Germline thymidylate synthase deficiency impacts nucleotide metabolism and causes dyskeratosis congenita.

Dyskeratosis congenita (DC) is an inherited bone-marrow-failure disorder characterized by a triad of mucocutaneous features that include abnormal skin pigmentation, nail dystrophy, and oral leucoplakia. Despite the identification of several genetic variants that cause DC, a significant proportion of probands remain without a molecular diagnosis. In a cohort of eight independent DC-affected families, we have identified a remarkable series of heterozygous germline variants in the gene encoding thymidylate synthase (TYMS). Although the inheritance appeared to be autosomal recessive, one parent in each family had a wild-type TYMS coding sequence. Targeted genomic sequencing identified a specific haplotype and rare variants in the naturally occurring TYMS antisense regulator ENOSF1 (enolase super family 1) inherited from the other parent. Lymphoblastoid cells from affected probands have severe TYMS deficiency, altered cellular deoxyribonucleotide triphosphate pools, and hypersensitivity to the TYMS-specific inhibitor 5-fluorouracil. These defects in the nucleotide metabolism pathway resulted in genotoxic stress, defective transcription, and abnormal telomere maintenance. Gene-rescue studies in cells from affected probands revealed that post-transcriptional epistatic silencing of TYMS is occurring via elevated ENOSF1. These cell and molecular abnormalities generated by the combination of germline digenic variants at the TYMS-ENOSF1 locus represent a unique pathogenetic pathway for DC causation in these affected individuals, whereas the parents who are carriers of either of these variants in a singular fashion remain unaffected.

Combination of Physicochemical Tropism and Affinity Moiety Targeting of Lipid Nanoparticles Enhances Organ Targeting.

Two camps have emerged for targeting nanoparticles to specific organs and cell types: affinity moiety targeting and physicochemical tropism. Here we directly compare and combine both using intravenous (IV) lipid nanoparticles (LNPs) designed to target the lungs. We utilized PECAM antibodies as affinity moieties and cationic lipids for physicochemical tropism. These methods yield nearly identical lung uptake, but aPECAM LNPs show higher endothelial specificity. LNPs combining these targeting methods had >2-fold higher lung uptake than either method alone and markedly enhanced epithelial uptake. To determine if lung uptake is because the lungs are the first organ downstream of IV injection, we compared IV vs intra-arterial (IA) injection into the carotid artery, finding that IA combined-targeting LNPs achieve 35% of the injected dose per gram (%ID/g) in the first-pass organ, the brain, among the highest reported. Thus, combining the affinity moiety and physicochemical strategies provides benefits that neither targeting method achieves alone.

An immunochemistry-based screen for chemical inhibitors of DNA-protein interactions and its application to human CGGBP1.

BACKGROUND: Inhibition of DNA-binding of proteins by small-molecule chemicals holds immense potential in manipulating the activities of DNA-binding proteins. Such a chemical inhibition of DNA-binding of proteins can be used to modulate processes such as replication, transcription, DNA repair and maintenance of epigenetic states. This prospect is currently challenged with the absence of robust and generic protocols to identify DNA-protein interactions. Additionally, much of the current approaches to designing inhibitors requires structural information of the target proteins. METHODS: We have developed a simple dot blot and immunodetection-based assay to screen chemical libraries for inhibitors of DNA-protein interactions. The assay has been applied to a library of 1685 FDA-approved chemicals to discover inhibitors of CGGBP1, a multifunctional DNA-binding protein with no known structure. Additional in vitro and in cellulo assays have been performed to verify and supplement the findings of the screen. RESULTS: Our primary screen has identified multiple inhibitors of direct or indirect interactions between CGGBP1 and genomic DNA. Of these, one inhibitor, Givinostat, was found to inhibit direct DNA-binding of CGGBP1 in the secondary screen using purified recombinant protein as the target. DNA and chromatin immunoprecipitation assays reinforced the findings of the screen that Givinostat inhibits CGGBP1-DNA binding. CONCLUSIONS: The assay we have described successfully identifies verifiable inhibitors of DNA-binding of protein; in this example, the human CGGBP1. This assay is customizable for a wide range of targets for which primary antibodies are available. It works with different sources of the target protein, cell lysates or purified recombinant preparations and does not require special equipment, DNA modifications or protein structural data. This assay is scalable and highly adaptable with the potential to discover inhibitors of transcription factors with implications in cancer biology.

CGGBP1-regulated cytosine methylation at CTCF-binding motifs resists stochasticity.

BACKGROUND: The human CGGBP1 binds to GC-rich regions and interspersed repeats, maintains homeostasis of stochastic cytosine methylation and determines DNA-binding of CTCF. Interdependence between regulation of cytosine methylation and CTCF occupancy by CGGBP1 remains unknown. RESULTS: By analyzing methylated DNA-sequencing data obtained from CGGBP1-depleted cells, we report that some transcription factor-binding sites, including CTCF, resist stochastic changes in cytosine methylation. By analysing CTCF-binding sites we show that cytosine methylation changes at CTCF motifs caused by CGGBP1 depletion resist stochastic changes. These CTCF-binding sites are positioned at locations where the spread of cytosine methylation in cis depends on the levels of CGGBP1. CONCLUSION: Our findings suggest that CTCF occupancy and functions are determined by CGGBP1-regulated cytosine methylation patterns.

PSIP1/LEDGF reduces R-loops at transcription sites to maintain genome integrity.

R-loops that accumulate at transcription sites pose a persistent threat to genome integrity. PSIP1 is a chromatin protein associated with transcriptional elongation complex, possesses histone chaperone activity, and is implicated in recruiting RNA processing and DNA repair factors to transcription sites. Here, we show that PSIP1 interacts with R-loops and other proteins involved in R-loop homeostasis, including PARP1. Genome-wide mapping of PSIP1, R-loops and γ-H2AX in PSIP1-depleted human and mouse cell lines revealed an accumulation of R-loops and DNA damage at gene promoters in the absence of PSIP1. R-loop accumulation causes local transcriptional arrest and transcription-replication conflict, leading to DNA damage. PSIP1 depletion increases 53BP1 foci and reduces RAD51 foci, suggesting altered DNA repair choice. Furthermore, PSIP1 depletion increases the sensitivity of cancer cells to PARP1 inhibitors and DNA-damaging agents that induce R-loop-induced DNA damage. These findings provide insights into the mechanism through which PSIP1 maintains genome integrity at the site of transcription.

G-quadruplex landscape and its regulation revealed by a new antibody capture method.

Our understanding of DNA G-quadruplexes (G4s) from in vitro studies has been complemented by genome-wide G4 landscapes from cultured cells. Conventionally, the formation of G4s is accepted to depend on G-repeats such that they form tetrads. However, genome-wide G4s characterized through high-throughput sequencing suggest that these structures form at a large number of regions with no such canonical G4-forming signatures. Many G4-binding proteins have been described with no evidence for any protein that binds to and stabilizes G4s. It remains unknown what fraction of G4s formed in human cells are protein-bound. The G4-chromatin immunoprecipitation (G4-ChIP) method hitherto employed to describe G4 landscapes preferentially reports G4s that get crosslinked to proteins in their proximity. Our current understanding of the G4 landscape is biased against representation of G4s which escape crosslinking as they are not stabilized by protein-binding and presumably transient. We report a protocol that captures G4s from the cells efficiently without any bias as well as eliminates the detection of G4s formed artifactually on crosslinked sheared chromatin post-fixation. We discover that G4s form sparingly at SINEs. An application of this method shows that depletion of a repeat-binding protein CGGBP1 enhances net G4 capture at CGGBP1-dependent CTCF-binding sites and regions of sharp interstrand G/C-skew transitions. Thus, we present an improved method for G4 landscape determination and by applying it we show that sequence property-specific constraints of the nuclear environment mitigate G4 formation.

Enabling non-viral DNA delivery using lipid nanoparticles co-loaded with endogenous anti-inflammatory lipids.

Lipid nanoparticles (LNPs) have transformed genetic medicine, recently shown by their use in COVID-19 mRNA vaccines. While loading LNPs with mRNA has many uses, loading DNA would provide additional advantages such as long-term expression and availability of promoter sequences. However, here we show that plasmid DNA (pDNA) delivery via LNPs (pDNA-LNPs) induces acute inflammation in naïve mice which we find is primarily driven by the cGAS-STING pathway. Inspired by DNA viruses that inhibit this pathway for replication, we co-loaded endogenous lipids that inhibit STING into pDNA-LNPs. Specifically, loading nitro-oleic acid (NOA) into pDNA-LNPs (NOA-pDNA-LNPs) ameliorates serious inflammatory responses in vivo enabling prolonged transgene expression (at least 1 month). Additionally, we demonstrate the ability to iteratively optimize NOA-pDNA-LNPs' expression by performing a small LNP formulation screen, driving up expression 50-fold in vitro. Thus, NOA-pDNA-LNPs, and pDNA-LNPs co-loaded with other bioactive molecules, will provide a major new tool in the genetic medicine toolbox, leveraging the power of DNA's long-term and promoter-controlled expression.

Physicochemical Targeting of Lipid Nanoparticles to the Lungs Induces Clotting: Mechanisms and Solutions.

Lipid nanoparticles (LNPs) have become the dominant drug delivery technology in industry, holding the promise to deliver RNA to up or down-regulate any protein of interest. LNPs have mostly been targeted to specific cell types or organs by physicochemical targeting in which LNP's lipid compositions are adjusted to find mixtures with the desired tropism. Here lung-tropic LNPs are examined, whose organ tropism derives from containing either a cationic or ionizable lipid conferring a positive zeta potential. Surprisingly, these LNPs are found to induce massive thrombosis. Such thrombosis is shown in the lungs and other organs, and it is shown that it is greatly exacerbated by pre-existing inflammation. This clotting is induced by a variety of formulations with cationic lipids, including LNPs and non-LNP nanoparticles, and even by lung-tropic ionizable lipids that do not have a permanent cationic charge. The mechanism depends on the LNPs binding to and then changing the conformation of fibrinogen, which then activates platelets and thrombin. Based on these mechanisms, multiple solutions are engineered that enable positively charged LNPs to target the lungs while ameliorating thrombosis. The findings illustrate how physicochemical targeting approaches must be investigated early for risks and re-engineered with a careful understanding of biological mechanisms.

Nanocarriers' repartitioning of drugs between blood subcompartments as a mechanism of improving pharmacokinetics, safety, and efficacy.

For medical emergencies, such as acute ischemic stroke, rapid drug delivery to the target site is essential. For many small molecule drugs, this goal is unachievable due to poor solubility that prevents intravenous administration, and less obviously, by extensive partitioning to plasma proteins and red blood cells (RBCs), which greatly slows delivery to the target. Here we study these effects and how they can be solved by loading into nanoscale drug carriers. We focus on fingolimod, a small molecule drug that is FDA-approved for treatment of multiple sclerosis, which has also shown promise in the treatment of stroke. Unfortunately, fingolimod has poor solubility and very extensive partitioning to plasma proteins and RBCs (in whole blood, 86% partitions to RBCs, 13.96% to plasma proteins, and 0.04% is free). We develop a liposomal formulation that slows the partitioning of fingolimod to RBCs and plasma proteins, enables intravenous delivery, and additionally prevents fingolimod toxicity to RBCs. The liposomal formulation nearly completely prevented fingolimod adsorption to plasma proteins (association with plasma proteins was 98.4 ± 0.4% for the free drug vs. 5.6 ± 0.4% for liposome-loaded drug). When incubated with whole blood in vitro, the liposomal formulation greatly slowed partitioning of fingolimod to RBCs and also eliminated deleterious effects of fingolimod on RBC rigidity, morphology, and hemolysis. In vivo, the liposomal formulation delayed fingolimod partitioning to RBCs for over 30 min, a critical time window for stroke. Fingolimod-loaded liposomes showed improved efficacy in a mouse model of post-stroke neuroinflammation, completely sealing the leaky blood-brain barrier (114 ± 11.5% reduction in albumin leak into the brain for targeted liposomes vs. 38 ± 16.5% reduction for free drug). This effect was only seen for liposomes modified with antibodies to enable targeted delivery to the site of action, and not in unmodified, long-circulating liposomes. Thus, loading fingolimod into liposomes prevented partitioning to RBCs and associated toxicities and enabled targeted delivery. This paradigm can be used for tuning the blood distribution of small molecule drugs for the treatment of acute illnesses requiring rapid pharmacologic intervention.

The evolving genetic landscape of telomere biology disorder dyskeratosis congenita.

Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome, caused by genetic mutations that principally affect telomere biology. Approximately 35% of cases remain uncharacterised at the genetic level. To explore the genetic landscape, we conducted genetic studies on a large collection of clinically diagnosed cases of DC as well as cases exhibiting features resembling DC, referred to as 'DC-like' (DCL). This led us to identify several novel pathogenic variants within known genetic loci and in the novel X-linked gene, POLA1. In addition, we have also identified several novel variants in POT1 and ZCCHC8 in multiple cases from different families expanding the allelic series of DC and DCL phenotypes. Functional characterisation of novel POLA1 and POT1 variants, revealed pathogenic effects on protein-protein interactions with primase, CTC1-STN1-TEN1 (CST) and shelterin subunit complexes, that are critical for telomere maintenance. ZCCHC8 variants demonstrated ZCCHC8 deficiency and signs of pervasive transcription, triggering inflammation in patients' blood. In conclusion, our studies expand the current genetic architecture and broaden our understanding of disease mechanisms underlying DC and DCL disorders.

Lipid Nanoparticle-Associated Inflammation is Triggered by Sensing of Endosomal Damage: Engineering Endosomal Escape Without Side Effects.

Lipid nanoparticles (LNPs) have emerged as the dominant platform for RNA delivery, based on their success in the COVID-19 vaccines and late-stage clinical studies in other indications. However, we and others have shown that LNPs induce severe inflammation, and massively aggravate pre-existing inflammation. Here, using structure-function screening of lipids and analyses of signaling pathways, we elucidate the mechanisms of LNP-associated inflammation and demonstrate solutions. We show that LNPs' hallmark feature, endosomal escape, which is necessary for RNA expression, also directly triggers inflammation by causing endosomal membrane damage. Large, irreparable, endosomal holes are recognized by cytosolic proteins called galectins, which bind to sugars on the inner endosomal membrane and then regulate downstream inflammation. We find that inhibition of galectins abrogates LNP-associated inflammation, both in vitro and in vivo . We show that rapidly biodegradable ionizable lipids can preferentially create endosomal holes that are smaller in size and reparable by the endosomal sorting complex required for transport (ESCRT) pathway. Ionizable lipids producing such ESCRT-recruiting endosomal holes can produce high expression from cargo mRNA with minimal inflammation. Finally, we show that both routes to non-inflammatory LNPs, either galectin inhibition or ESCRT-recruiting ionizable lipids, are compatible with therapeutic mRNAs that ameliorate inflammation in disease models. LNPs without galectin inhibition or biodegradable ionizable lipids lead to severe exacerbation of inflammation in these models. In summary, endosomal escape induces endosomal membrane damage that can lead to inflammation. However, the inflammation can be controlled by inhibiting galectins (large hole detectors) or by using biodegradable lipids, which create smaller holes that are reparable by the ESCRT pathway. These strategies should lead to generally safer LNPs that can be used to treat inflammatory diseases.

Marginated Neutrophils in the Lungs Effectively Compete for Nanoparticles Targeted to the Endothelium, Serving as a Part of the Reticuloendothelial System.

Nanomedicine has long pursued the goal of targeted delivery to specific organs and cell types but has yet to achieve this goal with the vast majority of targets. One rare example of success in this pursuit has been the 25+ years of studies targeting the lung endothelium using nanoparticles conjugated to antibodies against endothelial surface molecules. However, here we show that such "endothelial-targeted" nanocarriers also effectively target the lungs' numerous marginated neutrophils, which reside in the pulmonary capillaries and patrol for pathogens. We show that marginated neutrophils' uptake of many of these "endothelial-targeted" nanocarriers is on par with endothelial uptake. This generalizes across diverse nanomaterials and targeting moieties and was even found with physicochemical lung tropism (i.e., without targeting moieties). Further, we observed this in ex vivo human lungs and in vivo healthy mice, with an increase in marginated neutrophil uptake of nanoparticles caused by local or distant inflammation. These findings have implications for nanomedicine development for lung diseases. These data also suggest that marginated neutrophils, especially in the lungs, should be considered a major part of the reticuloendothelial system (RES), with a special role in clearing nanoparticles that adhere to the lumenal surfaces of blood vessels.

In vitro shear bond strength of three self-adhesive resin cements and a resin-modified glass ionomer cement to various prosthodontic substrates.

OBJECTIVE: To evaluate the shear bond strength (SBS) of three self-adhesive resin cements and a resin-modified glass ionomer cement (RMGIC) to different prosthodontic substrates. MATERIALS AND METHODS: The substrates base metal, noble metal, zirconia, ceramic, and resin composite were used for bonding with different cements (n=12). Specimens were placed in a bonding jig, which was filled with one of four cements (RelyX Unicem, Multilink Automix, Maxcem Elite, and FujiCEM Automix). Both light-polymerizing (LP) and self-polymerizing (SP) setting reactions were tested. Shear bond strength was measured at 15 minutes and 24 hours in a testing device at a test speed of 1 mm/min and expressed in MPa. A Student t-test and a one-way analysis of variance (ANOVA) were used to evaluate differences between setting reactions, between testing times, and among cements irrespective of other factors. Generalized linear regression model and Tukey tests were used for multifactorial analysis. RESULTS: Significantly higher mean SBS were demonstrated for LP mode relative to SP mode (p<0.001) and for 24 hours relative to 15 minutes (p<0.001). Multifactorial analysis revealed that all factors (cement, substrate, and setting reaction) and all their interactions had a significant effect on the bond strength (p<0.001). Resin showed significantly higher SBS than other substrates when bonded to RelyX Unicem and Multilink Automix in LP mode (p<0.05). Overall, FujiCEM demonstrated significantly lower SBS than the three self-adhesive resin cements (p<0.05). CONCLUSIONS: Overall, higher bond strengths were demonstrated for LP relative to SP mode, 24 hours relative to 15 minutes and self-adhesive resin cements compared to the RMGICs. Bond strengths also varied depending on the substrate, indicating that selection of luting cement should be partially dictated by the substrate and the setting reaction.

Meta-analysis of material properties influencing nanoparticle plasma pharmacokinetics.

Thorough characterization of the plasma pharmacokinetics (PK) is a critical step in clinical development of novel therapeutics and is routinely performed for small molecules and biologics. However, there is a paucity of even basic characterization of PK for nanoparticle-based drug delivery systems. This has led to untested generalizations about how nanoparticle properties govern PK. Here, we present a meta-analysis of 100 nanoparticle formulations following IV administration in mice to identify any correlations between four PK parameters derived by non-compartmental analysis (NCA) and four cardinal properties of nanoparticles: PEGylation, zeta potential, size, and material. There was a statistically significant difference between the PK of particles stratified by nanoparticle properties. However, single linear regression between these properties and PK parameters showed poor predictability (r2 < 0.10 for all analyses), while multivariate regressions showed improved predictability (r2 > 0.38, except for t1/2). This suggests that no single nanoparticle property alone is even moderately predictive of PK, while the combination of multiple nanoparticle features does provide moderate predictive power. Improved reporting of nanoparticle properties will enable more accurate comparison between nanoformulations and will enhance our ability to predict in vivo behavior and design optimal nanoparticles.

Physicochemical Targeting of Lipid Nanoparticles to the Lungs Induces Clotting: Mechanisms and Solutions.

Lipid nanoparticles (LNPs) have become the dominant drug delivery technology in industry, holding the promise to deliver RNA to up- or down-regulate any protein of interest. LNPs have been targeted to specific cell types or organs by physicochemical targeting, in which LNP's lipid compositions are adjusted to find mixtures with the desired tropism. In a popular approach, physicochemical targeting is accomplished by formulating with charged lipids. Negatively charged lipids localize LNPs to the spleen, and positively charged lipids to the lungs. Here we found that lung-tropic LNPs employing cationic lipids induce massive thrombosis. We demonstrate that thrombosis is induced in the lungs and other organs, and greatly exacerbated by pre-existing inflammation. This clotting is induced by a variety of formulations with cationic lipids, including LNPs and non-LNP nanoparticles. The mechanism depends on the LNPs binding to fibrinogen and inducing platelet and thrombin activation. Based on these mechanisms, we engineered multiple solutions which enable positively charged LNPs to target the lungs while not inducing thrombosis. Our findings implicate thrombosis as a major barrier that blood erects against LNPs with cationic components and illustrate how physicochemical targeting approaches must be investigated early for risks and re-engineered with a careful understanding of biological mechanisms.

H4K16ac activates the transcription of transposable elements and contributes to their cis-regulatory function.

Mammalian genomes harbor abundant transposable elements (TEs) and their remnants, with numerous epigenetic repression mechanisms enacted to silence TE transcription. However, TEs are upregulated during early development, neuronal lineage, and cancers, although the epigenetic factors contributing to the transcription of TEs have yet to be fully elucidated. Here, we demonstrate that the male-specific lethal (MSL)-complex-mediated histone H4 acetylation at lysine 16 (H4K16ac) is enriched at TEs in human embryonic stem cells (hESCs) and cancer cells. This in turn activates transcription of subsets of full-length long interspersed nuclear elements (LINE1s, L1s) and endogenous retrovirus (ERV) long terminal repeats (LTRs). Furthermore, we show that the H4K16ac-marked L1 and LTR subfamilies display enhancer-like functions and are enriched in genomic locations with chromatin features associated with active enhancers. Importantly, such regions often reside at boundaries of topologically associated domains and loop with genes. CRISPR-based epigenetic perturbation and genetic deletion of L1s reveal that H4K16ac-marked L1s and LTRs regulate the expression of genes in cis. Overall, TEs enriched with H4K16ac contribute to the cis-regulatory landscape at specific genomic locations by maintaining an active chromatin landscape at TEs.

Chimeric chromosome landscapes of human somatic cell cultures show dependence on stress and regulation of genomic repeats by CGGBP1.

Genomes of somatic cells in culture are prone to spontaneous mutations due to errors in replication and DNA repair. Some of these errors, such as chromosomal fusions, are not rectifiable and subject to selection or elimination in growing cultures. Somatic cell cultures are thus expected to generate background levels of potentially stable chromosomal chimeras. A description of the landscape of such spontaneously generated chromosomal chimeras in cultured cells will help understand the factors affecting somatic mosaicism. Here we show that short homology-associated non-homologous chromosomal chimeras occur in normal human fibroblasts and HEK293T cells at genomic repeats. The occurrence of chromosomal chimeras is enhanced by heat stress and depletion of a repeat regulatory protein CGGBP1. We also present evidence of homologous chromosomal chimeras between allelic copies in repeat-rich DNA obtained by methylcytosine immunoprecipitation. The formation of homologous chromosomal chimeras at Alu and L1 repeats increases upon depletion of CGGBP1. Our data are derived from de novo sequencing from three different cell lines under different experimental conditions and our chromosomal chimera detection pipeline is applicable to long as well as short read sequencing platforms. These findings present significant information about the generation, sensitivity and regulation of somatic mosaicism in human cell cultures.

Synthesis of cyclic α-pinane carbonate - a potential monomer for bio-based polymers.

This work reports the first known synthesis of α-pinane carbonate from an α-pinene derivative. Pinane carbonate is potentially useful as a monomer for poly(pinane carbonate), which would be a sustainable bio-based polymer. α-Pinene is a major waste product from the pulp and paper industries and the most naturally abundant monoterpene in turpentine oil. α-Pinene is routinely converted to pinene oxide and pinanediol, but no study has yet demonstrated the conversion of pinanediol into α-pinane carbonate. Here, α-pinane carbonate was synthesised via carboxylation of α-pinanediol with dimethyl carbonate under base catalysis using triazabicyclodecene guanidine (TBD). 81.1 ± 2.8% α-pinane carbonate yield was achieved at 98.7% purity. The produced α-pinane carbonate was a white crystalline solid with a melting point of 86 °C. It was characterised using FTIR, NMR, GCMS and a quadrupole time-of-flight (QTOF) mass spectrometer. The FTIR exhibited a C[double bond, length as m-dash]O peak at 1794 cm-1 confirming the presence of a cyclic carbonate. GCMS showed that the α-pinane carbonate fragments with loss of CO2, forming pinene epoxide. Base hydrolysis of the α-pinane carbonate using NaOH/ethanol/water regenerated the pinanediol with formations of Na2CO3.

CGGBP1-dependent CTCF-binding sites restrict ectopic transcription.

Binding sites of the chromatin regulator protein CTCF function as important landmarks in the human genome. The recently characterized CTCF-binding sites at LINE-1 repeats depend on another repeat-regulatory protein CGGBP1. These CGGBP1-dependent CTCF-binding sites serve as potential barrier elements for epigenetic marks such as H3K9me3. Such CTCF-binding sites are associated with asymmetric H3K9me3 levels as well as RNA levels in their flanks. The functions of these CGGBP1-dependent CTCF-binding sites remain unknown. By performing targeted studies on candidate CGGBP1-dependent CTCF-binding sites cloned in an SV40 promoter-enhancer episomal system we show that these regions act as inhibitors of ectopic transcription from the SV40 promoter. CGGBP1-dependent CTCF-binding sites that recapitulate their genomic function of loss of CTCF binding upon CGGBP1 depletion and H3K9me3 asymmetry in immediate flanks are also the ones that show the strongest inhibition of ectopic transcription. By performing a series of strand-specific reverse transcription PCRs we demonstrate that this ectopic transcription results in the synthesis of RNA from the SV40 promoter in a direction opposite to the downstream reporter gene in a strand-specific manner. The unleashing of the bidirectionality of the SV40 promoter activity and a breach of the transcription barrier seems to depend on depletion of CGGBP1 and loss of CTCF binding proximal to the SV40 promoter. RNA-sequencing reveals that CGGBP1-regulated CTCF-binding sites act as barriers to transcription at multiple locations genome-wide. These findings suggest a role of CGGBP1-dependent binding sites in restricting ectopic transcription.