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Methods Mol Biol ; 2351: 41-65, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382183


Enhancers are transcribed by RNA polymerase II (Pol II). In order to study the regulation of enhancer transcription and its function in target gene control, methods are required that track genome transcription with high precision in vivo. Here, we provide step-by-step guidance for performing native elongating transcript sequencing (NET-Seq) in mammalian cells. NET-Seq allows quantitative measurements of transcription genome-wide, including enhancer transcription, with single-nucleotide and DNA strand resolution. The approach consists of capturing and efficiently converting the 3'-ends of the nascent RNA into a sequencing library followed by next-generation sequencing and computational data analysis. The protocol includes quality control measurements to monitor the success of the main steps. Following this protocol, a NET-Seq library is obtained within 5 days.

Elementos Facilitadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de RNA/métodos , Transcrição Genética , Animais , Células Cultivadas , Cromatina/genética , Biologia Computacional/métodos , DNA , Biblioteca Gênica , Humanos , Reação em Cadeia da Polimerase , RNA , RNA Polimerase II/metabolismo , Software
Mol Cell ; 81(17): 3589-3603.e13, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34324863


Transcription elongation has emerged as a regulatory hub in gene expression of metazoans. A major control point occurs during early elongation before RNA polymerase II (Pol II) is released into productive elongation. Prior research has linked BRD4 with transcription elongation. Here, we use rapid BET protein and BRD4-selective degradation along with quantitative genome-wide approaches to investigate direct functions of BRD4 in Pol II transcription regulation. Notably, as an immediate consequence of acute BRD4 loss, promoter-proximal pause release is impaired, and transcriptionally engaged Pol II past this checkpoint undergoes readthrough transcription. An integrated proteome-wide analysis uncovers elongation and 3'-RNA processing factors as core BRD4 interactors. BRD4 ablation disrupts the recruitment of general 3'-RNA processing factors at the 5'-control region, which correlates with RNA cleavage and termination defects. These studies, performed in human cells, reveal a BRD4-mediated checkpoint and begin to establish a molecular link between 5'-elongation control and 3'-RNA processing.

Commun Biol ; 4(1): 485, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33859315


Female mammals achieve dosage compensation by inactivating one of their two X chromosomes during development, a process entirely dependent on Xist, an X-linked long non-coding RNA (lncRNA). At the onset of X chromosome inactivation (XCI), Xist is up-regulated and spreads along the future inactive X chromosome. Contextually, it recruits repressive histone and DNA modifiers that transcriptionally silence the X chromosome. Xist regulation is tightly coupled to differentiation and its expression is under the control of both pluripotency and epigenetic factors. Recent evidence has suggested that chromatin remodelers accumulate at the X Inactivation Center (XIC) and here we demonstrate a new role for Chd8 in Xist regulation in differentiating ES cells, linked to its control and prevention of spurious transcription factor interactions occurring within Xist regulatory regions. Our findings have a broader relevance, in the context of complex, developmentally-regulated gene expression.

Proteínas de Ligação a DNA/genética , Inativação do Cromossomo X , Cromossomo X/genética , Animais , Proteínas de Ligação a DNA/metabolismo , Compensação de Dosagem (Genética) , Feminino , Camundongos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo
BMC Vet Res ; 17(1): 6, 2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33407446


BACKGROUND: Actinobaculum suis is a bacterium known to cause infections of the urogenital tract of sows. Infection can occur through close contact to boars, who frequently carry the pathogen in their preputial diverticulum but do not become clinically diseased themselves. In the current case, Actinobaculum suis was isolated from pyogranuloma of inflamed epididymis in a boar with poor fertility. CASE PRESENTATION: Increased return to oestrus rate, which worsened after the purchase of a new boar, was reported in an organic farm in Switzerland. During herd examination, azoospermia of the boar was diagnosed, and slaughter, followed by examination of its urogenital tract, was carried out. Pathologically, pyogranuloma formation and epididymitis were diagnosed. Bacteriology of the pyogranulomas showed growth of Actinobaculum suis and mixed flora. After the boar was replaced, the return to oestrus rate improved tremendously. CONCLUSION: A close relative of Actinobaculum suis, namely Actinotignum schaalii, has already been associated with epididymitis in humans. Considering the present case and the parallels in human medicine, Actinobaculum suis should be included in the list of differentials of boars with poor fertility.

Actinomycetaceae , Infecções por Actinomycetales/veterinária , Azoospermia/veterinária , Epididimite/veterinária , Granuloma/veterinária , Doenças dos Suínos/microbiologia , Doenças dos Suínos/patologia , Infecções por Actinomycetales/patologia , Animais , Azoospermia/microbiologia , Azoospermia/patologia , Epididimite/microbiologia , Epididimite/patologia , Granuloma/diagnóstico , Granuloma/microbiologia , Masculino , Suínos
Porcine Health Manag ; 7(1): 13, 2021 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-33482877


BACKGROUND: Lawsonia intracellularis is causing diarrhea, poor growth and sudden death in pigs. It can be found in most pig populations leading to large economic losses worldwide. Many potential risk factors for the occurrence of disease or seropositivity have been described. The current study therefore focused on herd characteristics in European countries associated with direct detection of the pathogen determined by quantitative polymerase chain reaction. RESULTS: A median number of less than 30 nursery pigs per pen was correlated to less positive nursery pigs (p < 0.01) and generally less samples positive per herd (p < 0.05) as well as a lower median of genome equivalents determined per herd (p < 0.05). Routine use of zinc oxide at/ around weaning, which was mentioned by 41.0% of all farmers, was correlated to higher number of positive nursery pigs (p < 0.01) as well as higher median genome equivalents determined per herd (p < 0.05). Slatted flooring of more than 78.0% of the surface in nursery units was correlated to lower number of positive animals (p < 0.05) and a lower median of genome equivalents per herd (p < 0.05). A weight of more than 7.8 kg at weaning was correlated to a higher number of positive growing pigs (p < 0.05) as well as general higher number of positive samples/ herd (p < 0.01). CONCLUSIONS: Weaning and subsequent accommodation of nursery pigs seem to be of particular importance in prevention of infection with Lawsonia intracellularis and the spread of the pathogen within the herd.

Porcine Health Manag ; 5: 31, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31890255


Background: Lawsonia intracellularis causes large economic losses in the pig industry worldwide. Pigs suffer from reduced daily weight gain, poor feed conversion ratio and increased mortality. The number of affected animals and herds in Europe remains unknown. This study will provide an overview of the prevalence of Lawsonia intracellularis in herds with a history of diarrhoea in different European countries and thereby identify country specific differences. Results: Out of the 144 herds sampled in Germany, Denmark, Spain, the Netherlands and the United Kingdom, 90.3% (79.2-100.0%) contained at least one positive faecal sample on quantitative polymerase chain reaction (qPCR). Of the 6450 nursery, growing and finishing pigs of the previously mentioned herds, 26.2% (15.9-41.5%) of the animals were tested positive in faecal samples. Enzyme linked immunosorbent assay (ELISA) results of 60 herds were 91.7% (70-100%) positive. The percentage of positive samples in these 1791 blood samples was 31.6% (20.3-51.0%). Herd prevalence did not differ significantly by qPCR or ELISA. Significant differences between the countries were found regarding: Within-herd prevalence- qPCR: Samples from Denmark were more often positive than samples of Spain or the United Kingdom. Within-herd prevalence- ELISA: Samples from Denmark were more often positive than samples from Spain and the Netherlands. Affected age category- qPCR: Nursery pigs in Denmark were more often positive and shed more genome equivalents than nursery pigs in the other countries. Concentration of detected genome equivalents- qPCR: The concentration of genome equivalents from Lawsonia intracellularis in herds in Denmark was higher compared to all other countries. Conclusion: A widespread of Lawsonia intracellularis in the six European countries was confirmed, whereby a large part of the positive animals only excreted small amounts of genome equivalents. Country specific differences were found with Denmark in particular diagnosing more Lawsonia intracellularis then the other countries. Herd data collected in this study needs to be analysed to get more information about possible reasons for the differences found between the countries.