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Diarrhoeal disease caused by Cryptosporidium is a major cause of morbidity and mortality in young and malnourished children from low- and middle-income countries, with no vaccine or effective treatment. Here we describe the discovery of EDI048, a Cryptosporidium PI(4)K inhibitor, designed to be active at the infection site in the gastrointestinal tract and undergo rapid metabolism in the liver. By using mutational analysis and crystal structure, we show that EDI048 binds to highly conserved amino acid residues in the ATP-binding site. EDI048 is orally efficacious in an immunocompromised mouse model despite negligible circulating concentrations, thus demonstrating that gastrointestinal exposure is necessary and sufficient for efficacy. In neonatal calves, a clinical model of cryptosporidiosis, EDI048 treatment resulted in rapid resolution of diarrhoea and significant reduction in faecal oocyst shedding. Safety and pharmacological studies demonstrated predictable metabolism and low systemic exposure of EDI048, providing a substantial safety margin required for a paediatric indication. EDI048 is a promising clinical candidate for the treatment of life-threatening paediatric cryptosporidiosis., (© 2024. The Author(s).)

The PML::RARA fusion protein is the hallmark driver of Acute Promyelocytic Leukemia (APL) and disrupts retinoic acid signaling, leading to wide-scale gene expression changes and uncontrolled proliferation of myeloid precursor cells. While known to be recruited to binding sites across the genome, its impact on gene regulation and expression is under-explored. Using integrated multi-omics datasets, we characterize the influence of PML::RARA binding on gene expression and regulation in an inducible PML::RARA cell line model and APL patient ex vivo samples. We find that genes whose regulatory elements recruit PML::RARA are not uniformly transcriptionally repressed, as commonly suggested, but also may be upregulated or remain unchanged. We develop a computational machine learning implementation called Regulatory Element Behavior Extraction Learning to deconvolute the complex, local transcription factor binding site environment at PML::RARA bound positions to reveal distinct signatures that modulate how PML::RARA directs the transcriptional response., (© 2023. The Author(s).)

CUO246, a novel DNA gyrase/topoisomerase IV inhibitor, is active in vitro against a broad range of Gram-positive, fastidious Gram-negative, and atypical bacterial pathogens and retains activity against quinolone-resistant strains in circulation. The frequency of selection for single step mutants of wild-type S. aureus with reduced susceptibility to CUO246 was <4.64 × 10 -9 at 4× and 8× MIC and remained low when using an isogenic QRDR mutant (<5.24 × 10 -9 at 4× and 8× MIC). Biochemical assays indicated that CUO246 had potent inhibitory activity against both DNA gyrase (GyrAB) and topoisomerase IV (ParCE). Furthermore, CUO246 showed rapid bactericidal activity in time-kill assays and potent in vivo efficacy against S. aureus in a neutropenic murine thigh infection model. These results suggest that CUO246 may be useful in treating infections by various causative agents of acute skin and skin structure infections, respiratory tract infections, and sexually transmitted infections.

It is now evident that DNA forms an organized nuclear architecture, which is essential to maintain the structural and functional integrity of the genome. Chromatin organization can be systematically studied due to the recent boom in chromosome conformation capture technologies (e.g., 3C and its successors 4C, 5C and Hi-C), which is accompanied by the development of computational pipelines to identify biologically meaningful chromatin contacts in such data. However, not all tools are applicable to all experimental designs and all structural features. Capture Hi-C (CHi-C) is a method that uses an intermediate hybridization step to target and select predefined regions of interest in a Hi-C library, thereby increasing effective sequencing depth for those regions. It allows researchers to investigate fine chromatin structures at high resolution, for instance promoter-enhancer loops, but it introduces additional biases with the capture step, and therefore requires specialized pipelines. Here, we compare multiple analytical pipelines for CHi-C data analysis. We consider the effect of retaining multi-mapping reads and compare the efficiency of different statistical approaches in both identifying reproducible interactions and determining biologically significant interactions. At restriction fragment level resolution, the number of multi-mapping reads that could be rescued was negligible. The number of identified interactions varied widely, depending on the analytical method, indicating large differences in type I and type II error rates. The optimal pipeline depends on the project-specific tolerance level of false positive and false negative chromatin contacts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Aljogol, Thompson, Osborne and Mifsud.)

Altered transcription is a cardinal feature of acute myeloid leukemia (AML); however, exactly how mutations synergize to remodel the epigenetic landscape and rewire three-dimensional DNA topology is unknown. Here, we apply an integrated genomic approach to a murine allelic series that models the two most common mutations in AML: Flt3-ITD and Npm1c. We then deconvolute the contribution of each mutation to alterations of the epigenetic landscape and genome organization, and infer how mutations synergize in the induction of AML. Our studies demonstrate that Flt3-ITD signals to chromatin to alter the epigenetic environment and synergizes with mutations in Npm1c to alter gene expression and drive leukemia induction. These analyses also allow the identification of long-range cis-regulatory circuits, including a previously unknown superenhancer of Hoxa locus, as well as larger and more detailed gene-regulatory networks, driven by transcription factors including PU.1 and IRF8, whose importance we demonstrate through perturbation of network members., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Objectives: The neutropenic murine thigh infection model was used to assess the effectiveness of IID572, a novel β-lactamase inhibitor, in rescuing piperacillin activity against bacterial strains expressing various β-lactamase enzymes., Methods: Mice (n = 4/group) were inoculated with Enterobacteriaceae or Staphylococcus aureus bacterial strains expressing a range of β-lactamases via intramuscular injection. Two hours after bacterial inoculation, subcutaneous treatment with piperacillin/IID572 or piperacillin/tazobactam every 3 h was initiated. Animals were euthanized via CO2 24 h after the start of therapy and bacterial cfu (log10 cfu) per thigh was determined, and the static dose was calculated., Results: In a dose-dependent manner, piperacillin/IID572 reduced the thigh bacterial burden in models established with Enterobacteriaceae producing class A, C and D β-lactamases (e.g. ESBLs, KPC, CMY-2 and OXA-48). Piperacillin/IID572 was also efficacious against MSSA strains, including one producing β-lactamase. Static doses of piperacillin/IID572 were calculable from animals infected with all strains tested and the calculated static doses ranged from 195 to 4612 mg/kg/day piperacillin, the active component in the combination. Of the 13 strains investigated, a 1 log10 bacterial reduction was achieved for 9 isolates and a 2 log10 reduction was achieved for 3 isolates; piperacillin/tazobactam was not efficacious against 6 of the 13 isolates tested., Conclusions: In contrast to tazobactam, IID572 was able to rescue piperacillin efficacy in murine thigh infection models established with β-lactamase-producing strains of Enterobacteriaceae and S. aureus, including those expressing ESBLs or serine carbapenemases., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Cohesin complex disruption alters gene expression, and cohesin mutations are common in myeloid neoplasia, suggesting a critical role in hematopoiesis. Here, we explore cohesin dynamics and regulation of hematopoietic stem cell homeostasis and differentiation. Cohesin binding increases at active regulatory elements only during erythroid differentiation. Prior binding of the repressive Ets transcription factor Etv6 predicts cohesin binding at these elements and Etv6 interacts with cohesin at chromatin. Depletion of cohesin severely impairs erythroid differentiation, particularly at Etv6-prebound loci, but augments self-renewal programs. Together with corroborative findings in acute myeloid leukemia and myelodysplastic syndrome patient samples, these data suggest cohesin-mediated alleviation of Etv6 repression is required for dynamic expression at critical erythroid genes during differentiation and how this may be perturbed in myeloid malignancies., (© 2019 by The American Society of Hematology.)

Transcriptome analyses show a surprisingly large proportion of the mammalian genome is transcribed; much more than can be accounted for by genes and introns alone. Most of this transcription is non-coding in nature and arises from intergenic regions, often overlapping known protein-coding genes in sense or antisense orientation. The functional relevance of this widespread transcription is unknown. Here we characterize a promoter responsible for initiation of an intergenic transcript located approximately 3.3 kb and 10.7 kb upstream of the adult-specific human β-globin genes. Mutational analyses in β-YAC transgenic mice show that alteration of intergenic promoter activity results in ablation of H3K4 di- and tri-methylation and H3 hyperacetylation extending over a 30 kb region immediately downstream of the initiation site, containing the adult δ- and β-globin genes. This results in dramatically decreased expression of the adult genes through position effect variegation in which the vast majority of definitive erythroid cells harbor inactive adult globin genes. In contrast, expression of the neighboring ε- and γ-globin genes is completely normal in embryonic erythroid cells, indicating a developmentally specific variegation of the adult domain. Our results demonstrate a role for intergenic non-coding RNA transcription in the propagation of histone modifications over chromatin domains and epigenetic control of β-like globin gene transcription during development., Competing Interests: The authors have declared that no competing interests exist.

Kratom ( Mitragyna speciosa) is a prevalent medicinal plant used mainly for the stimulant and analgesic properties provided through multiple alkaloid compounds. Over the past decade, use of kratom has increased despite the limited knowledge of toxicities and adverse side effects. With the current opioid epidemic, both patients and providers are seeking alternative methods to treat both addiction and pain control, and kratom as an alternative means of treatment has increasingly entered the mainstream. In this article, we present the clinical course of a 47-year-old male who developed fatigue, pruritus, and abnormal liver tests (with a mixed hepatocellular/cholestatic pattern) approximately 21 days after beginning kratom. After extensive evaluation including a negligible alcohol history, negative hepatitis serologies, and inconclusive imaging, the patient was diagnosed with drug-induced liver injury (DILI) caused by kratom. Nine months after his liver tests returned to normal, he took kratom again, and after a latency of 2 days, he developed fatigue, pruritus, and loss of appetite along with abnormal liver tests (with the same biochemical profile as previously), consistent with a positive rechallenge. We believe, through the use of the Roussel-Uclaf Causality Assessment Method and expert opinion, that this is a highly likely or definite example of kratom-induced DILI. With the gaining popularity of this drug, it appears that DILI may be an important complication of kratom for providers to recognize.

Efforts are being directed to systematically analyze the non-coding regions of the genome for cancer-driving mutations 1-6 . cis-regulatory elements (CREs) represent a highly enriched subset of the non-coding regions of the genome in which to search for such mutations. Here we use high-throughput chromosome conformation capture techniques (Hi-C) for 19,023 promoter fragments to catalog the regulatory landscape of colorectal cancer in cell lines, mapping CREs and integrating these with whole-genome sequence and expression data from The Cancer Genome Atlas 7,8 . We identify a recurrently mutated CRE interacting with the ETV1 promoter affecting gene expression. ETV1 expression influences cell viability and is associated with patient survival. We further refine our understanding of the regulatory effects of copy-number variations, showing that RASL11A is targeted by a previously identified enhancer amplification 1 . This study reveals new insights into the complex genetic alterations driving tumor development, providing a paradigm for employing chromosome conformation capture to decipher non-coding CREs relevant to cancer biology.

Purpose: Influenza viruses are a common cause of human respiratory infections, resulting in epidemics of high morbidity and mortality. We investigated the effect of a novel mitogen-activated protein kinase (MAPK) inhibitor in vitro and in a murine influenza model to further explore whether p38 MAPK inhibition could reduce viral replication., Methods: In vitro, the antiviral effect of p38 MAPK inhibitor BCT194 was evaluated in differentiated human bronchial epithelial cells (HBECs); in vivo, female BALB/c mice were infected intranasally with 150 pfu of influenza H1N1 A/Puerto Rico/8/34 and treated with BCT197 (a closely related p38 MAPK inhibitor with an IC50 value of<1 µM, currently in clinical testing), dexamethasone or oseltamivir (Tamiflu) starting 24 h post infection. Body weight, bronchoalveolar lavage cells, cytokines, total protein and lactate dehydrogenase as well as serum cytokines were measured; a subset of animals was evaluated histopathologically.Results/Key findings. p38MAP kinase inhibition with BCT194 had no impact on influenza replication in HBECs. When examining BCT197 in vivo, and comparing to vehicle-treated animals, reduced weight loss, improvement in survival and lack of impaired viral control was observed at BCT197 concentrations relevant to those being used in clinical trials of acute exacerbations of chronic obstructive pulmonary disease; at higher concentrations of BCT197 these effects were reduced., Conclusions: Compared to vehicle treatment, BCT197 (administered at a clinically relevant concentration) improved outcomes in a mouse model of influenza. This is encouraging given that the use of innate inflammatory pathway inhibitors may raise concerns of negative effects on infection regulation.

Long-range cis- regulatory elements such as enhancers coordinate cell-specific transcriptional programmes by engaging in DNA looping interactions with target promoters. Deciphering the interplay between the promoter connectivity and activity of cis- regulatory elements during lineage commitment is crucial for understanding developmental transcriptional control. Here, we use Promoter Capture Hi-C to generate a high-resolution atlas of chromosomal interactions involving ~22,000 gene promoters in human pluripotent and lineage-committed cells, identifying putative target genes for known and predicted enhancer elements. We reveal extensive dynamics of cis -regulatory contacts upon lineage commitment, including the acquisition and loss of promoter interactions. This spatial rewiring occurs preferentially with predicted changes in the activity of cis- regulatory elements and is associated with changes in target gene expression. Our results provide a global and integrated view of promoter interactome dynamics during lineage commitment of human pluripotent cells.

There is a strong interrelationship within the cell nucleus between form and function of the genome. This connection is exhibited across multiple hierarchies, ranging from grand-scale positioning of chromosomes and their intersection with specific nuclear functional activities, the segregation of chromosome structure into distinct domains and long-range regulatory contacts that drive spatial and temporal expression patterns of genes. Fifteen years ago, the development of the chromosome conformation capture method placed the nature of specific, long-range regulatory interactions under scrutiny. However, its development and integration with next-generation sequencing technologies has greatly expanded the breadth and scope of what is detected. The sheer scale of data offered by these important advances has come with new and challenging bottlenecks that are both experimental and bioinformatical. Here, we discuss the recent and prospective development and implementation of new methodologies and analytical tools that are allowing an in-depth, yet focussed characterisation of genomic contacts that are associated with functional activities in the nucleus.

Gender dysphoria (GD), a feeling of persistent discomfort with one's biologic sex or assigned gender, is estimated to be more prevalent in male prison inmates than in nonincarcerated males; there may be 3000-4000 male inmates with GD in prisons in the United States. An increasing number of U.S. prison systems now offer gender dysphoric inmates diagnostic evaluation, psychotherapy, cross-sex hormone therapy, and opportunities, albeit limited, to enact their preferred gender role. Sex reassignment surgery (SRS), however, has not been offered to inmates except in response to litigation. In the first case of its kind, the California Department of Corrections and Rehabilitation recently agreed to provide SRS to an inmate and developed policy guidelines for its future provision. In other recent cases, U.S. courts have ruled that male inmates with GD are entitled to SRS when it is medically necessary. Although these decisions may facilitate the provision of SRS to inmates in the future, many U.S. prison systems will probably remain reluctant to offer SRS unless legally compelled to do so. In this review, we address the medical necessity of SRS for male inmates with GD. We also discuss eligibility criteria and the practical considerations involved in providing SRS to inmates. We conclude by offering recommendations for physicians, mental health professionals, and prison administrators, designed to facilitate provision of SRS to inmates with GD in a manner that provides humane treatment, maximizes the likelihood of successful outcomes, minimizes risk of regret, and generates data that can help inform future decisions.

Lymphomagenesis in the presence of deregulated MYC requires suppression of MYC-driven apoptosis, often through downregulation of the pro-apoptotic BCL2L11 gene (Bim). Transcription factors (EBNAs) encoded by the lymphoma-associated Epstein-Barr virus (EBV) activate MYC and silence BCL2L11. We show that the EBNA2 transactivator activates multiple MYC enhancers and reconfigures the MYC locus to increase upstream and decrease downstream enhancer-promoter interactions. EBNA2 recruits the BRG1 ATPase of the SWI/SNF remodeller to MYC enhancers and BRG1 is required for enhancer-promoter interactions in EBV-infected cells. At BCL2L11, we identify a haematopoietic enhancer hub that is inactivated by the EBV repressors EBNA3A and EBNA3C through recruitment of the H3K27 methyltransferase EZH2. Reversal of enhancer inactivation using an EZH2 inhibitor upregulates BCL2L11 and induces apoptosis. EBV therefore drives lymphomagenesis by hijacking long-range enhancer hubs and specific cellular co-factors. EBV-driven MYC enhancer activation may contribute to the genesis and localisation of MYC-Immunoglobulin translocation breakpoints in Burkitt's lymphoma., Competing Interests: The authors declare that no competing interests exist.

The Polycomb repressive complexes PRC1 and PRC2 maintain embryonic stem cell (ESC) pluripotency by silencing lineage-specifying developmental regulator genes. Emerging evidence suggests that Polycomb complexes act through controlling spatial genome organization. We show that PRC1 functions as a master regulator of mouse ESC genome architecture by organizing genes in three-dimensional interaction networks. The strongest spatial network is composed of the four Hox gene clusters and early developmental transcription factor genes, the majority of which contact poised enhancers. Removal of Polycomb repression leads to disruption of promoter-promoter contacts in the Hox gene network. In contrast, promoter-enhancer contacts are maintained in the absence of Polycomb repression, with accompanying widespread acquisition of active chromatin signatures at network enhancers and pronounced transcriptional upregulation of network genes. Thus, PRC1 physically constrains developmental transcription factor genes and their enhancers in a silenced but poised spatial network. We propose that the selective release of genes from this spatial network underlies cell fate specification during early embryonic development.

Transcriptional control in large genomes often requires looping interactions between distal DNA elements, such as enhancers and target promoters. Current chromosome conformation capture techniques do not offer sufficiently high resolution to interrogate these regulatory interactions on a genomic scale. Here we use Capture Hi-C (CHi-C), an adapted genome conformation assay, to examine the long-range interactions of almost 22,000 promoters in 2 human blood cell types. We identify over 1.6 million shared and cell type-restricted interactions spanning hundreds of kilobases between promoters and distal loci. Transcriptionally active genes contact enhancer-like elements, whereas transcriptionally inactive genes interact with previously uncharacterized elements marked by repressive features that may act as long-range silencers. Finally, we show that interacting loci are enriched for disease-associated SNPs, suggesting how distal mutations may disrupt the regulation of relevant genes. This study provides new insights and accessible tools to dissect the regulatory interactions that underlie normal and aberrant gene regulation.

The mammalian genome harbors up to one million regulatory elements often located at great distances from their target genes. Long-range elements control genes through physical contact with promoters and can be recognized by the presence of specific histone modifications and transcription factor binding. Linking regulatory elements to specific promoters genome-wide is currently impeded by the limited resolution of high-throughput chromatin interaction assays. Here we apply a sequence capture approach to enrich Hi-C libraries for >22,000 annotated mouse promoters to identify statistically significant, long-range interactions at restriction fragment resolution, assigning long-range interacting elements to their target genes genome-wide in embryonic stem cells and fetal liver cells. The distal sites contacting active genes are enriched in active histone modifications and transcription factor occupancy, whereas inactive genes contact distal sites with repressive histone marks, demonstrating the regulatory potential of the distal elements identified. Furthermore, we find that coregulated genes cluster nonrandomly in spatial interaction networks correlated with their biological function and expression level. Interestingly, we find the strongest gene clustering in ES cells between transcription factor genes that control key developmental processes in embryogenesis. The results provide the first genome-wide catalog linking gene promoters to their long-range interacting elements and highlight the complex spatial regulatory circuitry controlling mammalian gene expression., (© 2015 Schoenfelder et al.; Published by Cold Spring Harbor Laboratory Press.)

The mammalian nucleus is a highly complex structure that carries out a diverse range of functions such as DNA replication, cell division, RNA processing, and nuclear export/import. Many of these activities occur at discrete subcompartments that intersect with specific regions of the genome. Over the past few decades, evidence has accumulated to suggest that RNA transcription also occurs in specialized sites, called transcription factories, that may influence how the genome is organized. There may be certain efficiency benefits to cluster transcriptional activity in this way. However, the clustering of genes at transcription factories may have consequences for genome stability, and increase the susceptibility to recurrent chromosomal translocations that lead to cancer. The relationships between genome organization, transcription, and chromosomal translocation formation will have important implications in understanding the causes of therapy-related cancers., (©2013 AACR.)

Although somatic homologous pairing is common in Drosophila it is not generally observed in mammalian cells. However, a number of regions have recently been shown to come into close proximity with their homologous allele, and it has been proposed that pairing might be involved in the establishment or maintenance of monoallelic expression. Here, we investigate the pairing properties of various imprinted and non-imprinted regions in mouse tissues and ES cells. We find by allele-specific 4C-Seq and DNA FISH that the Kcnq1 imprinted region displays frequent pairing but that this is not dependent on monoallelic expression. We demonstrate that pairing involves larger chromosomal regions and that the two chromosome territories come close together. Frequent pairing is not associated with imprinted status or DNA repair, but is influenced by chromosomal location and transcription. We propose that homologous pairing is not exclusive to specialised regions or specific functional events, and speculate that it provides the cell with the opportunity of trans-allelic effects on gene regulation.

Massively parallel DNA sequencing is capable of sequencing tens of millions of DNA fragments at the same time. However, sequence bias in the initial cycles, which are used to determine the coordinates of individual clusters, causes a loss of fidelity in cluster identification on Illumina Genome Analysers. This can result in a significant reduction in the numbers of clusters that can be analysed. Such low sample diversity is an intrinsic problem of sequencing libraries that are generated by restriction enzyme digestion, such as e4C-seq or reduced-representation libraries. Similarly, this problem can also arise through the combined sequencing of barcoded, multiplexed libraries. We describe a procedure to defer the mapping of cluster coordinates until low-diversity sequences have been passed. This simple procedure can recover substantial amounts of next generation sequencing data that would otherwise be lost.

The eukaryotic cell nucleus displays a high degree of spatial organization, with discrete functional subcompartments that provide microenvironments where specialized processes take place. Concordantly, the genome also adopts defined conformations that, in part, enable specific genomic regions to interface with these functional centers. Yet the roles of many subcompartments and the genomic regions that contact them have not been explored fully. More fundamentally, it is not entirely clear how genome organization impacts function, and vice versa. The past decade has witnessed the development of a new breed of methods that are capable of assessing the spatial organization of the genome. These stand to further our understanding of the relationship between genome structure and function, and potentially assign function to various nuclear subcompartments. Here, we review the principal techniques used for analyzing genomic interactions, the functional insights they have afforded and discuss the outlook for future advances in nuclear structure and function dynamics.

LBM415 is an antibacterial agent belonging to the peptide deformylase inhibitor class of compounds. It has previously been shown to demonstrate good activity in vitro against a range of pathogens. In this study, the in vivo efficacy of LBM415 was evaluated in various mouse infection models. We investigated activity against a systemic infection model caused by intraperitoneal inoculation of Staphylococcus aureus (methicillin [meticillin] susceptible [MSSA] and methicillin resistant [MRSA]) and Streptococcus pneumoniae (penicillin susceptible [PSSP] and multidrug resistant [MDRSP]), a thigh infection model caused by intramuscular injection of MRSA, and a lung infection produced by intranasal inoculation of PSSP. In the systemic MSSA and MRSA infections, LBM415 was equivalent to linezolid and vancomycin. In the systemic PSSP infection, LBM415 was equivalent to linezolid, whereas against systemic MDRSP infection, the LBM415 50% effective dose (ED50) was 4.8 mg/kg (dosed subcutaneously) and 36.6 mg/kg (dosed orally), compared to 13.2 mg/kg for telithromycin and >60 mg/kg for penicillin V and clarithromycin. In the MRSA thigh infection, LBM415 significantly reduced thigh bacterial levels compared to those of untreated mice, with levels similar to those after treatment with linezolid at the same dose levels. In the pneumonia model, the ED50 to reduce the bacterial lung burden by >4 log10 in 50% of treated animals was 23.3 mg/kg for LBM415, whereas moxifloxacin showed an ED50 of 14.3 mg/kg. In summary, LBM415 showed in vivo efficacy in sepsis and specific organ infection models irrespective of resistance to other antibiotics. Results suggest the potential of peptide deformylase inhibitors as a novel class of therapeutic agents against antibiotic-resistant pathogens.

A recent spate of examples of specific interactions between loci on separate chromosomes in mammalian nuclei has illuminated another layer of complexity in gene regulation. As the specifics of the cross-talk between interacting loci are worked out, it is also important to consider exactly how, when and where loci can ever reliably find each other within such an intricate environment. Answers may lie in how the genome is organised in relation to itself and to specialised nuclear sub-compartments. Here, we discuss how such specialised nuclear bodies may have the potential to specifically sequester loci and provide a context where interchromosomal communications can occur.

Transcription in mammalian nuclei is highly compartmentalized in RNA polymerase II-enriched nuclear foci known as transcription factories. Genes in cis and trans can share the same factory, suggesting that genes migrate to preassembled transcription sites. We used fluorescent in situ hybridization to investigate the dynamics of gene association with transcription factories during immediate early (IE) gene induction in mouse B lymphocytes. Here, we show that induction involves rapid gene relocation to transcription factories. Importantly, we find that the Myc proto-oncogene on Chromosome 15 is preferentially recruited to the same transcription factory as the highly transcribed Igh gene located on Chromosome 12. Myc and Igh are the most frequent translocation partners in plasmacytoma and Burkitt lymphoma. Our results show that transcriptional activation of IE genes involves rapid relocation to preassembled transcription factories. Furthermore, the data imply a direct link between the nonrandom interchromosomal organization of transcribed genes at transcription factories and the incidence of specific chromosomal translocations.

Several lines of evidence have established strong links between transcriptional activity and specific post-translation modifications of histones. Here we show using RNA FISH that in erythroid cells, intergenic transcription in the human beta-globin locus occurs over a region of greater than 250 kb including several genes in the nearby olfactory receptor gene cluster. This entire region is transcribed during S phase of the cell cycle. However, within this region there are approximately 20 kb sub-domains of high intergenic transcription that occurs outside of S phase. These sub-domains are developmentally regulated and enriched with high levels of active modifications primarily to histone H3. The sub-domains correspond to the beta-globin locus control region, which is active at all developmental stages in erythroid cells, and the region flanking the developmentally regulated, active globin genes. These results correlate high levels of non-S phase intergenic transcription with domain-wide active histone modifications to histone H3.

The chromosome conformation capture technique is used to monitor intra- and intermolecular chromosomal associations. By introducing an adaptation of this technique, Ling and colleagues have identified an unexpected coassociation between two loci on separate chromosomes in mouse nuclei, the imprinted Igf2-H19 locus of chromosome 7 and the Wsb1-Nf1 locus of chromosome 11. Strikingly, this interaction is CCCTC-binding factor (CTCF)-dependent and strictly allele specific. These findings extend our appreciation for genome organization and its influence on gene expression and imprinting.

We have characterized a new clinical strain of Trichophyton rubrum highly resistant to terbinafine but exhibiting normal susceptibility to drugs with other mechanisms of action. Resistance to terbinafine in this strain is caused by a missense mutation in the squalene epoxidase gene leading to the amino acid substitution F397L.

The pre-B-cell receptor (pre-BCR), composed of Ig heavy and surrogate light chain (SLC), signals pre-BII-cell proliferative expansion. We have investigated whether the pre-BCR also signals downregulation of the SLC genes (VpreB and lambda5), thereby limiting this expansion. We demonstrate that, as BM cells progress from the pre-BI to large pre-BII-cell stage, there is a shift from bi- to mono-allelic lambda5 transcription, while the second allele is silenced in small pre-BII cells. A VpreB1-promoter-driven transgene shows the same pattern, therefore suggesting that VpreB1 is similarly regulated and thereby defines the promoter as a target for transcriptional silencing. Analyses of pre-BCR-deficient mice show a temporal delay in lambda5 downregulation, thereby demonstrating that the pre-BCR is essential for monoallelic silencing at the large pre-BII-cell stage. Our data also suggest that SLP-65 is one of the signaling components important for this process. Furthermore, the VpreB1/lambda5 alleles undergo dynamic changes with respect to nuclear positioning and heterochromatin association, thereby providing a possible mechanism for their transcriptional silencing.

As the relationship between nuclear structure and function begins to unfold, a picture is emerging of a dynamic landscape that is centred on the two main processes that execute the regulated use and propagation of the genome. Rather than being subservient enzymatic activities, the replication and transcriptional machineries provide potent forces that organize the genome in three-dimensional nuclear space. Their activities provide opportunities for epigenetic changes that are required for differentiation and development. In addition, they impose physical constraints on the genome that might help to shape its evolution.

There has only been one clinically confirmed case of terbinafine resistance in dermatophytes, where six sequential Trichophyton rubrum isolates from the same patient were found to be resistant to terbinafine and cross-resistant to other squalene epoxidase (SE) inhibitors. Microsomal SE activity from these resistant isolates was insensitive to terbinafine, suggesting a target-based mechanism of resistance (B. Favre, M. Ghannoum, and N. S. Ryder, Med. Mycol. 42:525-529, 2004). In this study, we have characterized at the molecular level the cause of the resistant phenotype of these clinical isolates. Cloning and sequencing of the SE gene and cDNA from T. rubrum revealed the presence of an intron in the gene and an open reading frame encoding a protein of 489 residues, with an equivalent similarity (57%) to both yeast and mammalian SEs. The nucleotide sequences of SE from two terbinafine-susceptible strains were identical whereas those of terbinafine-resistant strains, serially isolated from the same patient, each contained the same single missense introducing the amino acid substitution L393F. Introduction of the corresponding substitution in the Candida albicans SE gene (L398F) and expression of this gene in Saccharomyces cerevisiae conferred a resistant phenotype to the transformants when compared to those expressing the wild-type sequence. Terbinafine resistance in these T. rubrum clinical isolates appears to be due to a single amino acid substitution in SE.

The 7.2 kilobase (kb) Corfu deltabeta thalassemia mutation is the smallest known deletion encompassing a region upstream of the human delta gene that has been suggested to account for the vastly different phenotypes in hereditary persistence of fetal hemoglobin (HPFH) versus beta thalassemia. Fetal hemoglobin (HbF) expression in Corfu heterozygotes and homozygotes is paradoxically dissimilar, suggesting conflicting theories as to the function of the region on globin gene regulation. Here, we measure gamma- and beta-globin gene transcription, steady-state mRNA, and hemoglobin expression levels in primary erythroid cells cultured from several patients with Corfu deltabeta thalassemia. We show through RNA fluorescence in situ hybridization that the Corfu deletion results in high-level transcription of the fetal gamma genes in cis with a concomitant reduction in transcription of the downstream beta gene. Surprisingly, we find that elevated gamma gene transcription does not always result in a corresponding accumulation of gamma mRNA or fetal hemoglobin, indicating a post-transcriptional regulation of gamma gene expression. The data suggest that efficient gamma mRNA accumulation and HbF expression are blocked until beta mRNA levels fall below a critical threshold. These results explain the Corfu paradox and show that the deleted region harbors a critical element that functions in the developmentally regulated transcription of the beta-globin genes.

The process whereby the primitive vascular network develops into the mature vasculature, known as angiogenic vascular remodeling, is controlled by the Notch signaling pathway. Of the two mammalian Notch receptors expressed in vascular endothelium, Notch1 is broadly expressed in diverse cell types, whereas Notch4 is preferentially expressed in endothelial cells. As mechanisms that confer Notch4 expression were unknown, we investigated how NOTCH4 transcription is regulated in human endothelial cells and in transgenic mice. The NOTCH4 promoter and the 5' portion of NOTCH4 assembled into an endothelial cell-specific histone modification pattern. Analysis of NOTCH4 primary transcripts in human umbilical vein endothelial cells by RNA fluorescence in situ hybridization revealed that 36% of the cells transcribed one or both NOTCH4 alleles. The NOTCH4 promoter was sufficient to confer endothelial cell-specific transcription in transfection assays, but intron 1 or upstream sequences were required for expression in the vasculature of transgenic mouse embryos. Cell-type-specific activator protein 1 (AP-1) complexes occupied NOTCH4 chromatin and conferred endothelial cell-specific transcription. Vascular angiogenic factors activated AP-1 and reprogrammed the endogenous NOTCH4 gene in HeLa cells from a repressed to a transcriptionally active state. These results reveal an AP-1-Notch4 pathway, which we propose to be crucial for transducing angiogenic signals and to be deregulated upon aberrant signal transduction in cancer.

The intranuclear position of many genes has been correlated with their activity state, suggesting that migration to functional subcompartments may influence gene expression. Indeed, nascent RNA production and RNA polymerase II seem to be localized into discrete foci or 'transcription factories'. Current estimates from cultured cells indicate that multiple genes could occupy the same factory, although this has not yet been observed. Here we show that, during transcription in vivo, distal genes colocalize to the same transcription factory at high frequencies. Active genes are dynamically organized into shared nuclear subcompartments, and movement into or out of these factories results in activation or abatement of transcription. Thus, rather than recruiting and assembling transcription complexes, active genes migrate to preassembled transcription sites.

Despite terbinafine being fungicidal against Trichophyton rubrum in standard NCCLS assays and rapidly accumulating in nails in vivo, onychomycosis patients require prolonged terbinafine treatment to be cured. To investigate this, we developed a more clinically relevant onychomycosis in vitro test model. Human nail powder inoculated with T. rubrum and incubated in liquid RPMI 1640 salt medium, which did not support growth alone, developed extensive and invasive mycelial growth. Antifungal drugs were added at different concentrations and cultures incubated for 1 to 4 weeks. Fungal survival was determined by spreading cultures on PDA plates without drug and measuring CFU after 1 to 4 weeks incubation. Drug activity was expressed as the nail minimum fungicidal concentration (Nail-MFC) required for 99.9% elimination of viable fungus. Terbinafine Nail-MFC was 4 microg/ml after 1 week exposure, decreasing to 1 microg/ml after 4 weeks exposure, much higher than MFCs < or = 0.03 microg/ml determined in standard NCCLS MIC assays. In contrast, other clinically used drugs were unable to kill T. rubrum after 4 weeks incubation in this model. Invasive mycelial growth on nail appears to protect T. rubrum from the cidal action of systemic drugs, thus providing a rationale for the long treatment periods in onychomycosis.

This survey of 760 university students examined differences in Internet-related problems and psychosocial functioning between four patterns of participation in online sexual information and entertainment activities. Students who did not participate in either online sexual activity were more satisfied with their offline life and more connected to friends and family. Those who engaged in both online sexual activities were more dependent on the Internet and reported lower offline functioning. Students who only sought sexual information maintained strong offline affiliations. Those who only sought entertainment did not report lower offline functioning. Respondents most deficient in offline social support did not report compensatory online support. Despite students' common participation in online sexual activities (OSA) as a venue for social and sexual development, those relying on the Internet and the affiliations it provides appear at risk of decreased social integration. The authors discussed the finding's implications for social and sexual development.

In this study, we have investigated in vitro the resistance frequency and development of resistance to terbinafine of Trichophyton rubrum. Results demonstrated that naturally occurring mutants are rare and that T. rubrum appears to have little capacity to develop resistance to terbinafine even after prolonged exposure.

We have previously shown correction of X-linked severe combined immunodeficiency [SCID-X1, also known as gamma chain (gamma(c)) deficiency] in 9 out of 10 patients by retrovirus-mediated gamma(c) gene transfer into autologous CD34 bone marrow cells. However, almost 3 years after gene therapy, uncontrolled exponential clonal proliferation of mature T cells (with gammadelta+ or alphabeta+ T cell receptors) has occurred in the two youngest patients. Both patients' clones showed retrovirus vector integration in proximity to the LMO2 proto-oncogene promoter, leading to aberrant transcription and expression of LMO2. Thus, retrovirus vector insertion can trigger deregulated premalignant cell proliferation with unexpected frequency, most likely driven by retrovirus enhancer activity on the LMO2 gene promoter.

Silencing of retrovirus vectors poses a significant obstacle to genetic manipulation of stem cells and their use in gene therapy. We describe a mammalian silencer blocking assay using insulator elements positioned between retrovirus silencer elements and an LCRbeta-globin reporter transgene. In transgenic mice, we show that retrovirus silencers are blocked by the cHS4 insulator. Silencer blocking is independent of the CTCF binding site and is most effective when flanking the internal reporter transgene. These data distinguish silencer blocking activity by cHS4 from its enhancer blocking activity. Retrovirus vectors can be created at high titer with one but not two internal dimer cHS4 cores. cHS4 in the LTRs has no effect on expression in transduced F9 cells, suggesting that position effect blocking is not sufficient to escape silencing. The Drosophila insulators gypsy and Scs fail to block silencing in transgenic mice, but gypsy stimulates vector expression 2-fold when located in the LTRs of an infectious retrovirus. The silencer blocking assay complements existing insulator assays in mammalian cells, provides new insight into mechanisms of insulation and is a valuable tool to identify additional silencer blocking insulators that cooperate with cHS4 to improve stem cell retrovirus vector design.

An adult autochthonous Dutch patient who had exhibited severe perinatal anaemia, with partial recovery a few months after birth, was studied for the presence of beta-thalassaemia. Southern blotting showed that the patient was heterozygous for a novel deletion in the beta-globin gene cluster, leaving the beta-gene intact. Inverse polymerase chain reaction was used to determine the breakpoint sequence. The deletion removed 112 kb starting upstream of the HOR5'b6 gene to the second intron of the Agamma-globin gene, including the locus control region. The breakpoint fragment identified a 13-bp orphan sequence not present at either side of the breakpoint.

Communication between distal chromosomal elements is essential for control of many nuclear processes. For example, genes in higher eukaryotes often require distant enhancer sequences for high-level expression. The mechanisms proposed for long-range enhancer action fall into two basic categories. Non-contact models propose that enhancers act at a distance to create a favorable environment for gene transcription, or act as entry sites or nucleation points for factors that ultimately communicate with the gene. Contact models propose that communication occurs through direct interaction between the distant enhancer and the gene by various mechanisms that 'loop out' the intervening sequences. Although much attention has focused on contact models, the existence and nature of long-range interactions is still controversial and speculative, as there is no direct evidence that distant sequences physically interact in vivo. Here, we report the development of a widely applicable in situ technique to tag and recover chromatin in the immediate vicinity of an actively transcribed gene. We show that the classical enhancer element, HS2 of the prototypical locus control region (LCR) of the beta-globin gene cluster, is in close physical proximity to an actively transcribed HBB (beta-globin) gene located over 50 kb away in vivo, suggesting a direct regulatory interaction. The results give unprecedented insight into the in vivo structure of the LCR-gene interface and provide the first direct evidence of long-range enhancer communication.

Background: Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) have important functions during skin development, repair and maintenance. MMP-2 and MMP-9 (gelatinase A and gelatinase B) are involved in regulating keratinocyte migration., Objectives: To analyse whether Apligraf, a bilayered tissue-engineered human skin equivalent (HSE), produces gelatinases and TIMPs and whether or not epidermal-dermal interactions regulate MMP activity., Methods: The tissue distribution of MMP-2, MMP-9, TIMP-1, TIMP-2 and fibronectin was analysed by immunohistochemistry. Secreted MMP activity was quantified by a fluorimetric assay and gelatin zymography was used to monitor gelatinases in tissue culture supernatants., Results: Apligraf expressed MMP-2 and MMP-9 and contained immunohistochemically detectable amounts of TIMP-1 and TIMP-2. The gelatinases were predominantly produced in the epidermis, whereas immunostaining of TIMP-1 and TIMP-2 was largely confined to the dermal component of the HSE. Fibronectin was expressed only in the dermis. Gelatin zymography demonstrated that intact Apligraf produced both MMP-2 and MMP-9, the latter predominantly in its latent form. Separation of the dermis from the epidermis resulted in an enhanced production and activation of MMP-9 by the epidermal layer, and secretion of latent and active MMP-2 by the dermal layer. Moreover, the incubation media of the separated epidermis demonstrated significantly stronger MMP activity than did intact Apligraf or its dermal component., Conclusions: These observations provide evidence that epidermal-dermal interactions suppress epidermal gelatinase activity. In addition, coexpression of TIMPs and fibronectin in the Apligraf dermis suggests that the product has the potential to counteract the imbalance between matrix production and degradation in chronic wounds and thus may support wound re-epithelialization.

Retrovirus vectors are de novo methylated and transcriptionally silent in mammalian stem cells. Here, we identify epigenetic modifications that mark retrovirus-silenced transgenes. We show that murine stem cell virus (MSCV) and human immunodeficiency virus type 1 (HIV-1) vectors dominantly silence a linked locus control region (LCR) beta-globin reporter gene in transgenic mice. MSCV silencing blocks LCR hypersensitive site formation, and silent transgene chromatin is marked differentially by a histone code composed of abundant linker histone H1, deacetylated H3 and acetylated H4. Retrovirus-transduced embryonic stem (ES) cells are silenced predominantly 3 days post-infection, with a small subset expressing enhanced green fluorescent protein to low levels, and silencing is not relieved in de novo methylase-null [dnmt3a-/-;dnmt3b-/-] ES cells. MSCV and HIV-1 sequences also repress reporter transgene expression in Drosophila, demonstrating establishment of silencing in the absence of de novo and maintenance methylases. These findings provide mechanistic insight into a conserved gene silencing mechanism that is de novo methylase independent and that epigenetically marks retrovirus chromatin with a repressive histone code.

We have engineered the reporter gene plasmid pXPG by incorporating a novel high-copy origin of replication and a modified luciferase gene into a pXP1-derived vector that efficiently blocks read-through transcription in eukaryotic cells. pXPG contains the Luc+ luciferase gene derived from pGL3 that lacks a peroxisomal targeting sequence, thereby allowing accumulation of luciferase protein in the cytoplasm rather than subcellular organelles of transfected eukaryotic cells. pXPG has distinct advantages over pGL3, because it contains SV40 polyadenylation signals that appear to be more efficient at blocking read-through transcription than the synthetic polyadenylation signal present in pGL3. pXPG contains a novel mutation near the origin of replication that increases plasmid copy number in Escherichia coli. This mutation alters the -10 sequence in the RNA II promoter of the ColE1 origin of replication from TAATCT to TAATAT. As this sequence is a closer match to the consensus -10 element, we suggest that the mutation increases copy number by increasing the rate of transcription of the RNA II replication primer. This novel mechanism for increasing copy number may have more widespread applications than the commonly used pUC high-copy origin of replication mutation. Unlike pUC, which reverts to low copy number at 30 degrees C, the pXPG mutation supports a higher copy number at both 37 and 30 degrees C., (Copyright 2000 Academic Press.)

Retroviral vectors are transcriptionally silenced in hematopoietic stem cells, and this phenomenon must be overcome for effective gene therapy of blood diseases. The murine stem cell virus (MSCV) vector completely silences beta-globin reporter genes regulated by locus control region (LCR) elements 5'HS2 to 5'HS4 in seven of eight transgenic mice. Here, we show that no single known MSCV silencer element is sufficient for complete LCR beta-globin transgene silencing. However, partial silencing of high-copy transgenes is conveyed by the MSCV direct repeat and promoter elements. The CpG methylation pattern of silenced and expressed MSCV promoter transgenes is virtually identical, demonstrating that silencing does not absolutely correlate with methylation status. Combined mutations in all four MSCV silencer elements leads to expression of beta-globin in 6 of 10 transgenic mice. The same mutations incorporated into the HSC1 retrovirus vector direct neo gene expression in 71% of transduced F9 embryonic carcinoma cells. These studies demonstrate that combined mutation of four retroviral silencer elements relieves complete silencing in most transgenic mice and transduced F9 cells and suggests that novel silencer elements remain. Enhanced expression of the HSC1 vector in primitive stem cells is well suited for blood gene therapy applications.