GC-rich DNA Sequences Research Articles

Genome-wide chromatin remodeling identified at GC-rich long nucleosome-free regions.

To gain deeper insights into principles of cell biology, it is essential to understand how cells reorganize their genomes by chromatin remodeling. We analyzed chromatin remodeling on next generation sequencing data from resting and activated T cells to determine a whole-genome chromatin remodeling landscape. We consider chromatin remodeling in terms of nucleosome repositioning which can be observed most robustly in long nucleosome-free regions (LNFRs) that are occupied by nucleosomes in another cell state. We found that LNFR sequences are either AT-rich or GC-rich, where nucleosome repositioning was observed much more prominently in GC-rich LNFRs — a considerable proportion of them outside promoter regions. Using support vector machines with string kernels, we identified a GC-rich DNA sequence pattern indicating loci of nucleosome repositioning in resting T cells. This pattern appears to be also typical for CpG islands. We found out that nucleosome repositioning in GC-rich LNFRs is indeed associated with CpG islands and with binding sites of the CpG-island-binding ZF-CXXC proteins KDM2A and CFP1. That this association occurs prominently inside and also prominently outside of promoter regions hints at a mechanism governing nucleosome repositioning that acts on a whole-genome scale.

A short GC‐rich palindrome of human mannose receptor gene coding region displays a conformational switch

Conformational switching in DNA is fundamental to biological processes. The structural status of a palindromic GC-rich dodecamer DNA sequence, integral part of human MRC2 coding region, and a related sequence of opposite polarity from human FDX1 gene were characterized and compared. UV-melting, circular dichroism, and gel electrophoresis experiments demonstrated the formation of intermolecular structures. Although stability and molecularity of both the oligomeric structures were found to be almost identical, their secondary structures differed remarkably as A1 MRC2 sequence showed A-like and B-like DNA conformation, whereas the A2 FDX1 sequence exhibited only the A-like signatures. The study is relevant for understanding structural polymorphism at genomic locations depending on DNA sequence and solution environment.

Cytogenetic Analysis of Lagria villosa (Coleoptera, Tenebrionidae): Emphasis on the Mechanism of Association of the Xyp Sex Chromosomes

The Xy_p sex determination mechanism is the system most frequent and ancestral to Coleoptera. Moreover, the presence of argyrophilous material associated with the sex bivalent is described as being responsible for the maintenance and association of these chromosomes. There are no karyotype data available regarding the genus Lagria and no consensus in the literature regarding the argyrophilous material present in the lumen of sex bivalent. Therefore, the aim of this work was to investigate the mechanism of sex chromosome bivalent association in Lagria villosa by analyzing the argyrophilous nature of the material present in the Xy_p lumen. It was also intended to characterize L. villosa cytogenetically. The analysis of meiotic cells showed 2n = 18 = 16+Xy_p for males and 2n = 18 = 16+XX in females and the meiotic formula was 2n = 8^II+Xy_p. The C-banding showed blocks of pericentromeric heterochromatin in all chromosomes except in the y_p chromosome. In these regions, the use of fluorochromes revealed the presence of heterochromatin containing GC rich DNA sequences. The study of synaptonemal complex showed a gradual increase in the electron-density of the axial elements of the sex chromosomes and their association with strongly electron-dense material. The pepsin pretreatment revealed that the material impregnated by silver is protein.

Modulation of the Activity of Sp Transcription Factors by Mithramycin Analogues as a New Strategy for Treatment of Metastatic Prostate Cancer

Deregulated activity of transcription factors (TFs) of the Sp/KLF family, like Sp1, Sp3 and Sp4, and consequent over-expression of Sp-regulated genes occur frequently in human cancers. This provides the rationale for development of inhibitors of Sp TFs as cancer therapeutics. Mithramycin A (MTM-A) is a natural polyketide that binds GC-rich DNA sequences, inhibits activity of Sp TFs and exhibits potent antitumor activity in experimental systems. However, clinical use of MTM-A is limited by the severe toxicity of the compound. Here, we studied two MTM-A analogues, which had been generated by genetically engineering of the MTM-A biosynthetic pathway, and evaluated their activity in human prostate cancer in cell cultures and mouse models. The compounds, named MTM-SDK and MTM-SK, were highly effective in vitro inhibiting proliferation of prostate cancer cells and transcription of Sp-regulated genes by blocking binding of Sp proteins to the gene promoters When administered to mice, both compounds were well tolerated with maximum tolerated doses of MTM-SDK and MTM-SK, respectively, 4- and 32- fold higher than MTM-A. After systemic administration, both compounds were cleared rapidly from the bloodstream but maintained plasma levels well above the active concentrations required in vitro for inhibition of Sp TF activity and cell proliferation. Consistently, MTM-SDK and MTM-SK inhibited transcription of Sp-regulated genes in prostate tumor xenografts and exhibited potent antitumor activity in subcutaneous and metastatic tumor xenograft models with no or minimal toxicity. Taken together, these data indicate that MTM-SDK and MTM-SK possess significantly improved pharmacological and toxicological properties compared to MTM-A and represent promising drugs for treatment of advanced prostate cancer.

Karyotype analysis of three Solanum plants using combined PI-DAPI staining and double fluorescence in situ hybridization with 45S and 5S rDNA probes

In this study, mitotic metaphase chromosomes of Solanum surattense Burm ., Solanum lyratum Thunb. and Solanum photeinocarpum Nakam. were well prepared using an advanced chromosome preparation method. The chromosomes were distinguished by combined PI-DAPI (CPD) staining and double fluorescence in situ hybridization (FISH) with 45S and 5S rDNA probes and their molecular cytogenetic karyotypes were established. Although, the karyotype of S. surattense Burm. and S. photeinocarpum Nakam was first established, the karyotype formulas of the three Solanum plants ( S. surattense Burm., S. lyratum Thunb. and S. photeinocarpum Nakam) can be described as follows: 2n=24=18m +4sm+2st (2SAT), 2n=24=18m +4sm+2st (2SAT) and 2n=24=18m +6sm (2SAT). Moreover, the karyotype asymmetry of the three species belongs to 2A type. After CPD staining, the centromeres of all chromosomes in the three species were shown as red CPD bands, indicating the presence of GC-rich DNA sequences in the chromosomes. Subsequential double FISH shows that S. surattense Burm., S. lyratum Thunb. and S. photeinocarpum Nakam, all have a pair of 45S rDNA sites located on chromosomes, and all of these 45S rDNA sites correspond to the respective prominent CPD banded regions. S. surattense Burm., S. lyratum Thunb. and S. photeinocarpum Nakam correspond to the CPD banded regions at the ends of chromosomes 8, 10 and 12, respectively. The three Solanum plants all have a pair of 5S rDNA sites. In S. surattense Burm., the 5S rDNA sites are located on the long arm of the 6th chromosome pair, while the 5S rDNA sites of S. lyratum Thunb. and S. photeinocarpum Nakam are all located on the short arms of the 4th chromosome pair. Our study shows that the CPD bands and rDNA FISH signals provide effective chromosome markers allowing us to establish accurate molecular cytogenetic karyotypes of the three species tested. Key words: Solanum surattense Burm. Solanum lyratum Thunb., Solanum photeinocarpum Nakam , karyotype, CPD staining, fluorescence in situ hybridization (FISH).

Histone Deacetylase Inhibitors and Mithramycin A Impact a Similar Neuroprotective Pathway at a Crossroad between Cancer and Neurodegeneration

Mithramycin A (MTM) and histone deacetylase inhibitors (HDACi) are effective therapeutic agents for cancer and neurodegenerative diseases. MTM is a FDA approved aureolic acid-type antibiotic that binds to GC-rich DNA sequences and interferes with Sp1 transcription factor binding to its target sites (GC box). HDACi, on the other hand, modulate the activity of class I and II histone deacetylases. They mediate their protective function, in part, by regulating the acetylation status of histones or transcription factors, including Sp1, and in turn chromatin accessibility to the transcriptional machinery. Because these two classes of structurally and functionally diverse compounds mediate similar therapeutic functions, we investigated whether they act on redundant or synergistic pathways to protect neurons from oxidative death. Non-protective doses of each of the drugs do not synergize to create resistance to oxidative death suggesting that these distinct agents act via a similar pathway. Accordingly, we found that protection by MTM and HDACi is associated with diminished expression of the oncogene, Myc and enhanced expression of a tumor suppressor, p21waf1/cip1. We also find that neuroprotection by MTM or Myc knockdown is associated with downregulation of class I HDAC levels. Our results support a model in which the established antitumor drug MTM or canonical HDACi act via distinct mechanisms to converge on the downregulation of HDAC levels or activity respectively. These findings support the conclusion that an imbalance in histone acetylase and HDAC activity in favor of HDACs is key not only for oncogenic transformation, but also neurodegeneration.

Cytogenetics of the permanent translocation heterozygoteRhoeo spathaceavar.variegata. Implications for complex chromosome rearrangements inRhoeo

Abstract The chromosomal morphology, arrangement of AT- and GC-rich chromatin, 5S and 45S rDNA, NORs, and centromere behaviour during somatic interphase and during meiotic prophase I were studied for the first time in the variegated ring-forming Rhoeo variety – R. spathacea var. variegata. Fluorescence in situ hybridization (FISH) together with differential base-specific fluorescence revealed the existence of the highly conserved karyotype in Rhoeo. It was shown that the two existing ring-forming varieties -R. spathacea variegata and R. discolor (common Rhoeo variety) share essentially the same arrangement of the studied cytogenetic landmarks within the chromosomal arms, together with the spread of the GC-rich DNA sequences (including rDNA) across all the twelve pericentromeres. They both also exhibit the same profound trend for centromere clustering in soma- and germ-line. The obtained results together with the existing literature served as a basis to summarize the cytogenetic knowledge on the karyotype ...

Abstract 3683: Plant flavonoid apigenin preferentially binds with GC-rich DNA sequences and inhibits DNA methylation

Abstract Epigenetic modifications result in heritable changes in gene expression without changes to the DNA sequence. The most common forms of epigenetic regulation of gene expression are DNA methylation and histone modification, all of which are associated with chromatin remodeling. It is well established that DNA is the central target for both chemical carcinogens and anticancer drugs. Agents that target epigenetic regulators may represent an attractive target for drug development. In recent years, several studies have focused on small molecule interaction with DNA. Flavonoids are natural polyphenolic compounds with major antioxidant activity that exert a multitude of beneficial effects on human health. Apigenin (4’,5,7 trihydroxyflavone), an important bioactive common flavonoid present in a variety of plants, vegetables, fruits, and herbs is well known for its cancer preventive activity. Studies from our group have shown that apigenin preferentially retains in nuclear matrix with possibility of binding to nucleic acid bases. Although apigenin has shown its presence in the nucleus however it's binding with nucleic acid bases and mode of action has not been elucidated. In present study we explored the binding of apigenin with DNA bases, especially with GC versus AT rich sequences. We designed two different sets of 100 bp oligonucleotides probes with GC- and AT- rich sequences and the absorption spectrum changes were recorded by UV-VIS spectrophotometer at 230-450 nm range. UV-VIS spectral studies demonstrated that apigenin possesses high affinity for poly-ds-GC oligonucleotides compared to poly-ds-AT oligonucleotide sequence. Since DNA methylation primarily at the C5 position of cytosine affects gene expression at the promoter CpG islands, therefore, we next studied the effect of apigenin in reversing hypermethylation of cytosine bases in the DNA. For these studies we generated 100 bp highly GC-rich sequences from the CpG island of GSTP1 promoter, a gene which has been shown to be frequently silenced in prostate cancer due to promoter hypermethylation. Spectral studies exhibited that apigenin possess high affinity with CpG island of GSTP1 promoter sequence compared to randomly picked GSTP1 gene sequence. Furthermore, apigenin prevented the methylation of cytosine in the GC-rich promoter sequence incubated with MSsI enzyme. Similar results were obtained with transformed human prostate epithelial RWPE1 cells, where MSsI enzyme-mediated hypermethylation was reduced by apigenin pre-treatment, compared to the control group without apigenin. Taken together, our studies suggest that apigenin binds preferentially to the GC-rich sequence primarily positioned in the promoter region of various tumor suppressor genes and might prevent promoter hypermethylation, at least in part, accountable for its chemopreventive activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3683. doi:10.1158/1538-7445.AM2011-3683

A primer design strategy for PCR amplification of GC-rich DNA sequences

Objectives To establish a primer design method for amplification of GC-rich DNA sequences. Design and methods A group of 15 pairs of primers with higher T m (> 79.7 °C) and lower level Δ T m (< 1 °C) were designed to amplify GC-rich sequences (66.0%–84.0%). The statistical analysis of primer parameters and GC content of PCR products was performed and compared with literatures. Other control experiments were conducted using shortened primers for GC-rich PCR amplifications in this study, and the statistical analysis of shortened primer parameters and GC content of PCR products was performed compared with primers not shortened. A group of 26 pairs of primers were designed to test the applicability of this primer designing strategy in amplifications of non-GC-rich sequences (35.2%–53.5%). Results All the DNA sequences in this study were successfully amplified. Statistical analyses show that the T m and Δ T m were the main factors influencing amplifications. Conclusions This primer designing strategy offered a perfect tool for amplification of GC-rich sequences. It proves that the secondary structures cannot be formed at higher annealing temperature conditions (> 65 °C), and we can overcome this difficulty easily by designing primers and using higher annealing temperature.

Nuclear Tau, a Key Player in Neuronal DNA Protection

Tau, a neuronal protein involved in neurodegenerative disorders such as Alzheimer disease, which is primarily described as a microtubule-associated protein, has also been observed in the nuclei of neuronal and non-neuronal cells. However, the function of the nuclear form of Tau in neurons has not yet been elucidated. In this work, we demonstrate that acute oxidative stress and mild heat stress (HS) induce the accumulation of dephosphorylated Tau in neuronal nuclei. Using chromatin immunoprecipitation assays, we demonstrate that the capacity of endogenous Tau to interact with neuronal DNA increased following HS. Comet assays performed on both wild-type and Tau-deficient neuronal cultures showed that Tau fully protected neuronal genomic DNA against HS-induced damage. Interestingly, HS-induced DNA damage observed in Tau-deficient cells was completely rescued after the overexpression of human Tau targeted to the nucleus. These results highlight a novel role for nuclear Tau as a key player in early stress response.

Sole and Stable RNA Duplexes of G-Rich Sequences Located in the 5′-Untranslated Region of Protooncogenes

Guanine- (G-) rich nucleic acid sequences can form four-stranded structures called G-quadruplexes. It is widely held that the formation of a G-quadruplex in RNA is more feasible than in DNA because of the lack of a complementary strand in mRNA. Here, we analyzed sequences of 5'-untranslated regions of protooncogenes and surprisingly found that these regions showed an enrichment of not only guanine (G) but also cytosine (C) nucleotides. Since neighboring cytosine- (C-) rich regions can affect the formation and stability of a G-quadruplex structure, we further investigated the properties of DNA and RNA structures of G-rich and GC-rich regions. We selected typical GC-rich RNA sequences from protooncogenes and corresponding DNA sequences and investigated their structures. It was found that the GC-rich RNA sequences formed stable A-form duplexes as their major structure independent of the surrounding conditions, including the presence of different cations (Na(+), K(+), or Li(+)) or molecular crowding with 40 wt % poly(ethylene glycol) with an average molecular mass of 200 Da although there are a few exceptions in which only a combination of K(+) and molecular crowding induced a G-quadruplex structure of an extremely G-rich RNA sequence. In contrast, structural polymorphisms involving duplexes, G-quadruplexes, and i-motifs were observed for GC-rich DNA sequences depending on the surrounding factors. These results demonstrate the considerable structural and functional differences in GC-rich sequences of the genome (DNA) and transcriptosome (mRNA) with respect to the nucleic acid backbone. Moreover, it was suggested that structural study for a G-rich RNA sequence should be carried out under cell-mimicking condition where K(+) and crowding cosolutes exist.

Successful Pyrosequencing of GC-rich DNA Sequences by Partial Substitution of Deoxyguanosine With Deoxyinosine

We experienced significant problems while developing a PyrosequencingTM (Biotage, Uppsala, Sweden) assay to characterize the rifampin resistance-determining region of Mycobacteriumtuberculosis, a target with high guanosine-cytosine content. This paper describes the successful use of a modified pyrosequencing protocol through partial substitution of deoxyguanosine triphosphate with deoxyinosine triphosphate in the polymerase chain reaction.

Efficient amplification of genes involved in microbial secondary metabolism by an improved genome walking method

Genome walking is a commonly used technique for the identification of DNA sequences adjacent to known regions. Despite the development of various genome walking methods, nonspecific products are often produced in certain circumstances, especially when GC-rich DNA sequences are dealt with. To effectively resolve such technical issues, a simple nested polymerase chain reaction-based genome walking method has been developed by implementing a progressively decreased annealing temperature from 70 degrees C to 47.5 degrees C in the first round of amplification and a high annealing temperature of 65 degrees C in the second round of amplification. During the entire process, a lower ramp rate of 1.5 degrees C s(-1) and cooling rate of 2.5 degrees C s(-1) are performed to reach the annealing temperature. Using this method, we successfully obtained the upstream and downstream sequences of three GC-rich genes involved in the biosynthetic pathways of secondary metabolites from two bacterial genomes. The efficient amplification of DNA target longer than 1.5 Kb with GC content up to 75.0% indicates that the present technique could be a valuable tool for the investigation of biosynthetic pathways of various secondary metabolites.

HDAC4 Represses Matrix Metalloproteinase-13 Transcription in Osteoblastic Cells, and Parathyroid Hormone Controls This Repression

Parathyroid hormone (PTH) is a hormone regulating bone remodeling through its actions on both bone formation and bone resorption. Previously we reported that PTH induces matrix metalloproteinase-13 (MMP-13) transcription in osteoblastic cells. Here, we show that histone deacetylase 4 (HDAC4) interacts with Runx2, binds the MMP-13 promoter, and suppresses MMP-13 gene transcription in the rat osteoblastic cell line, UMR 106-01. PTH induces the rapid cAMP-dependent protein kinase-dependent release of HDAC4 from the MMP-13 promoter and subsequent transcription of MMP-13. Knock-out of HDAC4 either by siRNA in vitro or by gene deletion in vivo leads to an increase in MMP-13 expression, and overexpression of HDAC4 decreases the PTH induction of MMP-13. All of these observations indicate that HDAC4 represses MMP-13 gene transcription in bone. Moreover, PTH stimulates HDAC4 gene expression and enzymatic activity at times corresponding to the reassociation of HDAC4 with the MMP-13 promoter and a decline in its transcription. Thus, HDAC4 is a basal repressor of MMP-13 transcription, and PTH regulates HDAC4 to control MMP-13 promoter activity. These data identify a novel and discrete mechanism of regulating HDAC4 levels and, subsequently, gene expression.

Enhanced amplification of GC-rich DNA with two organic reagents

Amplification of GC-rich DNA sequences is still a difficult task worldwide. Two frequently seen and inexpensive reagents-ethylene glycol and 1,2-propanediol-were found to be more effective than betaine in the amplification of 104 randomly selected GC-rich human DNA sequences with GC contents of 60-80% and lengths of 700-800 bp.

Promoter analysis of intestinal genes induced during iron-deprivation reveals enrichment of conserved SP1-like binding sites.

BackgroundIron-deficiency leads to the induction of genes related to intestinal iron absorption and homeostasis. By analyzing a large GeneChip® dataset from the rat intestine, we identified a large cluster of 228 genes that was induced by iron-deprivation. Only 2 of these genes contained 3' iron-response elements, suggesting that other regulation including transcriptional may be involved. We therefore utilized computational methods to test the hypothesis that some of the genes within this large up-regulated cluster are co-ordinately regulated by common transcriptional mechanisms. We thus identified promoters from the up-regulated gene cluster from rat, mouse and human, and performed enrichment analyses with the Clover program and the TRANSFAC database.ResultsSurprisingly, we found a strong statistical enrichment for SP1 binding sites in our experimental promoters as compared to background sequences. As the TRANSFAC database cannot distinguish among SP/KLF family members, many of which bind similar GC-rich DNA sequences, we surmise that SP1 or an SP1-like factor could be involved in this response. In fact, we detected induction of SP6/KLF14 in the GeneChip® studies, and confirmed it by real-time PCR. Additional computational analyses suggested that an SP1-like factor may function synergistically with a FOX TF to regulate a subset of these genes. Furthermore, analysis of promoter sequences identified many genes with multiple, conserved SP1 and FOX binding sites, the relative location of which within orthologous promoters was highly conserved.ConclusionSP1 or a closely related factor may play a primary role in the genetic response to iron-deficiency in the mammalian intestine.

Copper-Activated DNA Photocleavage by a Pyridine-Linked Bis-Acridine Intercalator

We report the synthesis of new photonuclease 4 consisting of two acridine rings joined by a pyridine-based copper binding linker. We have shown that photocleavage of plasmid DNA is markedly enhanced when this ligand is irradiated in the presence of copper(II) (419 nm, 22 degrees C, pH 7.0). Viscometric data indicate that 4 binds to DNA by monofunctional intercalation, and equilibrium dialysis provides an estimated binding constant of 1.13 x 105 M-1 for its association with calf thymus DNA. In competition dialysis experiments, 4 exhibits preferential binding to GC-rich DNA sequences. When Cu(II) is added at a ligand to metal ratio of 1:1, electrospray ionization mass spectrometry demonstrates that compound 4 undergoes complex formation, while thermal melting studies show a 10 degrees C increase in the Tm of calf thymus DNA. Groove binding and intercalation are suggested by viscometric data. Finally, colorimetric and scavenger experiments indicate that the generation of Cu(I), H2O2, and superoxide contributes to the production of DNA frank strand breaks by the Cu(II) complex of 4. Whereas the strand breaks are distributed in a relatively uniform fashion over the four DNA bases, subsequent piperidine treatment of the photolysis reactions shows that alkaline labile lesions occur predominantly at guanine.

Disruption of an SP2/KLF6 Repression Complex by SHP Is Required for Farnesoid X Receptor-induced Endothelial Cell Migration

The farnesoid X receptor (FXR) signaling pathway regulates bile acid and cholesterol homeostasis. Here, we demonstrate, using a variety of gain- and loss-of-function approaches, a role of FXR in the process of cell motility, which involves the small heterodimeric partner (SHP)-dependent up-regulation of matrix metalloproteinase-9. We use this observation to reveal a transcriptional regulatory mechanism involving the SP/KLF transcription factors, SP2 and KLF6. Small interference RNA-based silencing studies in combination with promoter, gel shift, and chromatin immunoprecipitation assays indicate that SP2 and KLF6 bind to the matrix metalloproteinase-9 promoter and together function to maintain this gene in a silenced state. However, upon activation of FXR, SHP interacts with SP2 and KLF6, disrupting the SP2/KLF6 repressor complex. Thus, together, these studies identify a mechanism for antagonizing Sp/KLF protein repression function via SHP, with this process regulating endothelial cell motility.

Betaine, Dimethyl Sulfoxide, and 7-Deaza-dGTP, a Powerful Mixture for Amplification of GC-Rich DNA Sequences

Currently, polymerase chain reaction is the most used technique in many laboratories for either diagnostic or molecular biology purposes. Despite the large number of DNA sequences that can be easily analyzed, some GC-rich sequences are refractory to amplification due to the formation of secondary intramolecular structures. To overcome this problem, several molecules have been described to improve polymerization. Here we show that a combination of three additives--betaine, dimethyl sulfoxide, and 7-deaza-dGTP--was essential to achieve amplification of DNA sequences of three disease genes showing a GC content ranging from 67 to 79%.

DNA Recognition by the Brinker Repressor – An Extreme Case of Coupling Between Binding and Folding

The Brinker (Brk) nuclear repressor is a major element of the Drosophila Decapentaplegic morphogen signaling pathway. Its N-terminal part has weak homology to the Antennapedia homeodomain and binds to GC-rich DNA sequences. We have investigated the conformation and dynamics of the N-terminal 101 amino acid residues of Brk in the absence and in the presence of cognate DNA by solution NMR spectroscopy. In the absence of DNA, Brk is unfolded and highly flexible throughout the entire backbone. Addition of cognate DNA induces the formation of a well-folded structure for residues R46 to R95. This structure consists of four helices forming a helix-turn-helix motif that differs from homeodomains, but has similarities to the Tc3 transposase, the Pax-6 Paired domain, and the human centromere-binding protein. The GC-rich DNA recognition can be explained by specific major groove hydrogen bonds from the N-terminal end of helix α3. The transition from a highly flexible, completely unfolded conformation in the absence of DNA to a well-formed structure in the complex presents a very extreme case of the “coupling of binding and folding” phenomenon.