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Apurinic/apyrimidinic endonuclease 1 (APE1) is involved in DNA repair and transcriptional regulation mechanisms. This multifunctional activity of APE1 should be supported by specific structural properties of APE1 that have not yet been elucidated. Herein, we applied atomic force microscopy (AFM) to characterize the interactions of APE1 with DNA containing two well-separated G-rich segments. Complexes of APE1 with DNA containing G-rich segments were visualized, and analysis of the complexes revealed the affinity of APE1 to G-rich DNA sequences, and their yield was as high as 53%. Furthermore, APE1 is capable of binding two DNA segments leading to the formation of loops in the DNA–APE1 complexes. The analysis of looped APE1-DNA complexes revealed that APE1 can bridge G-rich segments of DNA. The yield of loops bridging two G-rich DNA segments was 41%. Analysis of protein size in various complexes was performed, and these data showed that loops are formed by APE1 monomer, suggesting that APE1 has two DNA binding sites. The data led us to a model for the interaction of APE1 with DNA and the search for the specific sites. The implication of these new APE1 properties in organizing DNA, by bringing two distant sites together, for facilitating the scanning for damage and coordinating repair and transcription is discussed. [ABSTRACT FROM AUTHOR]

Abstract Background Nuclear organization of interphase chromosomes involves individual chromosome territories, “open” and “closed” chromatin compartments, topologically associated domains (TADs) and chromatin loops. The DNA- and RNA-binding transcription factor CTCF together with the cohesin complex serve as major organizers of chromatin architecture. Cellular differentiation is driven by temporally and spatially coordinated gene expression that requires chromatin changes of individual loci of various complexities. Lens differentiation represents an advantageous system to probe transcriptional mechanisms underlying tissue-specific gene expression including high transcriptional outputs of individual crystallin genes until the mature lens fiber cells degrade their nuclei. Results Chromatin organization between mouse embryonic stem (ES) cells, newborn (P0.5) lens epithelium and fiber cells were analyzed using Hi-C. Localization of CTCF in both lens chromatins was determined by ChIP-seq and compared with ES cells. Quantitative analyses show major differences between number and size of TADs and chromatin loop size between these three cell types. In depth analyses show similarities between lens samples exemplified by overlaps between compartments A and B. Lens epithelium-specific CTCF peaks are found in mostly methylated genomic regions while lens fiber-specific and shared peaks occur mostly within unmethylated DNA regions. Major differences in TADs and loops are illustrated at the ~ 500 kb Pax6 locus, encoding the critical lens regulatory transcription factor and within a larger ~ 15 Mb WAGR locus, containing Pax6 and other loci linked to human congenital diseases. Lens and ES cell Hi-C data (TADs and loops) together with ATAC-seq, CTCF, H3K27ac, H3K27me3 and ENCODE cis-regulatory sites are shown in detail for the Pax6, Sox1 and Hif1a loci, multiple crystallin genes and other important loci required for lens morphogenesis. The majority of crystallin loci are marked by unexpectedly high CTCF-binding across their transcribed regions. Conclusions Our study has generated the first data on 3-dimensional (3D) nuclear organization in lens epithelium and lens fibers and directly compared these data with ES cells. These findings generate novel insights into lens-specific transcriptional gene control, open new research avenues to study transcriptional condensates in lens fiber cells, and enable studies of non-coding genetic variants linked to cataract and other lens and ocular abnormalities.

Background: Nuclear organization of interphase chromosomes involves individual chromosome territories, "open" and "closed" chromatin compartments, topologically associated domains (TADs) and chromatin loops. The DNA- and RNA-binding transcription factor CTCF together with the cohesin complex serve as major organizers of chromatin architecture. Cellular differentiation is driven by temporally and spatially coordinated gene expression that requires chromatin changes of individual loci of various complexities. Lens differentiation represents an advantageous system to probe transcriptional mechanisms underlying tissue-specific gene expression including high transcriptional outputs of individual crystallin genes until the mature lens fiber cells degrade their nuclei. Results: Chromatin organization between mouse embryonic stem (ES) cells, newborn (P0.5) lens epithelium and fiber cells were analyzed using Hi-C. Localization of CTCF in both lens chromatins was determined by ChIP-seq and compared with ES cells. Quantitative analyses show major differences between number and size of TADs and chromatin loop size between these three cell types. In depth analyses show similarities between lens samples exemplified by overlaps between compartments A and B. Lens epithelium-specific CTCF peaks are found in mostly methylated genomic regions while lens fiber-specific and shared peaks occur mostly within unmethylated DNA regions. Major differences in TADs and loops are illustrated at the ~ 500 kb Pax6 locus, encoding the critical lens regulatory transcription factor and within a larger ~ 15 Mb WAGR locus, containing Pax6 and other loci linked to human congenital diseases. Lens and ES cell Hi-C data (TADs and loops) together with ATAC-seq, CTCF, H3K27ac, H3K27me3 and ENCODE cis-regulatory sites are shown in detail for the Pax6, Sox1 and Hif1a loci, multiple crystallin genes and other important loci required for lens morphogenesis. The majority of crystallin loci are marked by unexpectedly high CTCF-binding across their transcribed regions. Conclusions: Our study has generated the first data on 3-dimensional (3D) nuclear organization in lens epithelium and lens fibers and directly compared these data with ES cells. These findings generate novel insights into lens-specific transcriptional gene control, open new research avenues to study transcriptional condensates in lens fiber cells, and enable studies of non-coding genetic variants linked to cataract and other lens and ocular abnormalities. [ABSTRACT FROM AUTHOR]

The bgl operon of Escherichia coli encodes proteins mediating the metabolism of aromatic beta-glucosides, but the operon is silent in wild type cells. Insertion of an insertion sequence (IS) element in the regulatory region upstream of the bgl promoter activates expression of the operon. The repression mechanism involves the histone-like nucleoid structuring (H-NS) protein with two DNA binding sites, one in the region upstream of the promoter, and the other within the first structural gene of the operon, bglG. The detailed mechanism of repression is not well understood. Here, we first show two terminators flanking bglG are not required for bgl operon silencing. Instead, several lines of experimental evidence clearly suggest that the silencing mechanism involves looping of the DNA between H-NS's two DNA binding sites. H-NS is known to preferentially bind to AT-rich curved DNA, and such regions are found in the vicinity of both sites. We show that strong repression is abolished by (1) preventing H-NS self-oligomerization while retaining DNA binding, (2) preventing or reducing H-NS binding to the bgl operon regulatory region, and (3) preventing or reducing H-NS binding to the binding site in the bglG gene. We also show that the phase of the DNA between these two binding sites is not important, and that large insertions of DNA in the putative loop region do not diminish repression. These results imply that H-NS depends on DNA looping to exert strong repression.

The establishment, maintenance and dynamic regulation of three-dimensional (3D) chromatin structures provide an important means for partitioning of genome into functionally distinctive domains, which helps to define specialized gene expression programs associated with developmental stages and cell types. Increasing evidence supports critical roles for intrinsically disordered regions (IDRs) harbored within transcription factors (TFs) and chromatin-modulatory proteins in inducing phase separation, a phenomenon of forming membrane-less condensates through partitioning of biomolecules. Such a process is also critically involved in the establishment of high-order chromatin structures and looping. IDR- and phase separation-driven 3D genome (re)organization often goes wrong in disease such as cancer. This review discusses about recent advances in understanding how phase separation of intrinsically disordered proteins (IDPs) modulates chromatin looping and gene expression. [ABSTRACT FROM AUTHOR]

Bacteriophage λ is a paradigm in the field of gene regulation and one of the best-understood systems in genetic regulatory biology. A so-called Genetic Switch determines the mechanisms by which λ transitions to its dual lifestyles-lytic or lysogenic. When λ initiates the lysogenic lifestyle, the phage-encoded CI repressor binds cooperatively to multi-partite operators in a defined pattern that autoregulates repression of phage lytic promoters as well as activation of the lysogenic promoter. The study of this genetic switch and related earlier research on phage λ revealed the main principles of gene expression and regulation in molecular biology. This article describes the underlying molecular details of λ lysogeny, as it is currently understood., Competing Interests: The authors declare no conflict of interest.

The bgl operon of Escherichia coli encodes proteins mediating the metabolism of aromatic beta-glucosides, but the operon is silent in wild type cells. Insertion of an insertion sequence (IS) element in the regulatory region upstream of the bgl promoter activates expression of the operon. The repression mechanism involves the histone-like nucleoid structuring (H-NS) protein with two DNA binding sites, one in the region upstream of the promoter, and the other within the first structural gene of the operon, bglG. The detailed mechanism of repression is not well understood. Here, we first show two terminators flanking bglG are not required for bgl operon silencing. Instead, several lines of experimental evidence clearly suggest that the silencing mechanism involves looping of the DNA between H-NS’s two DNA binding sites. H-NS is known to preferentially bind to AT-rich curved DNA, and such regions are found in the vicinity of both sites. We show that strong repression is abolished by (1) preventing H-NS self-oligomerization while retaining DNA binding, (2) preventing or reducing H-NS binding to the bgl operon regulatory region, and (3) preventing or reducing H-NS binding to the binding site in the bglG gene. We also show that the phase of the DNA between these two binding sites is not important, and that large insertions of DNA in the putative loop region do not diminish repression. These results imply that H-NS depends on DNA looping to exert strong repression.

In eukaryotic nuclei, chromatin loops mediated through cohesin are critical structures that regulate gene expression and DNA replication. Here, we demonstrate a new method to see endogenous genomic loci using synthetic zinc‐finger proteins harboring repeat epitope tags (ZF probes) for signal amplification via binding of tag‐specific intracellular antibodies, or frankenbodies, fused with fluorescent proteins. We achieve this in two steps: First, we develop an anti‐FLAG frankenbody that can bind FLAG‐tagged proteins in diverse live‐cell environments. The anti‐FLAG frankenbody complements the anti‐HA frankenbody, enabling two‐color signal amplification from FLAG‐ and HA‐tagged proteins. Second, we develop a pair of cell‐permeable ZF probes that specifically bind two endogenous chromatin loci predicted to be involved in chromatin looping. By coupling our anti‐FLAG and anti‐HA frankenbodies with FLAG‐ and HA‐tagged ZF probes, we simultaneously see the dynamics of the two loci in single living cells. This shows a close association between the two loci in the majority of cells, but the loci markedly separate from the triggered degradation of the cohesin subunit RAD21. Our ability to image two endogenous genomic loci simultaneously in single living cells provides a proof of principle that ZF probes coupled with frankenbodies are useful new tools for exploring genome dynamics in multiple colors. [ABSTRACT FROM AUTHOR]

Bacterial conjugation is the main horizontal gene transfer route responsible for the spread of antibiotic resistance, virulence and toxin genes. During conjugation, DNA is transferred from a donor to a recipient cell via a sophisticated channel connecting the two cells. Conjugation not only affects many different aspects of the plasmid and the host, ranging from the properties of the membrane and the cell surface of the donor, to other developmental processes such as competence, it probably also poses a burden on the donor cell due to the expression of the large number of genes involved in the conjugation process. Therefore, expression of the conjugation genes must be strictly controlled. Over the past decade, the regulation of the conjugation genes present on the conjugative Bacillus subtilis plasmid pLS20 has been studied using a variety of methods including genetic, biochemical, biophysical and structural approaches. This review focuses on the interplay between RcopLS20, RappLS20 and Phr*pLS20, the proteins that control the activity of the main conjugation promoter Pc located upstream of the conjugation operon. Proper expression of the conjugation genes requires the following two fundamental elements. First, conjugation is repressed by default and an intercellular quorum-signaling system is used to sense conditions favorable for conjugation. Second, different layers of regulation act together to repress the Pc promoter in a strict manner but allowing rapid activation. During conjugation, ssDNA is exported from the cell by a membrane-embedded DNA translocation machine. Another membrane-embedded DNA translocation machine imports ssDNA in competent cells. Evidences are reviewed indicating that conjugation and competence are probably mutually exclusive processes. Some of the questions that remain unanswered are discussed.

Abstract Genome-wide association studies (GWAS) and fine mapping studies in autoimmune diseases have identified thousands of genetic variants, the majority of which are located in non-protein-coding enhancer regions. Enhancers function within the context of the three-dimensional (3D) genome to form long-range DNA looping events with target gene promoters that spatially and temporally regulate gene expression. Investigating the functional significance of GWAS variants in the context of the 3D genome is essential for mechanistic understanding of these variants and how they influence disease pathology by altering DNA looping between enhancers and the target gene promoters they regulate. In this review, we discuss the functional complexity of the 3D genome and the technological approaches used to characterize DNA looping events. We then highlight examples from the literature that illustrate how functional mapping of the 3D genome can assist in defining mechanisms that influence pathogenic gene expression. We conclude by highlighting future advances necessary to fully integrate 3D genome analyses into the functional workup of GWAS variants in the continuing effort to improve the health of patients with autoimmune diseases.

Jasmonates (JAs) are essential plant hormones that play important roles in the regulation of plant growth and the response to environmental stress. In the JA signaling pathway, the core transcription factors are a class of basic helix-loop-helix (bHLH) proteins, including MYC2, MYC3, and MYC4, that have different regulatory capacities. Here, we report the 2.7 Å crystal structure of the MYC2 bHLH domain complexed with G-box DNA, showing a cis-tetrameric structure. Biochemical assays confirmed that full-length MYC2 forms a stable homo-tetramer both in solution and in DNA-bound states, whereas MYC3 forms only a homodimer. Isothermal titration calorimetry (ITC) assays demonstrated that tetramerization enhanced DNA binding affinity, and fluorescence resonance energy transfer (FRET) assay indicated DNA looping potential of tetrameric MYC2. Luciferase assay further confirmed the importance of tetramerization in transcriptional regulation. Our studies provide a mechanistic explanation for the regulatory differences of MYC transcription factors.

All forms of diabetes result from insufficient functional β-cell mass. Thus, achieving the therapeutic goal of expanding β-cell mass requires a better mechanistic understanding of how β-cells proliferate. Glucose is a natural β-cell mitogen that mediates its effects in part through the glucose-responsive transcription factor, carbohydrate response element binding protein (ChREBP) and the anabolic transcription factor, MYC. However, mechanistic details by which glucose activates Myc at the transcriptional level are poorly understood. Here, siRNA was used to test the role of ChREBP in the glucose response of MYC, ChIP and ChIPseq to identify potential regulatory binding sites, chromatin conformation capture to identify DNA/DNA interactions, and an adenovirus was constructed to expresses x-dCas9 and an sgRNA that specifically disrupts the recruitment of ChREBP to a specific targeted ChoRE. We found that ChREBP is essential for glucose-mediated transcriptional induction of Myc , and for increases in Myc mRNA and protein abundance. Further, ChIPseq revealed that the carbohydrate response element (ChoRE) nearest to the Myc transcriptional start site (TSS) is immediately upstream of the gene encoding the lncRNA, Pvt1 , 60,000 bp downstream of the Myc gene. Chromatin Conformation Capture (3C) confirmed a glucose-dependent interaction between these two sites. Transduction with an adenovirus expressing x-dCas9 and an sgRNA specifically targeting the highly conserved Pvt1 ChoRE, attenuates ChREBP recruitment, decreases Myc - Pvt1 DNA/DNA interaction, and decreases expression of the Pvt1 and Myc genes in response to glucose. Importantly, isolated and dispersed rat islet cells transduced with the ChoRE-disrupting adenovirus also display specific decreases in ChREBP-dependent, glucose-mediated expression of Pvt1 and Myc , as well as decreased glucose-stimulated β-cell proliferation. The mitogenic glucose response of Myc is mediated via glucose-dependent recruitment of ChREBP to the promoter of the Pvt1 gene and subsequent DNA looping with the Myc promoter. [Display omitted] • Expanding functional beta cell mass is a therapeutic goal for treating diabetes. • Glucose is a natural beta cell mitogen via ChREBP-dependent Myc induction. • ChREBP promotes a DNA loop between Pvt1 lncRNA and Myc. • Disruption of ChREBP binding to the Pvt1 promoter prevents glucose-mediated DNA looping, Myc induction, and beta cell proliferation. [ABSTRACT FROM AUTHOR]

Studying the regulation of transcription of the gal operon that encodes the amphibolic pathway of d-galactose metabolism in Escherichia coli discerned a plethora of principles that operate in prokaryotic gene regulatory processes. In this chapter, we have reviewed some of the more recent findings in gal that continues to reveal unexpected but important mechanistic details. Since the operon is transcribed from two overlapping promoters, P1 and P2, regulated by common regulatory factors, each genetic or biochemical experiment allowed simultaneous discernment of two promoters. Recent studies range from genetic, biochemical through biophysical experiments providing explanations at physiological, mechanistic and single molecule levels. The salient observations highlighted here are: the axiom of determining transcription start points, discovery of a new promoter element different from the known ones that influences promoter strength, occurrence of an intrinsic DNA sequence element that overrides the transcription elongation pause created by a DNA-bound protein roadblock, first observation of a DNA loop and determination its trajectory, and piggybacking proteins and delivering to their DNA target.

RNA polymerase II (RNAPII) transcribes DNA wrapped in the nucleosome by stepwise pausing, especially at nucleosomal superhelical locations -5 and -1 [SHL(-5) and SHL(-1), respectively]. In the present study, we performed cryo-electron microscopy analyses of RNAPII-nucleosome complexes paused at a major nucleosomal pausing site, SHL(-1). We determined two previously undetected structures, in which the transcribed DNA behind RNAPII is sharply kinked at the RNAPII exit tunnel and rewrapped around the nucleosomal histones in front of RNAPII by DNA looping. This DNA kink shifts the DNA orientation toward the nucleosome, and the transcribed DNA region interacts with basic amino acid residues of histones H2A, H2B, and H3 exposed by the RNAPII-mediated nucleosomal DNA peeling. The DNA loop structure was not observed in the presence of the transcription elongation factors Spt4/5 and Elf1. These RNAPII-nucleosome structures provide important information for understanding the functional relevance of DNA looping during transcription elongation in the nucleosome., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interests with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Long-range intrachromosomal interactions play an important role in 3D chromosome structure and function, but our understanding of how various factors contribute to the strength of these interactions remains poor. In this study we used a recently developed analysis framework for Bayesian network (BN) modeling to analyze publicly available datasets for intrachromosomal interactions. We investigated how 106 variables affect the pairwise interactions of over 10 million 5-kb DNA segments in the B-lymphocyte cell line GB12878. Strictly data-driven BN modeling indicates that the strength of intrachromosomal interactions (hic strength) is directly influenced by only four types of factors: distance between segments, Rad21 or SMC3 (cohesin components), transcription at transcription start sites (TSS), and the number of CCCTC-binding factor (CTCF)-cohesin complexes between the interacting DNA segments. Subsequent studies confirmed that most high-intensity interactions have a CTCF-cohesin complex in at least one of the interacting segments. However, 46% have CTCF on only one side, and 32% are without CTCF. As expected, high-intensity interactions are strongly dependent on the orientation of the ctcf motif, and, moreover, we find that the interaction between enhancers and promoters is similarly dependent on ctcf motif orientation. Dependency relationships between transcription factors were also revealed, including known lineage-determining B-cell transcription factors (e.g., Ebf1) as well as potential novel relationships. Thus, BN analysis of large intrachromosomal interaction datasets is a useful tool for gaining insight into DNA-DNA, protein-DNA, and protein-protein interactions. [ABSTRACT FROM AUTHOR]

Loops are ubiquitous topological elements formed when proteins simultaneously bind to two noncontiguous DNA sites. While a loop-mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM). An analysis of transcription elongation complexes, moving for 60 s at an average of 10 nt/s on unlooped DNA templates, revealed that they more often surpassed LacI bound to the lower affinity O2 operator than to the highest affinity Os operator. However, this difference was abrogated in looped DNA molecules where LacI became a strong roadblock independently of the affinity of the operator. Recordings of transcription elongation complexes, using magnetic tweezers, confirmed that they halted for several minutes upon encountering a LacI bound to a single operator. The average pause lifetime is compatible with RNAP waiting for LacI dissociation, however, the LacI open conformation visualized in the SFM images also suggests that LacI could straddle RNAP to let it pass. Independently of the mechanism by which RNAP bypasses the LacI roadblock, the data indicate that an obstacle with looped topology more effectively interferes with transcription. [ABSTRACT FROM AUTHOR]

Upstream stimulating factors (USFs), including USF1 and USF2, are key components of the transcription machinery that recruit coactivators and histone-modifying enzymes. Using the classic basic helix-loop-helix leucine zipper (bHLH-LZ) domain, USFs bind the E-box DNA and form tetramers that promote DNA looping for transcription initiation. The structural basis by which USFs tetramerize and bind DNA, however, remains unknown. Here, we report the crystal structure of the complete bHLH-LZ domain of USF2 in complex with E-box DNA. We observed that the leucine zipper (LZ) of USF2 is longer than that of other bHLH-LZ family transcription factors and that the C-terminus of USF2 forms an additional α-helix following the LZ region (denoted as LZ-Ext). We also found the elongated LZ-Ext facilitates compact tetramer formation. In addition to the classic interactions between the basic region and DNA, we show a highly conserved basic residue in the loop region, Lys271, participates in DNA interaction. Together, these findings suggest that USF2 forms a tetramer structure with a bent elongated LZ-Ext region, providing a molecular basis for its role as a key component of the transcription machinery., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Genes that are key to cell identity are generally regulated by cell-type-specific enhancer elements bound by transcription factors, some of which facilitate looping to distant gene promoters. In contrast, genes that encode housekeeping functions, whose regulation is essential for normal cell metabolism and growth, generally lack interactions with distal enhancers. We find that Ronin (Thap11) assembles multiple promoters of housekeeping and metabolic genes to regulate gene expression. This behavior is analogous to how enhancers are brought together with promoters to regulate cell identity genes. Thus, Ronin-dependent promoter assemblies provide a mechanism to explain why housekeeping genes can forgo distal enhancer elements and why Ronin is important for cellular metabolism and growth control. We propose that clustering of regulatory elements is a mechanism common to cell identity and housekeeping genes but is accomplished by different factors binding distinct control elements to establish enhancer-promoter or promoter-promoter interactions, respectively., Competing Interests: Declaration of interests T.P.Z. and R.A.Y. are founders and shareholders of Paratus Sciences, and R.A.Y. is a founder and shareholder of Syros Pharmaceuticals, Camp4 Therapeutics, Omega Therapeutics, and Dewpoint Therapeutics. A.D. is a consultant for Dewpoint Therapeutics., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Summary We analyzed the in silico purified DNA methylation signatures of 82 mantle cell lymphomas (MCL) in comparison with cell subpopulations spanning the entire B cell lineage. We identified two MCL subgroups, respectively carrying epigenetic imprints of germinal-center-inexperienced and germinal-center-experienced B cells, and we found that DNA methylation profiles during lymphomagenesis are largely influenced by the methylation dynamics in normal B cells. An integrative epigenomic approach revealed 10,504 differentially methylated regions in regulatory elements marked by H3K27ac in MCL primary cases, including a distant enhancer showing de novo looping to the MCL oncogene SOX11 . Finally, we observed that the magnitude of DNA methylation changes per case is highly variable and serves as an independent prognostic factor for MCL outcome. [ABSTRACT FROM AUTHOR]

Genomic DNA is vastly longer than the space allotted to it in a cell. The molecule must fold with a level of organization that satisfies the imposed spatial constraints as well as allow for the processing of genetic information. Key players in this organization include the negative supercoiling of DNA, which facilitates the unwinding of the double-helical molecule, and the associations of DNA with proteins, which partition the DNA into isolated loops, or domains. In order to gain insight into the principles of genome organization and to visualize the folding of spatially constrained DNA, we have developed new computational methods to identify the preferred three-dimensional pathways of protein-mediated DNA loops and to characterize the topological properties of these structures. Here, we focus on the levels of supercoiling and the spatial arrangements of DNA in model nucleoprotein systems with two topological domains. We construct these systems by anchoring DNA loops in opposing orientations on a common protein-DNA assembly, namely the Lac repressor protein with two bound DNA operators. The linked pieces of DNA form a covalently closed circle such that the protein attaches to two widely spaced sites along the DNA. We examine the effects of operator spacing, loop orientation, and long-range contacts on overall chain configuration and topology, and discuss our findings in the context of classic experiments on the effects of supercoiling and operator spacing on Lac repressor-mediated looping and recent work on the role of proteins as barriers that divide genomes into independent topological domains. [ABSTRACT FROM AUTHOR]

The function of DNA as a repository of genetic information is well-known. The post-genomic effort is to understand how this information-containing filament is chaperoned to manage its compaction and topological states. Indeed, the activities of enzymes that transcribe, replicate, or repair DNA are regulated to a large degree by access. Proteins that act at a distance along the filament by binding at one site and contacting another site, perhaps as part of a bigger complex, create loops that constitute topological domains and influence regulation. DNA loops and plectonemes are not necessarily spontaneous, especially large loops under tension for which high energy is required to bring their ends together, or small loops that require accessory proteins to facilitate DNA bending. However, the torsion in stiff filaments such as DNA dramatically modulates the topology, driving it from extended and genetically accessible to more looped and compact, genetically secured forms. Furthermore, there are accessory factors that bias the response of the DNA filament to supercoiling. For example, small molecules like polyamines, which neutralize the negative charge repulsions along the phosphate backbone, enhance flexibility and promote writhe over twist in response to torsion. Such increased flexibility likely pushes the topological equilibrium from twist toward writhe at tensions thought to exist in vivo. A predictable corollary is that stiffening DNA antagonizes looping and bending. Certain sequences are known to be more or less flexible or to exhibit curvature, and this may affect interactions with binding proteins. In vivo all of these factors operate simultaneously on DNA that is generally negatively supercoiled to some degree. Therefore, in order to better understand gene regulation that involves protein-mediated DNA loops, it is critical to understand the thermodynamics and kinetics of looping in DNA that is under tension, negatively supercoiled, and perhaps exposed to molecules that alter elasticity. Recent experiments quantitatively reveal how much negatively supercoiling DNA lowers the free energy of looping, possibly biasing the operation of genetic switches. [ABSTRACT FROM AUTHOR]

The transcription factor T-bet (Tbox protein expressed in T cells) is one of the master regulators of both the innate and adaptive immune responses. It plays a central role in T-cell lineage commitment, where it controls the TH¹ response, and in gene regulation in plasma B-cells and dendritic cells. T-bet is a member of the Tbox family of transcription factors; however, T-bet coordinately regulates the expression of many more genes than other Tbox proteins. A central unresolved question is how T-bet is able to simultaneously recognize distant Tbox binding sites, which may be located thousands of base pairs away. We have determined the crystal structure of the Tbox DNA binding domain (DBD) of T-bet in complex with a palindromic DNA. The structure shows a quaternary structure in which the T-bet dimer has its DNA binding regions splayed far apart, making it impossible for a single dimer to bind both sites of the DNA palindrome. In contrast to most other Tbox proteins, a single T-bet DBD dimer binds simultaneously to identical half-sites on two independent DNA. A fluorescencebased assay confirms that T-bet dimers are able to bring two independent DNA molecules into close juxtaposition. Furthermore, chromosome conformation capture assays confirm that T-bet functions in the direct formation of chromatin loops in vitro and in vivo. The data are consistent with a looping/synapsing model for transcriptional regulation by T-bet in which a single dimer of the transcription factor can recognize and coalesce distinct genetic elements, either a promoter plus a distant regulatory element, or promoters on two different genes. [ABSTRACT FROM AUTHOR]

Several studies have revealed that natural compounds are valuable resources to develop novel agents against dysregulation of the EGF/EGFR-mediated matrix metalloproteinase-9 (MMP-9) expression in cancer cells. In view of the findings that EGF/EGFR-mediated MMP-9 expression is closely related to invasion and metastasis of breast cancer. To determine the beneficial effects of gallic acid on the suppression of breast cancer metastasis, we explored the effect of gallic acid on MMP-9 expression in EGF-treated MCF-7 breast cancer cells. Treatment with EGF up-regulated MMP-9 mRNA and protein levels in MCF-7 cells. EGF treatment induced phosphorylation of EGFR and elicited Src activation, subsequently promoting Akt/NFκB (p65) and ERK/c-Jun phosphorylation in MCF-7 cells. Activation of Akt/p65 and ERK/c-Jun was responsible for the MMP-9 up-regulation in EGF-treated cells. Gallic acid repressed the EGF-induced activation of EGFR and Src; furthermore, inactivation of Akt/p65 and ERK/c-Jun was a result of the inhibitory effect of gallic acid on the EGF-induced MMP-9 up-regulation. Over-expression of constitutively active Akt and MEK1 or over-expression of constitutively active Src eradicated the inhibitory effect of gallic acid on the EGF-induced MMP-9 up-regulation. A chromosome conformation capture assay showed that EGF induced a chromosomal loop formation in the MMP-9 promoter via NFκB/p65 and AP-1/c-Jun activation. Treatment with gallic acid, EGFR inhibitor, or Src inhibitor reduced DNA looping. Taken together, our data suggest that gallic acid inhibits the activation of EGFR/Src-mediated Akt and ERK, leading to reduced levels of p65/c-Jun-mediated DNA looping and thus inhibiting MMP-9 expression in EGF-treated MCF-7 cells. [ABSTRACT FROM AUTHOR]

DNA cyclization, E. coli lac repressor binding to two spaced lac operators and repression enhancement can be successfully used for a better understanding of the conditions required for interaction between eukaryotic enhancers and the machinery of transcription initiation. Chronologically, the DNA looping model has first accounted for the properties initially defining enhancers, i.e. independence of action with distance or orientation with respect to the start of transcription. It has also predicted enhancer activity or its disruption at short distance (site orientation, alignment between promoter and enhancer sites), with high-order complexes of protein, or with transcription factor concentrations close or different from the w.t. situation. In another step, histones have been introduced into the model to further adapt it to eukaryotes. They in fact favor DNA cyclization in vitro. The resulting DNA compaction might explain the difference counted in base pairs in the distance of action between eukaryotic transcription enhancers and prokaryotic repression enhancers. The lac looping system provides a potential tool for analysis of this discrepancy and of chromatin state directly in situ. Furthermore, as predicted by the model, the contribution of operators O2 and O3 to repression of the lac operon clearly depends on the lac repressor level in the cell and is prevented in strains overproducing lac repressor. By extension, gene regulation especially that linked to cell fate, should also depend on transcription factor levels, providing a potential tool for cellular therapy. In parallel, a new function of the O1-O3 loop completes the picture of lac repression. The O1-O3 loop would at the same time ensure high efficiency of repression, inducibility through the low-affinity sites and limitation of the level of repressor through self-repression of the lac repressor. Last, the DNA looping model can be successfully adapted to the enhancer auxiliary elements known as insulators.

Brown adipose tissue (BAT) plays an essential role in metabolic homeostasis by dissipating energy via thermogenesis through uncoupling protein 1 (UCP1). Previously, we reported that the TATA-binding protein associated factor 7L (TAF7L) is an important regulator of white adipose tissue (WAT) differentiation. In this study, we show that TAF7L also serves as a molecular switch between brown fat and muscle lineages in vivo and in vitro. In adipose tissue, TAF7L-containing TFIID complexes associate with PPARγ to mediate DNA looping between distal enhancers and core promoter elements. Our findings suggest that the presence of the tissue-specific TAF7L subunit in TFIID functions to promote long-range chromatin interactions during BAT lineage specification.

Studying the regulation of transcription of the gal operon that encodes the amphibolic pathway of D-galactose metabolism in Escherichia coli discerned a plethora of principles that operate in prokaryotic gene regulatory processes. In this chapter, we have reviewed some of the more recent findings in gal that continues to reveal unexpected but important mechanistic details. Since the operon is transcribed from two overlapping promoters, P1 and P2, regulated by common regulatory factors, each genetic or biochemical experiment allowed simultaneous discernment of two promoters. Recent studies range from genetic, biochemical through biophysical experiments providing explanations at physiological, mechanistic and single molecule levels. The salient observations highlighted here are: the axiom of determining transcription start points, discovery of a new promoter element different from the known ones that influences promoter strength, occurrence of an intrinsic DNA sequence element that overrides the transcription elongation pause created by a DNA-bound protein roadblock, first observation of a DNA loop and determination its trajectory, and piggybacking proteins and delivering to their DNA target. [ABSTRACT FROM AUTHOR]

The aim of this study is to explore the spatial association of critical genomic elements in the effect of TNF-α on matrix metalloproteinase-9 (MMP-9) expression in human leukemia U937 cells. TNF-α up-regulated MMP-9 protein expression and mRNA level in U937 cells, and Akt-mediated-NFκB/p65 activation and JNK-mediated c-Jun activation were proven to be involved in TNF-α-induced MMP-9 up-regulation. Promoter luciferase activity assay revealed that NFκB (nt-600) and AP-1 (nt-79) binding sites were crucial for TNF-α-induced transcription of MMP-9 gene. The results of a chromatin immunoprecipitation assay indicated that TNF-α reduced histone deacetylase-1 (HDAC-1) recruitment but increased p300 (a histone acetyltransferase) recruitment to MMP-9 promoter regions surrounding NFκB and AP-1 binding sites. Consistently, TNF-α increased enrichment of the acetylated histone H3 mark on MMP-9 promoter regions. DNA affinity purification assay revealed that p300 and HDAC1 could bind oligonucleotides containing AP-1/c-Jun and NFκB/p65 binding sites. Chromosome conformation capture assay showed that TNF-α stimulated chromosomal loops in the MMP-9 promoter via NFκB/p65 and AP-1/c-Jun. The p300-associated acetyltransferase activity was crucial for p65/c-Jun-mediated DNA looping, and inhibition of HDAC activity increased the level of DNA looping. Reduction in the level of DNA looping eliminated all TNF-α-stimulated MMP-9 up-regulation. Taken together, our data suggest that p65/c-Jun-mediated DNA looping is involved in TNF-α-induced MMP-9 up-regulation and that the recruitment of p300 or HDAC1 to NFκB and AP-1 binding sites modifies the level of DNA looping. [ABSTRACT FROM AUTHOR]

Double-stranded DNA is a locally inflexible polymer that resists bending and twisting over hundreds of base pairs. Despite this, tight DNA bending is biologically important for DNA packaging in eukaryotic chromatin and tight DNA looping is important for gene repression in prokaryotes. We and others have previously shown that sequence nonspecific DNA kinking proteins, such as Escherichia coli heat unstable and Saccharomyces cerevisiae non-histone chromosomal protein 6A (Nhp6A), facilitate lac repressor (LacI) repression loops in E. coli. It has been unknown if this facilitation involves direct protein binding to the tightly bent DNA loop or an indirect effect promoting global negative supercoiling of DNA. Here we adapt two high-resolution in vivo protein-mapping techniques to demonstrate direct binding of the heterologous Nhp6A protein at a LacI repression loop in living E. coli cells. [ABSTRACT FROM AUTHOR]

Background: A variety of DNA binding proteins are involved in regulating and shaping the packing of chromatin. They aid the formation of loops in the DNA that function to isolate different structural domains. A recent experimental technique, Hi-C, provides a method for determining the frequency of such looping between all distant parts of the genome. Given that the binding locations of many chromatin associated proteins have also been measured, it has been possible to make estimates for their influence on the long-range interactions as measured by Hi-C. However, a challenge in this analysis is the predominance of non-specific contacts that mask out the specific interactions of interest. Results: We show that transforming the Hi-C contact frequencies into free energies gives a natural method for separating out the distance dependent non-specific interactions. In particular we apply Principal Component Analysis (PCA) to the transformed free energy matrix to identify the dominant modes of interaction. PCA identifies systematic effects as well as high frequency spatial noise in the Hi-C data which can be filtered out. Thus it can be used as a data driven approach for normalizing Hi-C data. We assess this PCA based normalization approach, along with several other normalization schemes, by fitting the transformed Hi-C data using a pairwise interaction model that takes as input the known locations of bound chromatin factors. The result of fitting is a set of predictions for the coupling energies between the various chromatin factors and their effect on the energetics of looping. We show that the quality of the fit can be used as a means to determine how much PCA filtering should be applied to the Hi-C data. Conclusions: We find that the different normalizations of the Hi-C data vary in the quality of fit to the pairwise interaction model. PCA filtering can improve the fit, and the predicted coupling energies lead to biologically meaningful insights for how various chromatin bound factors influence the stability of DNA loops in chromatin. [ABSTRACT FROM AUTHOR]

Low levels of λ repressor (CI) can autoenhance expression and lysogeny (quiescence) of bacteriophage λ infection. However, additional CI mediates an autorepressive DNA loop. Supercoiling catalyzes looping in unstretched plasmids, but whether supercoiling catalyzes looping under tension, and whether the cI promoter within the loop becomes superhelically isolated were investigated. CI efficiently partitioned plasmids into topological domains and formed barriers to the passage of supercoiling from one into another. Furthermore, in single DNA molecules twisted with magnetic tweezers, CI-mediated DNA loops confined as much as -15% to +11% supercoiling. Finally, under gentle tensions likely to occur in vivo, supercoiling was essential for looping. CI may exploit supercoil-driven looping events to regulate the lysogenic/lytic switch. [ABSTRACT FROM AUTHOR]

The authors discuss aspects of the bacteriophage lambda which mediates complex enhancer-like structure formation. They described the work by J. W. Little and C. B. Michalowski which shows that mutation of the integration host factor (IHF) site at OL decreased UP element-mediated PRM lysogenic promoter stimulation. It mentions that the distal OL operator of lambha has the determining properties of a complex enhancer.

The authors discuss the capability of bacteriophage lambda repressors to provide a model for long-range gene regulation, including the action of enhancer elements. They state that the statistical mechanical analysis of the operation of the lambda switch has permitted a quantitative description of the system in terms of binding free energies. They emphasize the potential of proteins recruited to enhancer-promoter complexes to possess enzymatic activity.

Within the nucleus of each cell lies DNA-an unfathomably long, twisted, and intricately coiled molecule-segments of which make up the genes that provide the instructions that a cell needs to operate. As we near the 60th anniversary of the discovery of the DNA double helix, crucial questions remain about how the physical arrangement of the DNA in cells affects how genes work. For example, how a cell stores the genetic information inside the nucleus is complicated by the necessity of maintaining accessibility to DNA for genetic processing. In order to gain insight into the roles played by various proteins in reading and compacting the genome, we have developed new methodologies to simulate the dynamic, three-dimensional structures of long, fluctuating, protein-decorated strands of DNA. Our a priori approach to the problem allows us to determine the effects of individual proteins and their chemical modifications on overall DNA structure and function. Here, we present our recent treatment of the communication between regulatory proteins attached to precisely constructed stretches of chromatin. Our simulations account for the enhancement in communication detected experimentally on chromatin compared to protein-free DNA of the same chain length, as well as the critical roles played by the cationic 'tails' of the histone proteins in this signaling. The states of chromatin captured in the simulations offer new insights into the ways that the DNA, histones, and regulatory proteins contribute to long-range communication along the genome. [ABSTRACT FROM AUTHOR]

Extraordinary single-cell diversity is generated in the vertebrate nervous system by the combinatorial expression of the clustered protocadherin genes (Pcdha, -ß, and -y). This diversity is generated by a combination of stochastic promoter choice and alternative premRNA splicing. Here we show that both the insulator-binding protein CTCF and the cohesin complex subunit Rad21 bind to two highly conserved DNA sequences, the first within and the second downstream of transcriptionally active Pcdha promoters. Both CTCF and Rad21 bind to these sites in vitro and in vivo, this binding directly correlates with alternative isoform expression, and knocking down CTCF expression reduces alternative isoform expression. Remarkably, a similarly spaced pair of CTCF/Rad21 binding sites was identified within a distant enhancer element (HS5-1), which is required for normal levels of alternative isoform expression. We also identify an additional, unique regulatory role for cohesin, as Rad21 binds to another enhancer (HS7) independently of CTCF, and knockdown of Rad21 reduces expression of the constitutive, biallelically expressed Pcdha isoforms ad and ac2. We propose that CTCF and the cohesin complex initiate and maintain Pcdha promoter choice by mediating interactions between Pcdha promoters and enhancers. [ABSTRACT FROM AUTHOR]

Abstract: Nuclear receptors (NRs) are key transcriptional regulators of metazoan physiology and metabolism. Different NRs bind to similar or even identical core response elements; however, they regulate transcription in a highly receptor- and gene-specific manner. These differences in gene activation can most likely be accounted for by mechanisms involving receptor-specific interactions with DNA as well as receptor-specific interactions with protein complexes binding to adjacent and distant DNA sequences. Here, we review key molecular aspects of transactivation by NRs with special emphasis on the recent advances in the molecular mechanisms responsible for receptor- and gene-specific transcriptional activation. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease. [Copyright &y& Elsevier]

We examined how remote enhancers establish physical communication with target promoters to activate gene transcription in response to environmental signals. Although the natural IFN-β enhancer is located immediately upstream of the core promoter, it also can function as a classical enhancer element conferring virus infection-dependent activation of heterologous promoters, even when it is placed several kilobases away from these promoters. We demonstrated that the remote IFN-β enhancer "loops out" the intervening DNA to reach the target promoter. These chromatin loops depend on sequence-specific transcription factors bound to the enhancer and the promoter and thus can explain the specificity observed in enhancer-promoter interactions, especially in complex genetic loci. Transcription factor binding sites scattered between an enhancer and a promoter can work as decoys trapping the enhancer in nonproductive loops, thus resembling insulator elements. Finally, replacement of the transcription factor binding sites involved in DNA looping with those of a heterologous prokaryotic protein, the λ repressor, which is capable of loop formation, rescues enhancer function from a distance by re-establishing enhancer-promoter loop formation. [ABSTRACT FROM AUTHOR]

Barrier-to-autointegration factor (BAF) is a protein that has been proposed to compact retroviral DNA, making it inaccessible as a target for self-destructive integration into itself (autointegration). BAF also plays an important role in nuclear organization. We studied the mechanism of DNA condensation by BAF using total internal reflection fluorescence microscopy. We found that BAF compacts DNA by a looping mechanism. Dissociation of BAF from DNA occurs with multiphasic kinetics; an initial fast phase is followed by a much slower dissociation phase. The mechanistic basis of the broad timescale of dissociation is discussed. This behavior mimics the dissociation of BAF from retroviral DNA within preintegration complexes as monitored by functional assays. Thus the DNA binding properties of BAF may alone be sufficient to account for its association with the preintegration complex. [ABSTRACT FROM AUTHOR]

Abstract: DNA molecules may form loops in response to binding with regulatory proteins that control the expression of genes. While DNA looping is a widely accepted gene regulatory mechanism, basic questions regarding the mechanics of the looping process remain open. The present paper contributes a computational rod model that accounts for non-homogeneous and discontinuous changes in stiffness to support the analysis of DNA looping. We pursue this objective in two steps. First, we illustrate the effects of non-homogeneous stiffness on the looping of generic rods under pure torsion. Results computed for this idealized case support our intuition that elastic deformation and strain energy localize in ‘soft’ regions and that equilibrium bifurcations are sensitive to non-homogenous stiffness. Next, we extend the formulation to describe the combined bending, torsion and compression induced on DNA by the LacR protein. We demonstrate that while moderate stiffness variations have only modest influence on LacR-mediated DNA looping, highly localized softening (e.g., ‘kinkable’ or ‘melted’ subdomains) may substantially reduce the energetic cost of looping and profoundly affect loop geometry. [Copyright &y& Elsevier]

EcoP15I is a type III restriction enzyme that requires two recognition sites in a defined orientation separated by up to 3.5 kbp to efficiently cleave DNA. The mechanism through which site-bound EcoP15I enzymes communicate between the two sites is unclear. Here, we use atomic force microscopy to study EcoP15I–DNA pre-cleavage complexes. From the number and size distribution of loops formed, we conclude that the loops observed do not result from translocation, but are instead formed by a contact between site-bound EcoP15I and a nonspecific region of DNA. This conclusion is confirmed by a theoretical polymer model. It is further shown that translocation must play some role, because when translocation is blocked by a Lac repressor protein, DNA cleavage is similarly blocked. On the basis of these results, we present a model for restriction by type III restriction enzymes and highlight the similarities between this and other classes of restriction enzymes. [ABSTRACT FROM AUTHOR]

The goal of systems biology is to understand the behavior of the whole in terms of knowledge of the parts. This is hard to achieve in many cases due to the difficulty of characterizing the many constituents involved in a biological system and their complex web of interactions. The lac promoter of Escherichia coil offers the possibility of confronting ‘system-level’ properties of transcriptional regulation with the known biochemistry of the molecular constituents and their mutual interactions. Such confrontations can reveal previously unknown constituents and interactions, as well as offer insight into how the components work together as a whole. Here we study the combinatorial control of the lac promoter by the regulators Lac repressor (LacR) and cAMP-receptor protein (CRP). A previous in vivo study [Setty Y, Mayo AE, Surette MG, Alon U (2003) Proc Natl Acad Sci USA 100:7702–7707] found gross disagreement between the observed promoter activities and the expected behavior based on the known molecular mechanisms. We repeated the study by identifying and removing several extraneous factors that significantly modulated the expression of the lac promoter. Through quantitative, systematic characterization of promoter activity for a number of key mutants and guided by the thermodynamic model of transcriptional regulation, we were able to account for the combinatorial control of the lac promoter quantitatively, in terms of a cooperative interaction between CRP and LacR-mediated DNA looping. Specifically, our analysis indicates that the sensitivity of the inducer response results from LacR-mediated DNA looping, which is significantly enhanced by CRP. [ABSTRACT FROM AUTHOR]

DNA looping occurs in many important protein-DNA interactions, including those regulating replication, transcription, and recombination. Recent theoretical studies predict that tension of only a few piconewtons acting on DNA would almost completely inhibit DNA looping. Here, we study restriction endnucleases that require interaction at two separated sites for efficient cleavage. Using optical tweezers we measured the dependence of cleavage activity on DNA tension with 15 known or suspected two-site enzymes (Bfil, Bpml, Bsgl, BspMl, Cfr9l, Cfr10l, Eco57l, EcoRll, Fokl, Hpall, MboIl, Nan, Sacll, Sau3Al, and SgrAI) and six one-site enzymes (BamHl, EcoRl, EcoRV, HaeIII, Hindlll, and DNasel). All of the one-site enzymes were virtually unaffected by 5 pN of tension, whereas all of the two-site enzymes were completely inhibited. These enzymes thus constitute a remarkable example of a tension sensing "molecular switch." A detailed study of one enzyme, Sau3Al, indicated that the activity decreased exponentially with tension and the decrease was ≈10-fold 0.7 pN. At higher forces (≈20-40 pN) cleavage by the one-site enzymes EcoRV and Haelll was partly inhibited and cleavage by Hindlll was enhanced, whereas BamHl, EcoRl, and DNasel were largely unaffected. These findings correlate with structural data Showing that EcoRV bends DNA sharply, whereas BamHl, EcoRl, and DNaseI do not. Thus, DNA-directed enzyme activity involving either DNA looping or bending can be modulated by tension, a mechanism that could facilitate mechanosensory transduction in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Tuc2009 is a temperate bacteriophage of Lactococcus lactis subsp. cremoris UC509 which encodes a CI- and Cro-type lysogenic–lytic switch region. A helix-swap of the α3 helices of the closely related CI-type proteins from the lactococcal phages r1t and Tuc2009 revealed the crucial elements involved in DNA recognition while also pointing to conserved functional properties of phage CI proteins infecting different hosts. CI-type proteins have been shown to bind to specific sequences located in the intergenic switch region, but to date, no detailed binding studies have been performed on lactococcal Cro analogues. Experiments shown here demonstrate alternative binding sites for these two proteins of Tuc2009. CI2009 binds to three inverted repeats, two within the intergenic region and one within the cro 2009 gene. This DNA-binding pattern appears to be conserved among repressors of lactococcal and streptococcal phages. The Cro2009 protein appears to bind to three direct repeats within the intergenic region causing distortion of the bound DNA. [Copyright &y& Elsevier]

The free energy of looping DNA by proteins and protein complexes determines to what extent distal DNA sites can affect each other. We inferred its in vivo value through a combined computational-experimental approach for different lengths of the loop and found that, in addition to the intrinsic periodicity of the DNA double helix, the free energy has an oscillatory component of about half the helical period. Moreover, the oscillations have such an amplitude that the effects of regulatory molecules become strongly dependent on their precise DNA positioning and yet easily tunable by their cooperative interactions. These unexpected results can confer to the physical properties of DNA a more prominent role at shaping the properties of gene regulation than previously thought. [ABSTRACT FROM AUTHOR]