Transcriptional provirus silencing as a crosstalk of de novo DNA methylation and epigenomic features at the integration site

The autonomous transcription of integrated retroviruses strongly depends on genetic and epigenetic effects of the chromatin at the site of integration. These effects are mostly suppressive and proviral activity can be finally silenced by mechanisms, such as DNA methylation and histone modifications. To address the role of the integration site at the whole-genome-scale, we performed clonal analysis of provirus silencing with an avian leucosis/sarcoma virus-based reporter vector and correlated the transcriptional silencing with the epigenomic landscape of respective integrations. We demonstrate efficient provirus silencing in human HCT116 cell line, which is strongly but not absolutely dependent on the de novo DNA methyltransferase activity, particularly of Dnmt3b. Proviruses integrated close to the transcription start sites of active genes into the regions enriched in H3K4 trimethylation display long-term stability of expression and are resistant to the transcriptional silencing after over-expression of Dnmt3a or Dnmt3b. In contrast, proviruses in the intergenic regions tend to spontaneous transcriptional silencing even in Dnmt3a−/− Dnmt3b−/− cells. The silencing of proviruses within genes is accompanied with DNA methylation of long terminal repeats, whereas silencing in intergenic regions is DNA methylation-independent. These findings indicate that the epigenomic features of integration sites are crucial for their permissivity to the proviral expression

The struggle for human dignity in the era of modern eugenics. Molecular genome editing tool CRISPR/CAS9 and its use in human genome therapy from the perspective of theological ethics

The recent breakthrough discovery of the molecular genome editing tool CRISPR/CAS9 represents a complete revolution in the field of molecular biology, biomedicine and other related fields. It is a highly effective biomolecular tool, derived from the bacterial immune system, with which it is possible to introduce precise changes in the genomes of all organisms. The thesis is limited to the ethical evaluation of the use of CRISPR/CAS9 exclusively in human gene therapy. Thanks to its efficiency, simplicity, accuracy and low financial costs, the CRISPR/CAS9 editing tool, in compliance with ethical parameters, already has a broad spectrum of use in therapeutic procedures on somatic or body cells in the treatment of human genetically determined diseases without introducing a change into the future offspring of the given individual. In addition to great therapeutic potential, the application of CRISPR/CAS9 raises many ethical questions related to the possibilities of its further use, possibly misuse. Ethically problematic genetic procedures include: human hereditary genome editing, i.e. the targeted alteration of the genome of sex cells, progenitor cells and cells of early embryonic development stages with the therapeutic goal of eliminating a genetically determined disease associated with the...Nedávný průlomový objev molekulárního nástroje editace genomu CRISPR/CAS9 představuje v oblasti molekulární biologie, biomedicíny a dalších přidružených oborů naprostou revoluci. Jedná se o vysoce efektivní biomolekulární nástroj, odvozený od bakteriálního imunitního systému, kterým je možné zavádět přesné změny v genomech všech organismů. Diplomová práce se omezuje na etické hodnocení použití CRISPR/CAS9 výlučně v lidské genové terapii. Díky efektivitě, jednoduchosti, přesnosti a nízkým finančním nákladům má již nyní editační nástroj CRISPR/CAS9, při dodrženi etických parametrů, širokospektrální využití v terapeutických postupech na somatických neboli tělních buňkách při léčení lidských geneticky podmíněných onemocnění bez zavedení změny do budoucího potomstva daného jedince. Kromě velkého terapeutického potenciálu, aplikace CRISPR/CAS9 vyvolává mnohé etické otázky spojené s možnostmi jejího dalšího využití, příp. zneužití. Mezi eticky problematické genetické postupy se řadí: lidská dědičná editace genomu, tedy cílené pozměňování genomu pohlavních buněk, progenitorových buněk a buněk raných embryonálních vývojových stadií s terapeutickým cílem eliminace geneticky podmíněného onemocnění spojeného se zavedením této změny do budoucího potomstva daného jedince. Dále jsou to kontroverzní neterapeutické...Department of systematic Theology and PhilosophyKatedra systematické teologie a filosofieKatolická teologická fakultaCatholic Theological Facult

Úloha de novo DNA methyltransferáz v transkripčním umlčování retrovirů a retrovirových vektorů odvozených od ptačího sarkomového a leukozového viru

Katedra genetiky a mikrobiologieDepartment of Genetics and MicrobiologyPřírodovědecká fakultaFaculty of Scienc

The struggle for human dignity in the era of modern eugenics. Molecular genome editing tool CRISPR/CAS9 and its use in human genome therapy from the perspective of theological ethics

The recent breakthrough discovery of the molecular genome editing tool CRISPR/CAS9 represents a complete revolution in the field of molecular biology, biomedicine and other related fields. It is a highly effective biomolecular tool, derived from the bacterial immune system, with which it is possible to introduce precise changes in the genomes of all organisms. The thesis is limited to the ethical evaluation of the use of CRISPR/CAS9 exclusively in human gene therapy. Thanks to its efficiency, simplicity, accuracy and low financial costs, the CRISPR/CAS9 editing tool, in compliance with ethical parameters, already has a broad spectrum of use in therapeutic procedures on somatic or body cells in the treatment of human genetically determined diseases without introducing a change into the future offspring of the given individual. In addition to great therapeutic potential, the application of CRISPR/CAS9 raises many ethical questions related to the possibilities of its further use, possibly misuse. Ethically problematic genetic procedures include: human hereditary genome editing, i.e. the targeted alteration of the genome of sex cells, progenitor cells and cells of early embryonic development stages with the therapeutic goal of eliminating a genetically determined disease associated with the..

The molecular mechanism of DNA methylation and its co-operation with histone modifications

Katedra genetiky a mikrobiologieDepartment of Genetics and MicrobiologyFaculty of SciencePřírodovědecká fakult