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The role of the p300/CBP complex components in the regulation of apoptosis under hypoxia

Georgia Xenaki

[Thesis].University of Manchester, School of Pharmacy and Pharmaceutical Sciences;2008.

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Abstract

Posttranslational modifications are of great importance in the mediation of transcriptional effects, necessary for signalling in cancer. A characteristic example of such modifications is acetylation of the p53 tumour suppressor, a transcription factor involved in several crucial cellular functions including cell-cycle arrest and apoptosis. p53 is stabilised under hypoxic and DNA damaging-conditions. However, only in the latter scenario is p53 fully capable of inducing the expression of its proapoptotic targets through acetylation. The hypoxia inducible factor 1 (HIF-1) transcription factor is stabilised at low oxygen levels to mediate a cellular adaptive response under these conditions, promoting cell survival. As these two opposing transcription factors share a common transcriptional regulator, p300/CBP, this study focused on deciphering the p300/CBP complex components under differential stress to determine its composition required for cellular responses elicited in response to DNA damage or hypoxia, in an effort to investigate a possible link between differential posttranslational modifications and the resulting cell fate. Hence, the aim of this study was to investigate the roles of p300/CBP components in dictating transcriptional regulation of both HIF-1 and p53 in hypoxic conditions.To carry out this study, the proapoptotic BID gene was the system used, as its promoter contains a p53 response element and a HIF-1 response element (HRE). The p300/CBP associated factors PCAF and Strap were appointed as potent candidates for posttranslational modifications under differential conditions, as they are stress-responsive cofactors. Under DNA damage, PCAF acetylates p53 at K320 and Strap augments p300 binding to p53, both of which amplify the p53 response. Evidence from this study demonstrates that under hypoxia-mimicking conditions PCAF-mediated p53 acetylation at K320 is reduced to a greater extent compared to p300/CBP acetylation at K382. The limited amounts of acetylated p53 at K320 are preferentially recruited to the promoter of the cell cycle arrest p21WAF-1/CIP-1 gene that appears to be unaffected by hypoxia, but fail to be recruited to the BID promoter, rendering p53 incapable of upregulating proapoptotic BID in hypoxic conditions. In addition, under the same conditions, PCAF was found to acetylate, and direct HIF-1 to a particular subset of its targets, leading to alterations in the net physiological effect. Moreover, the intrinsic acetyl transferase activity of PCAF was shown to increase the stability of HIF-1. An additional role was attributed to PCAF in relation to apoptosis, albeit from another angle. BID protein translocation to the cytoplasm in hypoxic conditions was facilitated by ectopically expressed PCAF.Strap was found to be preferentially recruited to the HRE of the BID promoter in hypoxic conditions, and to exert a transrepression effect that appeared to be p53-dependent. Strap also interacted with specific PCAF isoforms depending on the type of cellular stress. Contrary to PCAF, ectopically expressed Strap did not have any effect on BID subcellular distribution. This study has provided additional insight in the mechanisms by which cofactors are involved in cell fate, either by affecting activity and stability of HIF-1 and p53, or having a direct effect on Bcl-2 member subcellular distribution.

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Degree type:
PhD
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Total pages:
246
Table of contents:
TABLES 11DECLARATION 13COPYRIGHT STATEMENT 13DEDICATION 14THE AUTHOR 14ACKNOWLEDGEMENTS 15ABBREVIATIONS 161. INTRODUCTION 181.1. Transcription 181.1.1. Regulation of transcription 181.1.2. Transcription and Cancer 211.2. Apoptosis 221.2.1. Bcl-2 family members 231.2.1.1. BID, apoptosis and DNA damage response 261.2.2. Intrinsic pathway of apoptosis 261.2.3. Extrinsic pathway of apoptosis 271.2.4. Apoptosis and disease 291.3. Cell cycle 301.3.1. Transcription and cell cycle 321.3.2. Cell cycle checkpoints 341.3.3. Cell cycle, transcription and apoptosis 351.3.4. Cell cycle and cancer 361.4. Cancer 371.4.1. Tumourigenesis 371.4.2. Hypoxia and solid tumours 381.4.3. Hypoxia Inducible Factor-1, cell cycle and cancer 411.4.4. p53, cell cycle regulation and cancer 411.5. Hypoxia Inducible Factor-1 (HIF-1) 421.5.1. Identification of HIF-1 421.5.2. Structural characteristics of HIF-1 protein 431.5.3. Structural characteristics of HIF-2 protein 441.5.4. Structural characteristics of HIF-3 protein 451.5.5. Regulation of HIF-1 stability and activity. O2 dependent and O2-independent post-translational modifications of HIF-1 461.5.5.1. p300 461.5.5.2. Prolyl Hydoxylases and Factor Inhibiting HIF-1 471.5.5.3. p53 481.5.5.4. ARD-1 481.5.5.5. CITED2/CITED4 491.5.5.6. p14ARF 491.5.5.7. Phosphorylation cascades 501.5.5.8. Hsp90 501.5.5.9. Nitric oxide 511.5.6. HIF-1 transcriptional targets 541.5.6.1. Erythropoeisis/iron metabolism 541.5.6.2. Angiogenesis 541.5.6.3. Glucose metabolism 541.5.6.4. Cell proliferation and survival 551.5.6.5. Apoptosis 551.5.7. HIF-1 cofactors 561.5.8. HIF-1 and cancer therapy 561.6. p53 structure, function and regulation 581.6.1. The discovery of p53 581.6.2. Structural characteristics of p53 protein 581.6.3. Post-translational modifications of p53 591.6.4. Regulation of p53 stability 601.6.5. p53 transcriptional targets 611.6.6. p53 cofactors: acetylation and its role in disease/protein stability 641.6.6.1. The p300/CBP histone acetyltransferase 641.6.6.2. The p300/CBP-associated factor (PCAF) 651.6.6.3. The p300/CBP-associated protein Strap 671.6.7. p53 apoptosis and cancer therapy 681.7. The crosstalk between p53 and HIF-1 701.8. Common p53 and HIF-1 cofactors 731.9. Project aims 752. MATERIALS AND METHODS 772.1 Suppliers of reagents 772.2. Microbiology 772.2.1. Microbiology production of media 772.2.2. Transformation of competent bacteria 772.3. Preparation and handling of plasmid and genomic DNA 782.3.1. Maxiprep of plasmid DNA 782.3.2. Determination of DNA concentration 782.3.3. Miniprep 792.3.4. Genomic DNA extraction 802.3.4.1. Genomic DNA amplification for cloning into pGL3 luciferase promoter vector 802.3.4.2. BID cloning into a pEGFP-N1 vector 862.3.5. Restriction endonuclease digests 872.3.6. Agarose gel electrophoresis 882.3.7. DNA isolation 882.3.8. DNA ligation 882.3.9. Site-directed mutagenesis 892.4. Cell culture 902.4.1. Cell lines and culture conditions 902.4.2. Maintenance of mammalian cells 912.5. Plasmids used and transfection 912.5.1. Plasmids 912.5.2. HIF1- RNA interference 932.5.3. Transient transfection 932.6. Luciferase assays 942.7. Chromatin immunoprecipitation (ChIP) 952.7.1. Cross-linking and cell harvesting 952.7.2. SYBR Green qRT-PCR for ChIP-precipitated DNA detection 982.8. polyA+ RNA level detection 1022.8.1. RNA isolation 1022.8.2. Synthesis of cDNA using reverse transcriptase 1022.8.3. SYBR Green qRT-PCR for polyA+ RNA level detection 1032.9. Western blotting 1032.9.1. Preparation of cell lysates 1032.9.2. Coimmunoprecipitation 1042.9.3. Protein concentration determination 1052.9.4. Gel electrophoresis (SDS-PAGE) 1052.9.5. Protein transfer 1072.9.6. Immunoblotting and detection 1082.10. Immunofluorescence Microscopy 1092.11. Cell-cycle analysis 1102.12. Caspase 3 assay 1113. RESULTS 1133.1. The role of p300/CBP-associated factor (PCAF) in the p53 response under diverse types of stress 1133.1.1. BID expression is downregulated in hypoxia-mimicking conditions 1133.1.2. PCAF- and p300-dependent acetylation of p53 under hypoxia 1143.1.3. PCAF is not recruited to p53 proapoptotic targets under hypoxia 1163.1.4. p300 is differentially recruited to p53 target genes in DSFX-treated cells 1183.1.5. PCAF enhances the expression of the p21 p53 target in hypoxia 1193.1.6. PCAF recruitment to the promoters of p53 transcription affects their polyA+ RNA levels 1213.1.7. p53 acetylated at K320 differentially transactivates its transcription targets under DSFX-treatment 1243.1.8. PCAF does not induce the p53-mediated BID expression in DSFX treated cells 1253.1.9. PCAF augments p53-mediated transactivation of BID-p53bs-Luc in DNA-damage. 1263.1.10. Subcellular localisation of PCAF in hypoxia 1283.2. The role of PCAF in HIF-1 activity in hypoxic conditions 1303.2.1. BID is transcriptionally regulated by HIF-1 in hypoxic conditions 1303.2.2. PCAF is a HIF-1 cofactor 1333.2.3. PCAF overexpression upregulates BID transcription after DSFX treatment 1353.2.4. PCAF HAT activity regulates HIF-1-mediated transcriptional activity 1373.2.5. HIF-1 interacts with PCAF in hypoxic conditions 1403.2.6. PCAF overexpression promotes HIF-1 acetylation 1413.2.7. The PCAF HAT activity stabilises HIF-1 1443.2.8. Identification of the HIF-1 site that is targeted by PCAF for acetylation 1463.2.9. PCAF regulates cell cycle progression through p53 and HIF-1 activity 1493.2.10. HIF-1 subcellular distribution in DSFX treated cells 1523.2.11. BID subcellular localisation under diverse stress 1553.2.12. BID subcellular distribution in PCAF-overexpressing cells under diverse stress 1573.2.13. RNA interference towards silencing PCAF 1603.3. The role of the Stress Responsive Activator of p300 in the regulation of p53 and HIF-1 in hypoxic conditions 1623.3.1. Strap is unable to coactivate the p53-dependent BID gene expression in hypoxia 1623.3.2. p53 target promoters occupancy by Strap in hypoxia 1643.3.3. Strap modulates the HIF-1-mediated transcription in DSFX-treated cells 1653.3.4. Strap interacts with PCAF under hypoxic conditions 1693.3.5. Different PCAF isoforms interact with Strap under diverse stress 1703.3.6. Subcellular localisation of Strap under diverse types of cellular stress 1724. DISCUSSION 1784.1. Significance and key findings 1784.2. Detailed analysis of the results 1804.2.1. p53 is hypoacetylated at K320 in DSFX-treated cells 1804.2.2. PCAF promoter occupancy of p53 targets diminishes in DSFX-treated cells 1814.2.3. The p53 K320 acetylated isoform differentially occupies the BID and p21 promoters in hypoxia 1814.2.4. polyA+ RNA levels of p53 transcriptional targets are affected by DSFX-treatment and PCAF overexpression 1834.2.5. Flag-PCAF is recruited to promoters of HIF-1 targets in hypoxia 1834.2.6. PCAF overexpression upregulates BID expression in hypoxia 1844.2.7. PCAF HAT activity plays a role in HIF-1-mediated reporter transactivation 1844.2.8. PCAFwt overexpression increases levels of acetylated HIF-1 and has an effect on its stability 1854.2.9. PCAF plays a role in cell cycle progression under stress through p53 and HIF-1 activity 1874.2.10. BID localises in the cytoplasm in PCAF-overexpressing cells under DSFX treatment 1884.2.11. Strap overexpression is incapable of increasing p53-mediated reporter transactivation in DSFX-treated cells 1894.2.12. Strap overexpression has an effect on HIF-1-mediated BID transactivation in DSFX treatment 1894.2.13. Strap has a differential affinity to PCAF isoforms according to stress 1904.2.14. Strap overexpression does not shift BID cellular distribution 1914.3. Conclusions 1914.4. Future work 1955. BIBLIOGRAPHY 1986. APPENDIX 234
Abstract:
Posttranslational modifications are of great importance in the mediation of transcriptional effects, necessary for signalling in cancer. A characteristic example of such modifications is acetylation of the p53 tumour suppressor, a transcription factor involved in several crucial cellular functions including cell-cycle arrest and apoptosis. p53 is stabilised under hypoxic and DNA damaging-conditions. However, only in the latter scenario is p53 fully capable of inducing the expression of its proapoptotic targets through acetylation. The hypoxia inducible factor 1 (HIF-1) transcription factor is stabilised at low oxygen levels to mediate a cellular adaptive response under these conditions, promoting cell survival. As these two opposing transcription factors share a common transcriptional regulator, p300/CBP, this study focused on deciphering the p300/CBP complex components under differential stress to determine its composition required for cellular responses elicited in response to DNA damage or hypoxia, in an effort to investigate a possible link between differential posttranslational modifications and the resulting cell fate. Hence, the aim of this study was to investigate the roles of p300/CBP components in dictating transcriptional regulation of both HIF-1 and p53 in hypoxic conditions.To carry out this study, the proapoptotic BID gene was the system used, as its promoter contains a p53 response element and a HIF-1 response element (HRE). The p300/CBP associated factors PCAF and Strap were appointed as potent candidates for posttranslational modifications under differential conditions, as they are stress-responsive cofactors. Under DNA damage, PCAF acetylates p53 at K320 and Strap augments p300 binding to p53, both of which amplify the p53 response. Evidence from this study demonstrates that under hypoxia-mimicking conditions PCAF-mediated p53 acetylation at K320 is reduced to a greater extent compared to p300/CBP acetylation at K382. The limited amounts of acetylated p53 at K320 are preferentially recruited to the promoter of the cell cycle arrest p21WAF-1/CIP-1 gene that appears to be unaffected by hypoxia, but fail to be recruited to the BID promoter, rendering p53 incapable of upregulating proapoptotic BID in hypoxic conditions. In addition, under the same conditions, PCAF was found to acetylate, and direct HIF-1 to a particular subset of its targets, leading to alterations in the net physiological effect. Moreover, the intrinsic acetyl transferase activity of PCAF was shown to increase the stability of HIF-1. An additional role was attributed to PCAF in relation to apoptosis, albeit from another angle. BID protein translocation to the cytoplasm in hypoxic conditions was facilitated by ectopically expressed PCAF.Strap was found to be preferentially recruited to the HRE of the BID promoter in hypoxic conditions, and to exert a transrepression effect that appeared to be p53-dependent. Strap also interacted with specific PCAF isoforms depending on the type of cellular stress. Contrary to PCAF, ectopically expressed Strap did not have any effect on BID subcellular distribution. This study has provided additional insight in the mechanisms by which cofactors are involved in cell fate, either by affecting activity and stability of HIF-1 and p53, or having a direct effect on Bcl-2 member subcellular distribution.

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Manchester eScholar ID:
uk-ac-man-scw:152382
Created by:
Demonacos, Constantinos
Created:
16th January, 2012, 19:09:00
Last modified by:
Demonacos, Constantinos
Last modified:
16th January, 2012, 19:09:00

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