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... the split- ubiquitin system The main objective of this analysis is to demonstrate the feasibility of the split- ubiquitin system to detect interactions between human proteins inside human cells and... linker histone hH1 with the other core histone proteins, hH2A, hH2B, hH3 and hH4 have been analyzed inside human HT1080HPRT- cells with the help of the two-component split- ubiquitin system The. .. is the binding site for the TATA-binding protein (TBP) In some genes, the transcription initiation site consists of the initiator element (Inr) defined as an element encompassing the transcription
ANALYSIS OF TRANSCRIPTION FACTORS IN LIVING HUMAN CELLS WITH THE HELP OF SPLIT-UBIQUITIN SYSTEM RASHMI TRIPATHI B.Sc (Honors) Microbiology, University of Delhi THESIS SUBMITTED FOR THE DEGREE OF MASTERS DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2005 ACKNOWLEDGEMENTS I would like to express my gratitude towards Dr Norbert Lehming, my supervisor, for the astute guidance provided during the planning and execution stages of all the experiments He has been instrumental in enabling me to pursue independent work and provided me with the flexibility to try out new ideas His support has been invaluable during the course of this research I would also like to thank Yee Sun for introducing me to the science of tissue culture, her initial help in priming me to handle cell lines proved to be invaluable Her companionship during all these years has been a memorable experience for me I have obtained excellent technical support from Madam Chew, Wee Leng, Foo Chee and Cecilia They have ensured a very smooth running of the lab and have always been more than willing to share their expertise in handling new experimental situations I would like to thank Elicia, Hong Peng, Jin, Linh, Shin and Vivian for their encouragement, friendliness and camaraderie Last but not the least, I would like to express keen appreciation towards my family for the strength to carry on through the ups and downs and the unconditional love and support offered in times most needed They have always inspired me to strive towards the best in life TABLE OF CONTENTS Acknowledgements Table of Contents 3-6 List of Figures 7-9 List of Tables 10 List of Abbreviations 11-12 Measurement Units 13 Summary 14-15 CHAPTER ONE: INTRODUCTION 16 1.1 Aims and Objectives 17 1.2 Gene Expression in Humans: Mechanisms of 18 Transcription 1.2.1 Transcriptional Regulatory DNA Sequences 19 1.2.2 Gene Specific Activation and Repression 23 1.2.3 RNA Polymerase II and the Basic 25 Transcriptional Apparatus 1.2.4 Histones : DNA Packaging Proteins 27 Regulating Transcription 1.2.5 Human Histone Variants: Adding 32 Complexity to Transcriptional Regulation 1.2.6 Linker Histone: Forming Higher Order 37 Structure and Fine Tuning Chromatin Dynamics 1.3 Protein-Protein Interactions and Transcriptional 38 Networks 1.3.1 Current Technologies for Screeening 43 Human Protein-Protein Interactions 1.3.1.1 Mass Spectrometry 43 1.3.1.2 Yeast Two-Hybrid System 45 1.3.1.3 Critical Analysis of Yeast-Two-Hybrid 46 Assay and Mass Spectrometry as High Throughput Tools 1.3.1.4 Protein Chips: Automated Screening 1.3.2 Capturing Protein-Protein Interactions 47 50 Inside Living Human Cells: In Vivo Assays 1.3.2.1 Fluorescence Based Interaction 50 Assays 1.3.2.2 Cell-Signalling Based Interaction 51 Assays 1.4 Split-Ubiquitin System: A Unique Interaction 52 Assay for Transcriptional Proteins CHAPTER TWO: MATERIALS AND METHODS 56 2.1 Testing Interactions Between Linker Histone and 57 Core Histone Proteins inside Human Cells using the Split-Ubiquitin System 2.1.1 Cloning 57 2.1.1.1 PCR, Restriction and Ligation 57 2.1.1.2 Transformation of DH5 cells 58 2.1.1.3 Miniprep 59 2.1.1.4 Checking for Positive Clones 62 2.1.1.5 DNA Sequencing 62 2.1.2 Cloning into pCMV-myc Plasmid 64 2.1.3 Plasmid Purification using QIAfilter Plasmid 65 Midi Kit 2.1.4 Cell Lines and Transfections 2.1.4.1 Construction of Cell Lines Expressing 67 68 hH1-CubRGpt2 2.1.4.2 Co-expression of Nub Constructs with 69 hH1-CubRGpt2 2.1.5 Western-blot 70 2.1.6 Co-immunoprecipitation 71 2.1.7 Molecular Modeling 73 2.2 Using AAV Particles for Protein-Protein 73 Interaction Screening 2.2.1 Cloning 73 2.2.2 Cell Line and Transfection 74 2.2.3 Viral Stock Production 77 2.2.4 Viral infection 77 2.2.5 Fluorescence Activated Cell Sorting 78 2.2.6 PCR and Sequencing of Stably Integrated 78 Viral DNA CHAPTER THREE: ANALYSIS OF PROTEIN- 81 PROTEIN INTERACTIONS BETWEEN THE LINKER HISTONE AND THE CORE HISTONES USING THE SPLIT-UBIQUITIN SYSTEM 3.1 Theory 82 3.2 Results 84 3.3 Discussion 96 3.4 Conclusion 100 3.5 Future Work 100 CHAPTER FOUR: USING AAV PARTICLES FOR 102 SCREENING PROTEIN-PROTEIN INTERACTIONS INSIDE LIVING HUMAN CELLS 4.1 Theory 103 4.2 Results 108 4.3 Discussion 113 4.4 Conclusion 118 4.5 Future Work 118 BIBLIOGRAPHY 120 LIST OF FIGURES CHAPTER ONE 1.1 Steps in chromatin assembly 21 1.2 Promoter architecture in yeast and mammals 22 1.3 Regulating nucleosomal mobility 24 1.4 A model for transcriptional initiation involving RNA 26 polymerase II holoenzyme complex 1.5 Nucleosome core particle) 28 1.6 Annotation map of known histone modifications on 30 the surface of the X laevis nucleosomal core particle 1.7 Human histone gene clusters spread over seven 34 human chromosomes 1.8 Known histone variants and their functions 1.9 35 40 Modular Nature of Protein Complexes 1.10 Tandem Affinity Purification strategy showing the 48 design of C-terminal and N-terminal TAP tags (left) and the overall complex 1.11 The Yeast Two Hybrid System 48 1.12 Design of the Split-Ubiquitin System 55 CHAPTER TWO 2.1 Map of pcDNA3.1(+) /Zeocin vector 60 2.2 Map of pcDNA3.1/Neomycin vector 61 2.3 Map of pCMV-myc Vector used for co- 66 immunoprecipitation 2.4 Vector Maps of pAAV-IRES-hrGFP, pAAV-RC and 76 pHelper CHAPTER THREE 3.1 Phenotype testing and Western-blot analysis of 86 HT1080HPRT- cells expressing hH1-CubRGpt2 3.2 hH1 interacts with core histones hH3 and hH4 and 87 not hH2A and hH2B 3.3 Western-blot analysis showing in vivo Interaction 90 between linker and core histones hH3 and hH4 3.4 Co-immunoprecipitation showing binding of linker 91 histone with core histones hH3 and hH4 3.5 Molecular modeling confirms contact of linker 94-95 histone at the dyad axis of symmetry CHAPTER FOUR 4.1 Schematic representation of protocol for screening 111 of protein-protein interactions inside human HT1080HPRT- cells expressing hH1-CubRGpt2 4.2 AAV-293 cells 114 4.3 Isolation of genomic DNA from clones 115 4.4 PCR amplification to detect viral plasmid DNA 115 inserted into genomic DNA List of Tables CHAPTER ONE 1.1 Interaction coverage of protein-protein interactions 42 by species (Adapted from Bork et al., 2004) CHAPTER TWO 2.1 Human histones amplified by PCR from the 63 indicated human cDNA sources 2.2 PCR reaction mix per sample 63 2.3 Ligation reaction per sample 63 2.4 Restriction digests to screen for positive inserts 63 CHAPTER FOUR 4.1 Viral titer estimation after infection of 116 HT1080HPRT- cells with pAAV-LacZ stocks 4.2 Recovery and selection of cells in 6-TG after 116 FACS 10 cells were appropriately gated to isolate them Cells were sorted into 24-well plates at a density of 50 cells per well After allowing a recovery period of two days, the cells were trypsinized and transferred into medium containing 6-TG and zeocin to select for positive protein-protein interactions Table 4.2 highlights the recovery rate of cells after FACS in these 24-well plates Selection in 6-TG for Protein-Protein Interactions-Cells from each well were transferred into medium containing 6-TG and zeocin and selected for protein 110 Nub-X Inserted pAAV-NubX-IRES-hrGFP pHelper pAAV-RC AAV293 Packaging Cell Line AAV particles carrying pAAV-NubX-IRES-hrGFP HT1080 HPRT- + cells expressing hH1-CubRGpt2 and hrGFP di-cistronically FACS Selection of positively transduced hrGFP clones in 6-TG Amplify clones in 75cm2 flasks Isolate Genomic DNA PCR Amplification of Integrated viral DNA, column purification of PCR product and sequencing to identify inserts Figure 4.1 Schematic representation of protocol for screening of protein-protein interactions inside human HT1080HPRT- cells expressing hH1-CubRGpt2 111 interactions in vivo Genomic DNA was extracted from the cell cultures by phenol-chloroform extraction (See Figure 4.3) The integrated viral DNA was amplified specifically by using primers hybridizing within the ITR cassette As a control, PCR was also carried out to amplify histone hH3 as an indication of the quality and intactness of the genomic DNA isolated (Figure 4.4) For the positive control, a PCR Product of 312 bp was detected in lanes (1, 3, 5, 7, 9, 11, 13 and 15) Histone hH4 was amplified from the genomic DNA in lanes and (533 base pairs), histone hH3 in lanes and (632 base pairs) and hProthymosin in lanes 10 and 12 (544 base pairs) Nub (expected size 372 base pairs) was not amplified from any of the genomic DNA isolated This indicated that that there were no stable integrations of the viral DNA containing Nub (See Figure 4.4.) As can be seen from Table 4.1., the flow sorting procedure was highly efficient in enriching cells expressing Nub fusion proteins di-cistronically with GFP Moreover all the candidates that were tested for stable integration of the viral plasmid DNA into the genome using PCR showed positive amplifications For pAAV-Nub-IRES-GFP, only three colonies showed growth in 6-TG and zeocin To rule out the possibility of any interaction of Nub with the bait protein, PCR was carried out to detect integrations that might have taken place into the genomic DNA of HT1080HPRT- cells However, no integrants were detected, indicating that the colonies which grew in 6-TG and zeocin actually arose because of antibiotic resistance developing in these rapidly dividing cells due to a certain low level spontaneous mutations Sequencing PCR Products- The PCR products were purified and sequenced using the Nub129 primer Sequences of human histones hH3, hH4 and 112 hProthymosin were retrieved by performing a Nucleotide-nucleotide BLAST (blastn) available in the NCBI website 4.3 DISCUSSION AAV-2 or Adeno-associated viruses are replication deficient viruses which have the highest bio safety ratings among all other viruses currently being used for gene delivery and expression in human cell lines Moreover these viruses have a high rate of integration into the human genome, specifically into Chromosome 17 (Waltz and Schlehofer, 1992) These reasons justify the utilization of the AAV-Helper-Free-System for screening of protein-protein interactions inside human HT1080HPRT cells As part of the split-ubiquitin system, hH1-CubRgpt2 has been employed as a bait protein Using this novel protocol for screening a potential human cDNA library (Figure 4.1.), potential interacting partners fused to Nub can be isolated by selection of HT1080HPRT cells expressing hH1- CubRGpt2 in a medium containing 6-TG and zeocin The pAAV-IRES-hrGFP plasmid contains a di-cistronically expressed hrGFP protein which is a useful marker for selection of positively infected HT1080HPRT cells using Fluorescence Activated Cell Sorting Procedure This process is highly efficient in enriching cells expressing the Nub- fusion proteins along with hrGFP These cells are then subjected to the second selection step involving 6-TG and zeocin Isolation of genomic DNA and PCR amplification of the integrated DNA further eliminates any false positives obtained in 6-TG and zeocin 113 AAV-293 cells (Uninfected) AAV-293 cells (with cytopathic effects AAV-293 cells (hrGFP expression) Figure 4.2 AAV-293 cells Uninfected AAV-293 cells (top); AAV-293 cells showing cytopathic effects indicating AAV-2 production after transfection with pAAV-Nub-IRES-hrGFP plasmid, pAAV-RC and pHelper plasmids (middle); AAV-293 cells showing hrGFP expression under the inverted fluorescence microscope (bottom) 114 Figure 4.3 Isolation of genomic DNA from clones 10 11 12 13 14 15 16 750bp 500bp 250bp Figure 4.4 PCR Amplification to Detect Viral Plasmid DNA Inserted into Genomic DNA Two independent colonies were picked following selection in 6-TG after infecting hH1-CubRgpt2 expressing HT1080HPRT- cells with pAAV-Nub-hH4IRES-hrGFP (lanes 1-4), pAAV-Nub-hH3-hrGFP (lanes 5-8), pAAV-NubhProthymosin-hrGFP (lanes 9-12), and pAAV-Nub-hrGFP (lanes 13-16) carrying AAV particles PCR was performed using either primers for the gene encoding human histone hH3 as a positive control (Size 411 base pairs) for isolation of intact human genomic DNA from the HT1080HPRT- cell line clones infected as mentioned above (Lanes 1, 3, 5, 7, 11, 15) or using primers lying in the BetaGlobin intron and IRES sequences to amplify the viral plasmid DNA integrated into the genome Histone hH4 was amplified from the genomic DNA in lanes and (533 base pairs), histone hH3 in lanes and (632 base pairs) and hProthymosin in lanes 10 and 12 (544 base pairs) Nub (expected size 372 base pairs) was not amplified from any of the genomic DNA isolated indicating that that there were no stable integrations of the viral DNA 115 Table 4.1 Viral Titer Estimation after Infection of HT1080HPRT- Cells with pAAV-LacZ stocks Dilution Number of Infected Cells per Field IFU/ml TNTC~ 68 56 33 1.22x 107 5.01x 107 1.48x 107 1x 10-2 2x 10-3 4x 10-4 8x 10-5 Table 4.2 Recovery and Selection of Cells in 6-TG after FACS Candidate pAAV-Nub-IRES-hrGFP pAAV-Nub-Hh3-IRES-hrGFP pAAV-Nub-hH4-IRES-hrGFP pAAV-Nub-hProthymosinIRES-hrGFP Number of Wells which showed growth in DMEM Number of which showed growth in DMEM+6-TG 22/24 19/24 21/24 18/24 19 21 18 Number of candidates showing integrations by PCR 2/3 2/2 2/2 116 6-thioguanine or 6-TG is a cytotoxic purine and spontaneous resistance to this compound has been shown to arise on a number of occasions in various cell lines This resistance is mediated by loss or reduction in hypoxanthine phosphoribosyltransferase (HPRT) activity This phenomenon has been recorded in human lymphoid cells (Duncan, 1977) and in Chinese Hamster Ovary (CHO) cells (Manjanatha et al., 1994) Mutants isolated in CHO cells were shown to contain certain exon-deletions, point deletions, missense or nonsense mutations in the HPRT gene Hence it was not surprising that a few false positives were obtained for cells growing in medium containing this selecting agent Posttranslational modifications of human proteins play an important role in governing their folding and hence determine the possible interacting partners for a given protein expressed inside human cells This fact has been shown to be especially relevant for histone proteins whose modification status can lead to the recruitment of different chromatin binding proteins Hence making use of the split-ubiquitin system inside human cells for screening of interacting partners for human proteins, presents itself as a powerful tool for identifying relationships between proteins involved in regulating chromatin architecture like the histones The efficiency of isolation of interacting partners depends on the successful execution of Fluorescence Activated Cell Sorting (FACS) procedure to purify positively transduced cells The population of cells expressing GFP currently is not very high (about to 7%) The percentage of infected cells could further be increased by devising certain viral purification procedures to concentrate viral stocks like affinity and ion-exchange chromatography 117 4.4 CONCLUSION Utilizing Adeno-Associated Viruses to deliver Nub fusions into HT1080HPRTcells expressing hH1-CubRGpt2 as a bait protein is an efficient and a reliable method for determining novel protein-protein interactions for human proteins This protocol has a low rate of obtaining false positives which can nevertheless be easily identified by PCR amplification of integrated viral DNA This AAV based method can hence be used to deliver a human cDNA library into cells and colonies obtained in 6-TG would reveal interacting partners for any chosen bait protein Subsequent isolation of genomic DNA, PCR and sequencing would yield the identity of interacting partners 4.5 FUTURE WORK This protocol is ideal for screening a cDNA library constructed from RNA isolated from any of the human cell-lines in common use A cDNA library could be cloned in pAAV-Nub-IRES-hrGFP vector containing Nub in three reading frames One very exciting option would be to construct cDNA libraries based on RNA isolated from embryonic stem cell lines Screening of interacting partners using these cDNA libraries could yield interesting insights regarding mechanisms that govern changes in chromatin accessibility and dynamics during various stages of development and differentiation Using cancer cell line-based cDNA libraries could also help identify valuable interactions for key regulatory proteins involved in cancer signaling and manifestation of tumors To further validate any newly 118 discovered interactions, alternative in vitro procedures like GST pull-down and co-immunoprecipitation would have to be carried 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