The Role of BCL-3 Feedback Loops in Regulating NF-Κb Signalling
Total Page：16
File Type：pdf, Size：1020Kb
The role of BCL-3 feedback loops in regulating NF-κB signalling A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Engineering and Physical Sciences 2012 Thomas Walker School of Chemical Engineering and Analytical Sciences Integrative Systems Biology Contents Contents……………………………………………………………………………….... 1 Word count………………………………………………………………………………. 9 List of figures……………………………………………………………………………. 9 List of tables…………………………………………………………………………….. 11 Abbreviations………………………………………………………………………........ 11 Abstract………………………………………………………………………………...... 15 Declaration & Copyright Statement…………………………………………..…...... 16 Acknowledgements……………………………………………………………..……… 17 Chapter 1 Introduction……………………………………………………………………..……….. 18 1.1. The inflammatory response…………………………………………………………...……………. 18 1.1.1. PAMPs: initial indicators of infection…………………………………………………………….. 18 1.1.2. The inflammatory response………………………………………………………………………. 19 1.1.3. Cytokines…………………………………………………………………………………………… 19 1.1.3.1. The TNF family of cytokines…………………………………………………………..... 20 1.1.3.2. The TNFR family………………………………………………………………………… 20 1.1.3.3. Diverse cell types produce and are responsive to TNF α……………………………. 21 1.1.4. Fibroblasts as inflammation mediators……………………………………………………………. 21 1.2. NF-κB transcription factors………………………………………………………………………… 22 1.2.1. NF-κB subunits and dimer combinations……………………………………………………..... 22 1.2.2. Canonical NF-κB signalling………………………………………………………………………. 23 1.2.3. p50 homodimers………………………………………………………………………………….. 25 1.2.4. Non-canonical NF-κB signalling…………………………………………………………………. 26 1.2.5. Post transcriptional modification of NF-κB factors…………………………………………...... 27 1.2.6. DNA sequence specific binding of NF-κB………………………………………………………. 27 1.2.6.1. Variant κB sites have different affinities for NF-κB dimers…………………………... 27 1.2.6.2. The dynamic nature of κB site binding………………………………………………… 28 1.2.6.3. Dynamic NF-κB DNA binding is made possible by active removal mechanisms……………………………………………………………………………………….. 28 1.2.7. NF-κB as a transcription mediator…………………………………………………………………. 29 1.2.8. NF-κB and other cytokine induced signalling pathways…………………………………………. 30 1.3. The I κB family of proteins………………………………………………………………………….. 30 1.3.1. BCL-3: A distinct member of the I κB family…………………………………………………..... 30 1.3.1.1. BCL-3……………………………………………………………………………………… 31 1.3.2. BCL-3 binds a specific subset of NF-κB dimers……………………………………………….. 31 1.3.3. Sub-cellular localisation of BCL-3……………………………………………………………….. 32 1.3.4. Post-transcriptional modification of BCL-3……………………………………………............. 32 1.3.5. The cellular function of BCL-3………………………………………………………………….. 32 1.3.6. BCL-3 effects on NF-κB binding…………………………………………………………………. 33 1.3.6.1. BCL-3 enhances the DNA binding abilty of p50 and p52 homodimers……………. 33 1.3.6.2. Negative effects of BCL-3 on p50/p52 homodimer DNA binding…………………… 33 1.3.7. Functional effects of BCL-3 complexes………………………………………………………….. 34 1.3.7.1. Negative effects of BCL-3 on transcription: HDAC recruitment…………………….. 34 1.3.7.2. BCL-3 as a positive transcription factor……………………………………………….. 35 - 1 - 1.3.7.3. The contrary nature of BCL-3 activity…………………………………………………. 36 1.3.8. NF-κB mediated induction of BCL-3…………………………………………………………......... 37 1.3.9. Anti-inflammaoty cytokines and BCL3 expression: IL-9 and -10……………………………….. 37 1.3.10. Negative feedback and BCL-3……………………………………………………………………. 37 1.4. TNF α: An inducer and target of NF-κB signalling……………………………………………… 38 1.4.1. NF-κB induces TNF Α promoter activity………………………………………………………… 38 1.4.2. Cytokine overexpression………………………………………………………………………… 38 1.4.3. Mechanisms to reduce the extent of NF-κB signalling……………………………………….. 38 1.4.3.1. I κB negative feedback………………………………………………………………….. 38 1.4.3.2. A20………………………………………………………………………………………… 39 1.4.3.3. Post-transcriptional modifcation of NF-κB………………………………………......... 39 1.4.4. Limiting TNF Α transcript induction to inflammatory stimuli..……………………………………. 40 1.4.4.1. TNF Α mRNA stability……………………………………………………………………. 40 1.4.4.2. BCL-3 as a direct inhibitor of TNF α self induced transcription……………………… 40 1.4.5. The dynamic nature of the TNF Α gene promoter……………………………………………….. 40 1.4.5.1. TNF Α promoters across species: From mouse to human………………………….. 41 1.4.5.2. κB sites within the human TNF Α promoter: Spatial segregation of ……………….. 42 contrary roles………………………………………………………………………………….. … 43 1.4.5.3. Competition at distal binding sites…………………………………………………….. 43 1.5. Chromatin strutre and dynamics…………………………………………………………………. 44 1.5.1. Chromatin structure………………………………………………………………………………. 44 1.5.2. Nucleosomes and transcription factor binding……………………………………………....... 45 1.5.3. Nucleosome positioning………………………………………………………………………….. 46 1.5.4. Chromatin remodelling………………………………………………………………………........ 47 1.5.4.1. Nucleosome binding activity of chromatin remodelling complexes………………… 48 1.5.5. Post transcriptional modification of histones……………………………………………………... 48 1.5.6. Inducible HAT recruitment…………………………………………………………………………. 49 1.6. RNA polymerase II dynamics and binding………………………………………………………. 50 1.6.1. Pre-initiaion complex assembly……………………………………………………………………. 50 1.6.2. Core promoter elements……………………………………………………………………………. 51 1.6.3. TBP induced DNA curvature………………………………………………………………………. 52 1.6.4. Nucleosome Depleted Regions (NDRs)………………………………………………………….. 53 1.6.4.1. Flanking nucleosomes………………………………………………………………….. 53 1.6.4.2. Histone modification and open chromatin at the TSS………………………………... 54 1.6.4.3. Sequence mediated NDRs………………………………………….………………….. 54 1.6.4.4. Inducible or constitutive chromatin marks at gene TSSs………….………………… 55 1.6.5. The RNA polymerase II transcription cycle…………………………………….…………………. 55 1.6.5.1. Pre-RNAP binding transcription control……………………………………………….. 56 1.6.5.2. Post-RNAP binding transcription control……………………………………………… 56 1.6.5.2.1. Overcoming nucleosome obstacles……………………………………….. 57 1.6.5.2.2. Non-nucleosome mediated pausing mechanisms……………………….. 57 1.6.5.2.2.1. Transcription initiation and promoter escape…………………. 58 1.6.5.3. DSIF/NELF mediated arrest……………………………………………………………. 58 1.6.5.4. P-TEFb mediated release from pausing……………………………………………… 59 1.6.5.5. P-TEFb and recruitment of RNA processing factors………………………………… 60 1.6.5.6. RNAP backtracking and TFIIS mediated release…………………………………….. 60 1.6.6. Functions of pre-stimulus bound and paused RNAP……………………………………………. 61 1.6.7. Transcription activators – differential points of activity………………………………………….. 63 1.6.8. NF-κB conducts transcription activation roles at multiple sites in the transcription cycle…………………………………………………………………………………………... 63 1.6.8.1. Timing of NF-κB transcription induction……………………………………………….. 63 1.7. Aims of the work…………………………………………………………………………………….. 63 - 2 - Chapter 2 Materials and Methods………………………………………………………………… 65 2.1. Cell culture…………………………………………………………………………………………….. 65 2.1.1. Cell lines used……………………………………………………………………………………….. 65 2.1.1.1. SK-N-AS……………………………………………………………………………......... 65 2.1.1.2. HT1080…………………………………………………………………………………… 65 2.1.2. Cell culture…………………………………………………………………………………………… 65 2.1.2.1. Cell growth conditions…………………………………………………………………... 65 2.1.2.2. Adherent cell detachment……………………………………………………………… 66 2.1.2.3. Cell sub-culturing………………………………………………………………………… 66 2.1.3. Cell stimulation with TNF α………………………….. …………………………………………….. 66 2.1.4. Cell treatment reagents…………………………………………………………………………….. 66 2.1.5. Counting cells………………………………………………………………………………………… 67 2.1.6. Cryopreservation of HT1080 cell line……………………………………………………………… 67 2.1.7. Cell viability assay…………………………………………………………………………………… 67 2.2. Quantitative real time reverse transcriptase PCR (qRT-PCR)………………………………... 68 2.2.1. RNA extraction……………………………………………………………………………………….. 68 2.2.2. cDNA synthesis………………………………………………………………………………………. 68 2.2.3. qRT-PCR conditions…………………………………………………………………………………. 68 2.2.4. Ct method and Statistical comparison of data……………………………………………………. 69 2.3. Human cell transfection…………………………………………………………………………….. 70 2.3.1. Plasmid transfection of HT1080 cells……………………………………………………………… 70 2.3.1.1. ExGen500………………………………………………………………………………... 70 2.3.1.2. FuGene 6………………………………………………………………………………… 70 2.3.2. siRNA transfection…………………………………………………………………………………... 71 2.3.2.1. Lipofectamine 2000……………………………………………………………………… 71 2.3.3. BAC transfection…………………………………………………………………………………….. 71 2.4. Live imaging of human cells………………………………………………………………………. 71 2.4.1. Cell culture…………………………………………………………………………………………… 71 2.4.2. Micropscopy…………………………………………………………………………………………. 72 2.4.3. Cell tracker…………………………………………………………………………………………… 72 2.5. Western blots…………………………………………………………………………………………. 72 2.5.1. Protein extraction…………………………………………………………………………………… 72 2.5.2. Protein quantification……………………………………………………………………………….. 72 2.5.3. SDS-PAGE…………………………………………………………………………………………… 73 2.5.4. Blotting and blocking……………………………………………………………………………….. 73 2.5.5. Antibody binding and detection……………………………………………………………………. 74 2.6. Immunocytochemistry………………………………………………………………………………. 76 2.7. Chromatin immunoprecipatation (ChIP)…………………………………………………………. 77 2.7.1. Cell fixation and chromatin extraction…………………………………………………………….. 77 2.7.2. Chromatin sonication……………………………………………………………………………….. 77 2.7.3. Antibody binding of chromatin……………………………………………………………………… 77 2.7.4. Immunoprecipiation of chromatin, washes and elution………………………………………….. 78 2.7.5. Quantification of eluted DNA fragments…………………………………………………………… 78 2.8. Cloning/Molecular Biology techniques………………………………………………………….