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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., Ministry of Science and Innovation of the Spanish Government (PID2019 108778GB C21 (AEI/FEDER, EU)) to WM, (BIO2016-77883-C2-2-P and FIS2015-72574-EXP (AEI/FEDER, EU)), to DB, (PID2019-104544GBI00 (AEI/FEDER/EU)) to CA, (PID2019-109320GB-100/AEI/10.13039/501100011033) to SA, (PGC2018-094143-B-I00, (AEI/FEDER, EU)) to FR, and (FIS2016-78313-P) to SA. The Ministry of Science and Innovation of the Spanish Government also funded the Centro Nacional de Biotecnología (CNB) by means of the “Severo Ochoa” Center of Excellence grant SEV 2017-0712. A Wellcome Investigator grant (209500) to Jeff Errington supported LW. Institutional grants from the ‘Fundación Ramón Areces’ and ‘Banco de Santander’ supported

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 A 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.

SummaryWe 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.

Significance We report here that a recently developed Bayesian network (BN) methodology and software platform yield useful information when applied to the analysis of intrachromosomal interaction datasets combined with Encyclopedia of DNA Elements publicly available datasets for the B-lymphocyte cell line GM12878. Of 106 variables analyzed, interaction strength between DNA segments was found to be directly dependent on only four types of variables: distance, Rad21 or SMC3 (cohesin components), transcription at transcription start sites, and the number of CCCTC-binding factor (CTCF)–cohesin complexes between interacting DNA segments. The importance of directionally oriented ctcf motifs was confirmed not only for loops but also for enhancer–promoter interactions. Purely data-driven BN analyses also identified known critical, lineage-determining transcription factors (TFs) as well as some potentially new dependencies between TFs., 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.

Transcriptional regulation has a critical role in the coordinate and context-specific expression of a cluster of genes encoding members of the tumour necrosis factor (TNF) superfamily found at chromosome 6p21, comprising TNF, LTA (encoding lymphotoxin-α) and LTB (encoding lymphotoxin-Β). This is important, as dysregulated expression of these genes is implicated in susceptibility to many autoimmune, inflammatory and infectious diseases. We describe here a novel regulatory element in the fourth exon of LTB, which is highly conserved, localises to the only CpG island in the locus, and is associated with a DNase I hypersensitive site and specific histone modifications. We find evidence of binding by Yin Yang 1 (YY1), cyclic AMP response element (CRE)-binding protein (CREB) and CCCTC-binding factor (CTCF) to this region in Jurkat T cells, which is associated with transcriptional repression on reporter gene analysis. Chromatin conformation capture experiments show evidence of DNA looping, involving interaction of this element with the LTB promoter, LTA promoter and TNF 3′ untranslated region (UTR). Small interfering RNA (siRNA) experiments demonstrate a functional role for YY1 and CREB in LTB expression. Our findings provide evidence of additional complexity in the transcriptional regulation of LTB with implications for coordinate expression of genes in this important genomic locus. © 2011 Macmillan Publishers Limited All rights reserved.

Most restriction endonucleases bridge two target sites before cleaving DNA: examples include all of the translocating Type I and Type III systems, and many Type II nucleases acting at their sites. A subset of Type II enzymes, the IIB systems, recognise bipartite sequences, like Type I sites, but cut specified phosphodiester bonds near their sites, like Type IIS enzymes. However, they make two double-strand breaks, one either side of the site, to release the recognition sequence on a short DNA fragment; 34 bp long in the case of the archetype, BcgI. It has been suggested that BcgI needs to interact with two recognition sites to cleave DNA but whether this is a general requirement for Type IIB enzymes had yet to be established. Ten Type IIB nucleases were tested against DNA substrates with one or two copies of the requisite sequences. With one exception, they all bridged two sites before cutting the DNA, usually in concerted reactions at both sites. The sites were ideally positioned in cis rather than in trans and were bridged through 3-D space, like Type II enzymes, rather than along the 1-D contour of the DNA, as seen with Type I enzymes. The standard mode of action for the restriction enzymes that excise their recognition sites from DNA thus involves concurrent action at two DNA sites.

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 0 helices of the closely related Cl-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 Cl 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 Cro(2009) protein appears to bind to three direct repeats within the intergenic region causing distortion of the bound DNA. (c) 2005 Elsevier Inc. All rights reserved.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12859-015-0584-2) contains supplementary material, which is available to authorized users.

Deoxyribonucleic acid cyclization, Escherichia 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 wild-type 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.

The looping of DNA provides a means of communication between sequentially distant genomic sites that operate in tandem to express, copy, and repair the information encoded in the DNA base sequence. The short loops implicated in the expression of bacterial genes suggest that molecular factors other than the naturally stiff double helix are involved in bringing the interacting sites into close spatial proximity. New computational techniques that take direct account of the three-dimensional structures and fluctuations of protein and DNA allow us to examine the likely means of enhancing such communication. Here, we describe the application of these approaches to the looping of a 92 base-pair DNA segment between the headpieces of the tetrameric Escherichia coli Lac repressor protein. The distortions of the double helix induced by a second protein--the nonspecific nucleoid protein HU--increase the computed likelihood of looping by several orders of magnitude over that of DNA alone. Large-scale deformations of the repressor, sequence-dependent features in the DNA loop, and deformability of the DNA operators also enhance looping, although to lesser degrees. The correspondence between the predicted looping propensities and the ease of looping derived from gene-expression and single-molecule measurements lends credence to the derived structural picture.

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. DOI: http://dx.doi.org/10.7554/eLife.02811.001, eLife digest Mammals produce two distinct types of adipose tissue: white adipose tissue (white fat) is the more common type and is used to store energy; brown adipose tissue (brown fat) is mostly found in young animals and infants, and it plays an important role in dissipating energy as heat rather than storing it in fat for future use. In adults, higher levels of brown fat are associated with lower levels of fat overall, so there is considerable interest in learning more about this form of fat to help address rising levels of obesity in the world. Building on previous work in which they showed that a gene control protein called TAF7L has a central role in the development of the cells that make up white adipose tissue, Zhou et al. now show that this protein also helps to regulate the development of brown adipose tissue. Mice lacking the gene for this protein developed embryos with 40% less brown fat than wild-type mice with the gene. Moreover, these mice developed muscle-like cells in the regions that should have contained brown fat. Gene expression analysis revealed that ‘knocking out’ the gene for TAF7L changed the expression of more than a thousand genes in these mice. Zhou et al. suggest that TAF7L works as a ‘molecular switch’ that determines whether certain precursor cells (called mesenchymal stem cells) go on to become brown fat cells or muscle cells. A future challenge will be to devise interventions to regulate the activity or levels of TAF7L as a potential means of modulating brown fat depots in animals and humans. DOI: http://dx.doi.org/10.7554/eLife.02811.002

ATP-dependent chromatin remodeling enzymes are highly abundant and play pivotal roles regulating DNA-dependent processes. The mechanisms by which they are targeted to specific loci have not been well understood on a genome-wide scale. Here we present evidence that a major targeting mechanism for the Isw2 chromatin remodeling enzyme to specific genomic loci is through sequence-specific transcription factor (TF)-dependent recruitment. Unexpectedly, Isw2 is recruited in a TF-dependent fashion to a large number of loci without TF binding sites. Using the 3C assay, we show that Isw2 can be targeted by Ume6- and TFIIB-dependent DNA looping. These results identify DNA looping as a previously unknown mechanism for the recruitment of a chromatin remodeling enzyme and defines a novel function for DNA looping. We also present evidence suggesting that Ume6-dependent DNA looping is involved in chromatin remodeling and transcriptional repression, revealing a mechanism by which the three-dimensional folding of chromatin affects DNA-dependent processes.

"Growing evidence supports the concept that dynamic intra-and inter-chromosomal links between specific loci contribute to the creation of cell type-specific gene expression profiles. Therefore, analysis of the establishment of peculiar functional correlations between sites, also distant on linear DNA, that govern the transcriptional process appears to be of fundamental relevance. We propose here an experimental approach showing that 17 beta-estradiol-induced transcription associates to formation of loops between the promoter and termination regions of hormone-responsive genes. This strategy reveals as a tool to be also suitably used, in conjunction with automated techniques, for an extensive analysis of sites shared by multiple genes for induced expression."

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.

Snf2 related chromatin remodelling enzymes possess an ATPase subunit similar to that of the SF-II helicases which hydrolyzes ATP to track along DNA. Translocation and any resulting torque in the DNA could drive chromatin remodeling. To determine whether the ISWI protein can translocate and generate torque, tethered particle motion experiments and atomic force microscopy have been performed using recombinant ISWI expressed in E. coli. In the absence of ATP, ISWI bound to and wrapped DNA thereby shortening the overall contour length measured in atomic force micrographs. Although naked DNA only weakly stimulates ATP hydrolysis by ISWI, both atomic force microscopy and tethered particle motion data indicate that the protein generated loops in the presence of ATP. The duration of the looped state of the DNA measured using tethered particle motion was ATP-dependent. Finally, ISWI relaxed positively supercoiled plasmids visualized by atomic force microscopy. While other chromatin remodeling ATPases catalyze either DNA wrapping or looping, both are catalyzed by ISWI.