles of the transcriptional factors pannier and tramtrack in blocking long-range contacts. In human, the results suggest that the blocking effects of the well-known architectural proteins CTCF, cohesin and ZNF143 depend on the distance between loci, where each protein may participate at different scales of the 3D chromatin organization.

The metazoan genome is compartmentalized in areas of highly interacting chromatin known as topologically associating domains (TADs). TADs are demarcated by boundaries mostly conserved across cell types and even across species. However, a genome-wide characterization of TAD boundary strength in mammals is still lacking. In this study, we first use fused two-dimensional lasso as a machine learning method to improve Hi-C contact matrix reproducibility, and, subsequently, we categorize TAD boundaries based on their insulation score. We demonstrate that higher TAD boundary insulation scores are associated with elevated CTCF levels and that they may differ across cell types. Intriguingly, we observe that super-enhancers are preferentially insulated by strong boundaries. Furthermore, we demonstrate that strong TAD boundaries and super-enhancer elements are frequently co-duplicated in cancer patients. Taken together, our findings suggest that super-enhancers insulated by strong TAD boundaries may be exploited, as a functional unit, by cancer cells to promote oncogenesis.

Enhancers and insulators are involved in the regulation of gene expression, but the basic underlying mechanisms of action of these elements are unknown. We analyzed the individual effects of the enhancer and the insulator from Drosophila mobile elements copia [enh(copia)] and gypsy using transfected genetic constructs in S2 cells. This system excludes the influence of genomic cis regulatory elements. The enhancer-induced synthesis of 350-1050-nt-long enhancer RNAs (eRNAs) and H3K4me3 and H3K18ac marks, mainly in the region located about 300bp downstream of the enhancer. Insertion of the insulator between the enhancer and the promoter reduced these effects. We also observed the binding of dCTCF to the enhancer and to gypsy insulator. Our data indicate that a single gypsy insulator interacts with both the enhancer and the promoter, while two copies of the gypsy insulator preferentially interact with each other. Our results suggest the formation of chromatin loops that are shaped by the enhancer and the insulator., (Copyright © 2017 Elsevier B.V. All rights reserved.)

NOMe-seq (nucleosome occupancy and methylome sequencing) identifies nucleosome-depleted regions that correspond to promoters, enhancers, and insulators. The NOMe-seq method is based on the treatment of chromatin with the M.CviPI methyltransferase, which methylates GpC dinucleotides that are not protected by nucleosomes or other proteins that are tightly bound to the chromatin (GpC m does not occur in the human genome and therefore there is no endogenous background of GpC m ). Following bisulfite treatment of the M.CviPI-methylated chromatin (which converts unmethylated Cs to Ts and thus allows the distinction of GpC from GpC m ) and subsequent genomic sequencing, nucleosome-depleted regions can be ascertained on a genome-wide scale. The bisulfite treatment also allows the distinction of CpG from C m pG (most endogenous methylation occurs at CpG dinucleotides) and thus the endogenous methylation status of the genome can also be obtained in the same sequencing reaction. Importantly, open chromatin is expected to have high levels of GpC m but low levels of C m pG; thus, each of the two separate methylation analyses serve as independent (but opposite) measures which provide matching chromatin designations for each regulatory element.NOMe-seq has advantages over ChIP-seq for identification of regulatory elements because it is not reliant upon knowing the exact modifications on the surrounding nucleosomes. Also, NOMe-seq has advantages over DHS (DNase hypersensitive site)-seq, FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements)-seq, and ATAC (Assay for Transposase-Accessible Chromatin)-seq because it also gives positioning information for several nucleosomes on either side of each open regulatory element. Here, we provide a detailed protocol for NOMe-seq that begins with the isolation of chromatin, followed by methylation of GpCs with M.CviPI and treatment with bisulfite, and ending with the creation of next generation sequencing libraries. We also include sequencing QC analysis metrics and bioinformatics steps that can be used to identify nucleosome-depleted regions throughout the genome.

The ability to mutate a promoter in situ is potentially a very useful approach for gaining insights into endogenous gene regulation mechanisms. The advent of CRISPR/Cas systems has provided simple, efficient, and targeted genetic manipulation in eukaryotes, which can be applied to studying genome structure and function.The basic CRISPR toolkit comprises an endonuclease, Cas9, and a short DNA-targeting sequence, made up of a single guide RNA (sgRNA). The catalytic domains of Cas9 are rendered active upon dimerization of Cas9 with sgRNA, resulting in targeted double stranded DNA breaks. Among other applications, this method of DNA cleavage can be coupled to endogenous homology-directed repair (HDR) mechanisms for the generation of site-specific editing or knockin mutations, at both promoter regulatory and gene coding sequences.A well-characterized regulatory feature of promoter regions is the high abundance of CpGs. These CpG islands tend to be unmethylated, ensuring a euchromatic environment that promotes gene transcription. Here, we demonstrate CRISPR-mediated editing of two CpG islands located within the promoter region of the MDR1 gene (Multi Drug Resistance 1). Cas9 is used to generate double stranded breaks across multiple target sites, which are then repaired while inserting the beta globin (β-globin) insulator, 5'HS5. Thus, we are screening through promoter regulatory sequences with a chromatin barrier element to identify functional regions via "insulator scanning." Transcriptional and functional assessment of MDR1 expression provides evidence of genome engineering. Overall, this method allows the scanning of CpG islands to identify their promoter functions.

We use an oligonucleotide library of >10,000 variants to identify an insulation mechanism encoded within a subset of σ 54 promoters. Insulation manifests itself as reduced protein expression for a downstream gene that is expressed by transcriptional readthrough. It is strongly associated with the presence of short CT-rich motifs (3-5 bp), positioned within 25 bp upstream of the Shine-Dalgarno (SD) motif of the silenced gene. We provide evidence that insulation is triggered by binding of the ribosome binding site (RBS) to the upstream CT-rich motif. We also show that, in E. coli, insulator sequences are preferentially encoded within σ 54 promoters, suggesting an important regulatory role for these sequences in natural contexts. Our findings imply that sequence-specific regulatory effects that are sparsely encoded by short motifs may not be easily detected by lower throughput studies. Such sequence-specific phenomena can be uncovered with a focused oligo library (OL) design that mitigates sequence-related variance, as exemplified herein., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Rational engineering of biological systems is often complicated by the complex but unwanted interactions between cellular components at multiple levels. Here we address this issue at the level of prokaryotic transcription by insulating minimal promoters and operators to prevent their interaction and enable the biophysical modeling of synthetic transcription without free parameters. This approach allows genetic circuit design with extraordinary precision and diversity, and consequently simplifies the design-build-test-learn cycle of circuit engineering to a mix-and-match workflow. As a demonstration, combinatorial promoters encoding NOT-gate functions were designed from scratch with mean errors of <1.5-fold and a success rate of >96% using our insulated transcription elements. Furthermore, four-node transcriptional networks with incoherent feed-forward loops that execute stripe-forming functions were obtained without any trial-and-error work. This insulation-based engineering strategy improves the resolution of genetic circuit technology and provides a simple approach for designing genetic circuits for systems and synthetic biology.Unwanted interactions between cellular components can complicate rational engineering of biological systems. Here the authors design insulated minimal promoters and operators that enable biophysical modeling of bacterial transcription without free parameters for precise circuit design.

The Shelterin component Rif1 has emerged as a global regulator of the replication-timing program in all eukaryotes examined to date, possibly by modulating the 3D-organization of the genome. In fission yeast a second Shelterin component, Taz1, might share similar functions. Here, we identified unexpected properties for Rif1 and Taz1 by conducting high-throughput genetic screens designed to identify cis- and trans-acting factors capable of creating heterochromatin-euchromatin boundaries in fission yeast. The preponderance of cis-acting elements identified in the screens originated from genomic loci bound by Taz1 and associated with origins of replication whose firing is repressed by Taz1 and Rif1. Boundary formation and gene silencing by these elements required Taz1 and Rif1 and coincided with altered replication timing in the region. Thus, small chromosomal elements sensitive to Taz1 and Rif1 (STAR) could simultaneously regulate gene expression and DNA replication over a large domain, at the edge of which they established a heterochromatin-euchromatin boundary. Taz1, Rif1, and Rif1-associated protein phosphatases Sds21 and Dis2 were each sufficient to establish a boundary when tethered to DNA. Moreover, efficient boundary formation required the amino-terminal domain of the Mcm4 replicative helicase onto which the antagonistic activities of the replication-promoting Dbf4-dependent kinase and Rif1-recruited phosphatases are believed to converge to control replication origin firing. Altogether these observations provide an insight into a coordinated control of DNA replication and organization of the genome into expression domains., Competing Interests: The authors declare no conflict of interest.

Background: Gene therapy in mammalian cells requires vectors exhibiting long-term stability and high expression. Episomal gene expression vectors offer a safe and attractive alternative to those that integrate into the host cell genome., Materials & Methods: In the present study, we developed a new episomal vector based on the insulator, chicken hypersensitive site 4 (cHS4). The cHS4 element was artificially synthesized, cloned into the pEGFP-C1 vector, and used to transfect Chinese hamster ovary (CHO) and human Chang liver cells. The stably transfected cell colonies were further cultured in either the presence or absence of G418 selection. Fluorescence in situ hybridization (FISH) analysis and vector rescue experiments demonstrated that the vector replicated episomally in both CHO and human Chang liver cells. Compared with episomal vectors mediated by matrix attachment region sequences, the cHS4 element-containing vector yielded increased transgene expression levels, transfection efficiency, and stability during long-term culture. The vector was present at a very low copy number in the cells and was stably maintained over more than 100 generations without selection pressure., Conclusion: In conclusion, apart from a few free vector forms, the cHS4-containing vector mainly replicates episomally in mammalian cells and out- performs comparable systems in terms of yielding both higher expression levels and stability levels., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)