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The FNR protein of Neisseria gonorrhoeaeOverton, Timothy William January 2002 (has links)
No description available.
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Replication and Transcription Activation by Polyomavirus Enhancer Motifs PEA1, PEA2, and PEA3 / Replication and Transcription Activation by Polyomavirus Enhancer MotifsMcWilliams, H. M. 08 1900 (has links)
This thesis is missing page 157, the other copies of the thesis did not have the page either. -Digitization Centr / The polyomavirus enhancer is organized into three elements. One of these elements, Element 2, is particularly interesting because the activities of the factors which interact with it are highly regulated. There are at least three cellular proteins, PEA1, PEA2, and PEA3, which bind to adjacent sites in Element 2. These proteins are differentially active in mouse cells at different developmental stages and their activity is modulated by serum, tumor promoting agents and the products of several oncogenes. It is likely, therefore, that these cellular proteins play an important role in interpreting growth stimuli and other physiological cues in the mouse. A plasmid was contructed which can be used to test enhancer elements for their ability to activate both transcription and DNA replication. This plasmid includes the Py origin of replication and a minimal promoter, consisting of a TATA box only, controlling expression of a reporter gene. The activity of the PEA factors was studied by cloning the binding sites for these factors into this reporter plasmid as monomers, multiple tandem copies, and in paired combinations, and testing their ability to activate transcription and DNA replication in vivo. The results of these studies show that PEA1 and PEA3 can function independently and cooperatively to activate both replication and transcription. By contrast, PEA2 is unable to independently activate transcription and represses PEA1-activated transcription when the binding sites for these factors are located adjacent to one another. However, PEA2 functions cooperatively with PEA1 to activate DNA replication, and can weakly activate replication on its own. / Thesis / Master of Science (MS)
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The role of Histone H3 Lysine 4 trimethylation in zebrafish embryonic developmentKrause, Maximilian 06 April 2017 (has links) (PDF)
Cells within multicellular organisms share the same genetic information, yet their shape and function can differ dramatically. This diversity of form and function is established by differential use of the genetic information. Early embryonic development describes the processes that lead to a fully differentiated embryo starting from a single fertilized cell - the zygote. Interestingly, in metazoan species this early development is governed by maternally provided factors (nutrients, RNA, protein), while the zygotic genome is transcriptionally inactive. Only at a specific developmental stage, the zygotic genome becomes transcriptionally active, and zygotic transcripts drive further embryonic development. This major change is called zygotic genome activation (ZGA). While major regulators of activation of early zygotic genes could be identified recently, the molecular mechanisms that contribute to robust global genome activation during embryonic development is not fully understood.
In this study, I investigated whether the establishment of histone H3 lysine 4 trimethylation (H3K4me3) is involved in zebrafish zygotic transcription activation and early embryonic development. H3K4me3 is a chromatin modification that is implicated in transcription regulation. H3K4me3 has been shown to be enriched at Transcription Start Sites (TSS) of genes prior to their activation, and is postulated facilitate transcription activation of developmentally important genes. To interfere with H3K4me3 establishment, I generated histone methyltransferase mutants. I further inhibited H3K4me3 establishment by introduction of histones with lysine 4-to-methionine (K4-to-M) substitution, which act as dominant-negative inhibitors of H3K4me3 establishment. Upon H3K4me3 reduction, I studied the resulting effect on early transcription activation. I found that H3K4me3 is not involved in transcription activation during early zebrafish embryogenesis. Finally I analyzed possible cues in DNA sequence and chromatin environment that might favor early H3K4me3 establishment.
These studies show that H3K4me3 is established during ZGA, yet it is not involved in transcription activation during early zebrafish development. Establishment of H3K4me3 might be a consequence of histone methyltransferase recruitment during a permissive chromatin state, and might be targeted to CpG-rich promoter elements that are enriched for the histone variant H2A.z. / Jede Zelle eines multizellulären Organismus enthält dieselbe Erbinformation, und doch können Form und Funktion von Zellen untereinander sehr unterschiedlich sein. Diese Diversität wird durch unterschiedliches Auslesen - Transkribieren - der Erbinformation erreicht. Embryogenese beschreibt den Prozess, der aus einer einzelnen Zelle - der Zygote - einen multizellulären Embryo entstehen lässt. Interessanterweise laufen frühe Stadien der Embryogenese ohne Transkription der embryonalen Erbinformation ab, sondern werden durch maternal bereitgestellte Faktoren ermöglicht. Erst nach einer spezies-spezifischen Entwicklungsphase wird das Erbgut der Zygote aktiv transkribiert und ermöglicht die weitere Embryonalentwicklung. Obwohl bereits wichtige Regulatoren dieser globalen Genomaktivierung identifiziert werden konnten, sind viele molekulare Mechanismen, die zur Aktivierung des zygotischen Genoms beitragen, bisher unbekannt.
In der hier vorliegenden Doktorarbeit habe ich die Rolle von Histon H3 Lysin 4 Trimethylierung (H3K4me3) während der frühen Embryogenese des Zebrafischs untersucht. H3K4me3 ist eine Chromatinmodifikation, die mit aktiver Transkription in Verbindung gebracht wird. H3K4me3 ist an Transkriptions-Start-Stellen von aktiv ausgelesenen Genen angereichert und es wird vermutet, dass diese Modifikation das Binden von Transkriptionsfaktoren und der Transkriptionsmaschinerie erleichtert. Während meiner Arbeit habe ich durch Mutation verschiedener Histon-Methyltransferasen beziehungsweise die Überexpression eines dominant-negativen Histonsubstrats versucht, die Etablierung von H3K4me3 in frühen Entwicklungsstadien des Zebrafischs zu verhindern. Anschliessend habe untersucht, welchen Effekt H3K4me3-Reduktion auf Tranksriptionsaktivität entsprechender Gene hat. Allerdings konnte ich keinen Zusammenhang zwischen H3K4me3-Reduktion und Transkriptionsaktivität beobachten. Um herauszufinden, weshalb H3K4me3 dennoch während früher Embryonalstadien etabliert wird, habe ich nachfolgend untersucht, ob möglicherweise bestimmte DNASequenzen oder Chromatin-Modifikationen zur Etablierung von H3K4me3 wahrend der Embryogenese des Zebrafischs beitragen.
Aus der hier vorliegenden Arbeit lässt sich schlussfolgern, dass H3K4me3 in Tranksriptionsaktivierung während früher Embryonalstadien des Zebrafischs nicht involviert ist. Möglicherweise wird H3K4me3 in diesen Stadien in einer permissiven Chromatinumgebung etabliert, bevorzugt an Promotoren mit starker H2A.z-Anreicherung und CpG-reichen DNA-Elementen.
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Chemical Biology Approaches for the Molecular Recognition of DNA Double Helix / DNA二重らせんの分子認識に関するケミカルバイオロジー研究Abhijit, Saha 23 March 2015 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18807号 / 理博第4065号 / 新制||理||1585(附属図書館) / 31758 / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM
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The role of Histone H3 Lysine 4 trimethylation in zebrafish embryonic developmentKrause, Maximilian 09 March 2017 (has links)
Cells within multicellular organisms share the same genetic information, yet their shape and function can differ dramatically. This diversity of form and function is established by differential use of the genetic information. Early embryonic development describes the processes that lead to a fully differentiated embryo starting from a single fertilized cell - the zygote. Interestingly, in metazoan species this early development is governed by maternally provided factors (nutrients, RNA, protein), while the zygotic genome is transcriptionally inactive. Only at a specific developmental stage, the zygotic genome becomes transcriptionally active, and zygotic transcripts drive further embryonic development. This major change is called zygotic genome activation (ZGA). While major regulators of activation of early zygotic genes could be identified recently, the molecular mechanisms that contribute to robust global genome activation during embryonic development is not fully understood.
In this study, I investigated whether the establishment of histone H3 lysine 4 trimethylation (H3K4me3) is involved in zebrafish zygotic transcription activation and early embryonic development. H3K4me3 is a chromatin modification that is implicated in transcription regulation. H3K4me3 has been shown to be enriched at Transcription Start Sites (TSS) of genes prior to their activation, and is postulated facilitate transcription activation of developmentally important genes. To interfere with H3K4me3 establishment, I generated histone methyltransferase mutants. I further inhibited H3K4me3 establishment by introduction of histones with lysine 4-to-methionine (K4-to-M) substitution, which act as dominant-negative inhibitors of H3K4me3 establishment. Upon H3K4me3 reduction, I studied the resulting effect on early transcription activation. I found that H3K4me3 is not involved in transcription activation during early zebrafish embryogenesis. Finally I analyzed possible cues in DNA sequence and chromatin environment that might favor early H3K4me3 establishment.
These studies show that H3K4me3 is established during ZGA, yet it is not involved in transcription activation during early zebrafish development. Establishment of H3K4me3 might be a consequence of histone methyltransferase recruitment during a permissive chromatin state, and might be targeted to CpG-rich promoter elements that are enriched for the histone variant H2A.z.:Frontmatter II
Acknowledgements VII
Thesis Summary (English) IX
Thesis Summary (German) X
Table of Contents XIV
List of Figures XVI
List of Tables XVII
List of Abbreviations XXIII
1 Introduction 1
1.1 Transcription regulation 2
1.1.1 Promoter elements - genetic information that guides transcription initiation 2
1.1.2 Enhancers - fine-tuning of transcription by distal DNA elements 3
1.1.3 CpG islands - DNA sequences that allow for epigenetic regulation 4
1.2 Chromatin 4
1.2.1 Histone variants 7
1.2.2 Posttranslational histone modifications 7
1.2.3 Histone Lysine methylation 8
1.2.4 H3K4me3 in embryonic development 10
1.3 Establishment and removal of H3K4me3 10
1.3.1 Set1 homologs - Set1a and Set1b 11
1.3.2 Trithorax homologs - Mll1 and Mll2 11
1.3.3 Homologs of Trithorax-related - Mll3 and Mll4 13
1.3.4 COMPASS complex proteins 13
1.3.5 H3K4me3 removal 14
1.4 Transcription activation in embryos 14
1.4.1 Zebrafish early embryonic development 15
1.4.2 H3K4me3 during early zebrafish development 17
1.5 Thesis aim 17
2 Materials and Methods 19
2.1 Materials 19
2.2 Methods 36
2.2.1 Zebrafish husbandry and care 36
2.2.2 Generation of zebrafish knock-out lines by TALEN mutagenesis 36
2.2.3 Generation of plasmids for mRNA production 38
2.2.4 Microinjection 39
2.2.5 Germline transplantation 39
2.2.6 Western Blot Assays 40
2.2.7 RNA extraction and quantification assays 41
2.2.8 Chromatin immunoprecipitation (ChIP) 43
2.3 Bioinformatics Analyses 46
2.3.1 Quality control, alignment and peak calling 46
2.3.2 Lambda normalization 46
2.3.3 Differential ChIP enrichment analysis 47
2.3.4 Data integration 47
2.3.5 Gene classification 48
3 Results I: H3K4me3 interference by Histone methyltransferase mutation 49
3.1 Generation and phenotypic description of histone methyl-transferase mutants 49
3.1.1 HMT TALEN mutagenesis workflow 49
3.1.2 Ash2l TALEN mutation does not result in a larval or adult phenotype 52
3.1.3 Mll2 mutation results in increased larval mortality, while adult fish are healthy and fertile 54
3.1.4 Mll1 mutation results in increased larval mortality and a severe adult phenotype 56
3.2 HMT mutations do not affect global H3K4me3 levels in early zebrafish embryos 60
3.3 Mll1 mutation results in local H3K4me3 reduction of a small subset of genes 62
3.4 Early embryonic transcription is not altered in mll1 maternal-zygotic mutants 67
3.5 Conclusion 70
4 Results II: H3K4me3 interference by introduction of HMT inhibitors 71
4.1 Establishing a Western Blot assay to monitor H3K4me3 reduction 71
4.2 Overexpression of H3K4-specific histone demethylases does not result in global H3K4me3 reduction 73
4.3 Global reduction of H3K4me3 could not be achieved by small-molecule inhibition of HMT activity 75
4.4 Overexpression of K4-specific methylation-defective H3 results in global H3K4me3 reduction 76
4.4.1 Overexpression of H3K4-to-E constructs does not affect global H3K4me3 establishment 76
4.4.2 H3K4-to-M constructs act as dominant-negative substrate for H3K4me3 establishment 77
4.5 H3K4me3 levels at gene promoters are reduced upon introduction of
methylation-defective Histone H3 79
4.6 Early transcription activation is not altered upon K4M overexpression 88
4.7 Conclusion 92
5 Results III: Promoters rich in CpG and H2A.z gain H3K4me3 early 93
5.1 H3K4me3 levels increase over developmental time at all gene classes 93
5.2 H3K4me3 is gained at CpG-rich elements 98
5.3 H2A.z marks overlaps with H3K4me3 at promoters of non-transcribed genes 100
5.4 High CpG density and H2A.z enrichment are predictive for H3K4me3 establishment 101
5.5 Maternally provided genes are enriched for H2A.z and CpG content 103
5.6 Conclusion 104
6 Discussion 105
6.1 Neither Mll1 nor Mll2 are the main histone methyltransferase for H3K4me3 establishment in early zebrafish development 106
6.2 H3K4me3 reduction does not affect transcription initiation during genome activation 107
6.3 The timing of H3K4me3 establishment might be determined by a permissive chromatin state 109
6.4 H3K4me3 potentially gains importance during later developmental stages 111
6.5 CpG-content and H2A.z enrichment might be predictive for H3K4me3 establishment during genome activation 112
6.6 Conclusion 115
Appendix 117
Bibliography 139
Authorship Declaration 159 / Jede Zelle eines multizellulären Organismus enthält dieselbe Erbinformation, und doch können Form und Funktion von Zellen untereinander sehr unterschiedlich sein. Diese Diversität wird durch unterschiedliches Auslesen - Transkribieren - der Erbinformation erreicht. Embryogenese beschreibt den Prozess, der aus einer einzelnen Zelle - der Zygote - einen multizellulären Embryo entstehen lässt. Interessanterweise laufen frühe Stadien der Embryogenese ohne Transkription der embryonalen Erbinformation ab, sondern werden durch maternal bereitgestellte Faktoren ermöglicht. Erst nach einer spezies-spezifischen Entwicklungsphase wird das Erbgut der Zygote aktiv transkribiert und ermöglicht die weitere Embryonalentwicklung. Obwohl bereits wichtige Regulatoren dieser globalen Genomaktivierung identifiziert werden konnten, sind viele molekulare Mechanismen, die zur Aktivierung des zygotischen Genoms beitragen, bisher unbekannt.
In der hier vorliegenden Doktorarbeit habe ich die Rolle von Histon H3 Lysin 4 Trimethylierung (H3K4me3) während der frühen Embryogenese des Zebrafischs untersucht. H3K4me3 ist eine Chromatinmodifikation, die mit aktiver Transkription in Verbindung gebracht wird. H3K4me3 ist an Transkriptions-Start-Stellen von aktiv ausgelesenen Genen angereichert und es wird vermutet, dass diese Modifikation das Binden von Transkriptionsfaktoren und der Transkriptionsmaschinerie erleichtert. Während meiner Arbeit habe ich durch Mutation verschiedener Histon-Methyltransferasen beziehungsweise die Überexpression eines dominant-negativen Histonsubstrats versucht, die Etablierung von H3K4me3 in frühen Entwicklungsstadien des Zebrafischs zu verhindern. Anschliessend habe untersucht, welchen Effekt H3K4me3-Reduktion auf Tranksriptionsaktivität entsprechender Gene hat. Allerdings konnte ich keinen Zusammenhang zwischen H3K4me3-Reduktion und Transkriptionsaktivität beobachten. Um herauszufinden, weshalb H3K4me3 dennoch während früher Embryonalstadien etabliert wird, habe ich nachfolgend untersucht, ob möglicherweise bestimmte DNASequenzen oder Chromatin-Modifikationen zur Etablierung von H3K4me3 wahrend der Embryogenese des Zebrafischs beitragen.
Aus der hier vorliegenden Arbeit lässt sich schlussfolgern, dass H3K4me3 in Tranksriptionsaktivierung während früher Embryonalstadien des Zebrafischs nicht involviert ist. Möglicherweise wird H3K4me3 in diesen Stadien in einer permissiven Chromatinumgebung etabliert, bevorzugt an Promotoren mit starker H2A.z-Anreicherung und CpG-reichen DNA-Elementen.:Frontmatter II
Acknowledgements VII
Thesis Summary (English) IX
Thesis Summary (German) X
Table of Contents XIV
List of Figures XVI
List of Tables XVII
List of Abbreviations XXIII
1 Introduction 1
1.1 Transcription regulation 2
1.1.1 Promoter elements - genetic information that guides transcription initiation 2
1.1.2 Enhancers - fine-tuning of transcription by distal DNA elements 3
1.1.3 CpG islands - DNA sequences that allow for epigenetic regulation 4
1.2 Chromatin 4
1.2.1 Histone variants 7
1.2.2 Posttranslational histone modifications 7
1.2.3 Histone Lysine methylation 8
1.2.4 H3K4me3 in embryonic development 10
1.3 Establishment and removal of H3K4me3 10
1.3.1 Set1 homologs - Set1a and Set1b 11
1.3.2 Trithorax homologs - Mll1 and Mll2 11
1.3.3 Homologs of Trithorax-related - Mll3 and Mll4 13
1.3.4 COMPASS complex proteins 13
1.3.5 H3K4me3 removal 14
1.4 Transcription activation in embryos 14
1.4.1 Zebrafish early embryonic development 15
1.4.2 H3K4me3 during early zebrafish development 17
1.5 Thesis aim 17
2 Materials and Methods 19
2.1 Materials 19
2.2 Methods 36
2.2.1 Zebrafish husbandry and care 36
2.2.2 Generation of zebrafish knock-out lines by TALEN mutagenesis 36
2.2.3 Generation of plasmids for mRNA production 38
2.2.4 Microinjection 39
2.2.5 Germline transplantation 39
2.2.6 Western Blot Assays 40
2.2.7 RNA extraction and quantification assays 41
2.2.8 Chromatin immunoprecipitation (ChIP) 43
2.3 Bioinformatics Analyses 46
2.3.1 Quality control, alignment and peak calling 46
2.3.2 Lambda normalization 46
2.3.3 Differential ChIP enrichment analysis 47
2.3.4 Data integration 47
2.3.5 Gene classification 48
3 Results I: H3K4me3 interference by Histone methyltransferase mutation 49
3.1 Generation and phenotypic description of histone methyl-transferase mutants 49
3.1.1 HMT TALEN mutagenesis workflow 49
3.1.2 Ash2l TALEN mutation does not result in a larval or adult phenotype 52
3.1.3 Mll2 mutation results in increased larval mortality, while adult fish are healthy and fertile 54
3.1.4 Mll1 mutation results in increased larval mortality and a severe adult phenotype 56
3.2 HMT mutations do not affect global H3K4me3 levels in early zebrafish embryos 60
3.3 Mll1 mutation results in local H3K4me3 reduction of a small subset of genes 62
3.4 Early embryonic transcription is not altered in mll1 maternal-zygotic mutants 67
3.5 Conclusion 70
4 Results II: H3K4me3 interference by introduction of HMT inhibitors 71
4.1 Establishing a Western Blot assay to monitor H3K4me3 reduction 71
4.2 Overexpression of H3K4-specific histone demethylases does not result in global H3K4me3 reduction 73
4.3 Global reduction of H3K4me3 could not be achieved by small-molecule inhibition of HMT activity 75
4.4 Overexpression of K4-specific methylation-defective H3 results in global H3K4me3 reduction 76
4.4.1 Overexpression of H3K4-to-E constructs does not affect global H3K4me3 establishment 76
4.4.2 H3K4-to-M constructs act as dominant-negative substrate for H3K4me3 establishment 77
4.5 H3K4me3 levels at gene promoters are reduced upon introduction of
methylation-defective Histone H3 79
4.6 Early transcription activation is not altered upon K4M overexpression 88
4.7 Conclusion 92
5 Results III: Promoters rich in CpG and H2A.z gain H3K4me3 early 93
5.1 H3K4me3 levels increase over developmental time at all gene classes 93
5.2 H3K4me3 is gained at CpG-rich elements 98
5.3 H2A.z marks overlaps with H3K4me3 at promoters of non-transcribed genes 100
5.4 High CpG density and H2A.z enrichment are predictive for H3K4me3 establishment 101
5.5 Maternally provided genes are enriched for H2A.z and CpG content 103
5.6 Conclusion 104
6 Discussion 105
6.1 Neither Mll1 nor Mll2 are the main histone methyltransferase for H3K4me3 establishment in early zebrafish development 106
6.2 H3K4me3 reduction does not affect transcription initiation during genome activation 107
6.3 The timing of H3K4me3 establishment might be determined by a permissive chromatin state 109
6.4 H3K4me3 potentially gains importance during later developmental stages 111
6.5 CpG-content and H2A.z enrichment might be predictive for H3K4me3 establishment during genome activation 112
6.6 Conclusion 115
Appendix 117
Bibliography 139
Authorship Declaration 159
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Mechanism Of mom Gene Transactivation By Transcription Factor C Of Phage MUChakraborty, Atanu 05 1900 (has links)
Regulation of transcription initiation is the major determining event employed by the cell to control gene expression and subsequent cellular processes. The weak promoters, with low basal transcription activities, are activated by activators. Bacteriophage Mu mom gene, which encodes a unique DNA modification function, is detrimental to cell when expressed early or in large quantities. Mu has designed a complex, well-controlled and orchestrated regulatory network for mom expression to ensure its synthesis only in late lytic cycle. The phage encoded transcription activator protein C activates the gene by promoter unwinding of the DNA and thereby recruiting of RNAP to the promoter.
C protein functions as a dimer for DNA binding and transcription activation. Mutagenesis and chemical crosslinking studies revealed that the leucine zipper motif, and not the coiled coil motif in the N terminal region, is responsible for C dimerization. The DNA binding domain of C is a HTH domain which is preceded by the leucine zipper motif. The C protein is one of the few examples in the bacterial proteins containing both leucine zipper and HTH domain.
Most of the transcription activators either influence initial binding of RNAP or conversion of closed to open complex formation. Very few activators act at subsequent steps of promoter-polymerase interaction. Earlier studies showed high level of transcription from a mutant mom promoter, tin7. Addition of C further increased transcription from Ptin7 indicating that C may have a role beyond polymerase recruitment. Each steps of transcription initiation have been dissected using the Ptin7 and a positive control (pc) mutant of C, R105D. The results revealed multi-step transcription activation mechanism for C protein at Pmom. C recruits RNAP at Pmom and subsequently increases the productive RNAP-promoter complex and enhances promoter clearance.
To further understand the C mediated transactivation mechanism, interaction between C and RNAP was assessed. C interacts with holo and core RNAP only in presence of DNA. Positive control mutants of C, F95A and R015D, were found to be compromised in RNAP interactions. These mutants were efficient in RNAP recruitment to Pmom but do not enhance promoter clearance. Trypsin cleavage protection experiment indicated that probably C protein interacts with b¢ subunit of RNAP. Interaction between C and RNAP appears to enhance the formation of productive RNAP-promoter complex leading to promoter clearance.
The connection between activator-polymerase interaction and transcription activation is well documented where the recruitment of RNAP is influenced. In case of activators acting at post recruitment steps of initiation, the role of polymerase contact is poorly understood. Our study shows that activator-polymerase interaction can lead to increased promoter clearance at Pmom by overcoming abortive initiation.
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