Investigation Of DNA Repair Gene Expression And Protein .

Investigation of DNA repair gene expressionand protein function in human oocytes andpreimplantation embryosbyPinar TulayThesis submitted for the degree of Doctor of Philosophy atUniversity College LondonJune 2013Supervisors: Dr. Sioban SenGuptaDr. Joyce HarperUCL Centre for PGDInstitute for Women’s HealthUniversity College London1

I, Pinar Tulay, confirm that the work presented in this thesis is my own. Whereinformation has been derived from other sources, I confirm that this has beenindicated in the thesis.2

AcknowledgementsACKNOWLEDGEMENTSIt is my pleasure to thank to those who made this project possible and helpedthrough my PhD. First of all, I would like to thank my dear supervisor, SiobanSenGupta. Sioban, you have helped and guided me throughout my PhD andPGD training. You have patiently listened to me several times to talk about howthings with the experimental work are not progressing as I wanted. You alwayssupported me. It was a great pleasure to be your PhD student. Without yourhelp, guidance and continuous support, I wouldn’t have been at this stage now.I have already missed our 3-hour PGD and PhD meetings. Thank you verymuch for making me a better scientist.I would like to thank to my secondary supervisor, Dr. Joyce Harper. Joyce, youwere the first person who introduced me to preimplantation genetics during myMaster’s degree. Your passion in this field made me grow a great interest inpreimplantation genetics and embryology. I would like to thank you for yourwarm welcoming to this field and all your support and guidance throughout myPhD. I would also like to thank to Professor Joy Delhanty for her expert adviceand guidance through my PhD and PGD training. I would like to thank to thewhole PGD team; Seema Dhanjal, Thalia Mamas, Leoni Xanthopoulou andHarita Gheveria. Seema, you have helped me though my PGD training. I havelearned so much from you. Thank you for being patient with me and being thereto support both the experimental side of things and personal things. Thank youvery much for reading this thesis- your comments were very much appreciated.I miss our tube rides to Waterloo. Thalia, you were the first person at the UCL(starting from my Master’s) to help me. You were extremely patient with me. Ihave learned what I know about aCGH from you. It was very nice to know that Icould turn to you when I felt down. Leoni, thank you very much for helping mewith the FISH work-ups. I am very glad that you were there with me at CMduring long hours at the weekends- it was very nice to come to the office inbetween running around in the lab to have lunch with you and take shortbreaks. Harita, thank you for your help in FISH work-ups and being a friend. I3

Acknowledgementswill miss going to conferences with you. Roy, thank you for all your guidanceand statistical advice for my expression study. You have been always a goodfriend and supported me when I felt down- thank you very much. I also wouldlike to thank to the rest of the group members; Aisha, Razan and Amanthi- itwas nice working with you all. Razan, thank you for helping me sort out labthings after I left London.I also would like to thank to my friends outside the lab for all their support andmotivation- Didem, Melek and Acelya. Didem, you are one of my best friends.You were always there to support me and calm me when I felt much stressed. Itwas very comforting to know that I could turn to you when I needed.I also would like to acknowledge all the patients who made this project possibleby giving consent to use their samples.Finally and most importantly, I would like to thank to my family. My dear sister,Bahar, I am extremely lucky to have you as a sister. You always supported me.No matter what time I needed you, you were there to keep me motivated andcheer me up. My brother in law, Necati, thank you for cheering me up when Ithought the end would never come. My little niece, Melisa, even though you arevery little to know that you were a great courage to me, you have motivated meand kept me going by saying: “auntie finish school and come home”. Mygrandmother, Fikriye, and my aunt, Semral, thank you very much to you both.You have called me and checked on me constantly to keep me company while Iwas far away from home.And finally, my parents, Muyesser and Erdal, there are not enough words toexplain how grateful I am to you. You have supported me until this age withoutthinking twice. You stood by all my decisions. You encouraged me to start myPhD and complete it. You have motivated me all the time. I hope in return I hadmade you proud. I could not have accomplished any of this without you. Thankyou for loving me and supporting me.4

AcknowledgementsI am dedicating this PhD to my parents, Muyesser and Erdal.5

AbstractAbstractThis study investigated microRNA and mRNA expression and protein functionassociated with DNA repair in human oocytes and embryos.MicroRNAs have been shown to down-regulate and in some cases to stabilisethe expression of several genes including repair genes. The first aim of thisstudy was to analyse the differences in the expression of microRNAs and theirtarget mRNAs involved in repair. This study showed that the levels ofexpression of most of the repair gene mRNAs were higher in oocytes thanblastocysts and this was also the case for many of the miRNAs. The correlationanalysis of the miRNA and their target expression levels in the oocyte andblastocyst samples were restricted by the limited size and number of availablesamples, but indicated no clear cut pattern of differences in relative expression.Differential methylation of parental genomes that may lead to differentialparental gene expression had been observed previously in mouse embryos.The second aim of this study was to investigate differential parental expressionof BRCA1 rnal BRCA1 expression exists in the early developing embryos. Moreover,embryos with paternally inherited BRCA mutations were shown to develop moreslowly compared to embryos with maternally inherited BRCA1 mutations. Bothdifferential expression and the developmental delay may be associatedwith differential methylation of parental genomes.Expression analyses are crucial to investigate the potential function of genes.However these analyses do not provide information on the functionalityof biological processes. Therefore, the final part of this study aimed to develop ficiencyinpreimplantation embryos. In this study a unique assay was developed to detectmismatch repair efficiency using small amounts of nuclear/whole cell extractsand experiments demonstrated that mismatch repair is active in mouse andhuman blastocysts.6

Table of ContentsTable of Contents1. Introduction. 241.1.DNA repair in human oocytes and preimplantation embryos . 251.2.Gametogenesis and preimplantation embryo development . 261.3. Origin of DNA damage and DNA repair in gametes and preimplantationembryos . 291.3.1. Current knowledge on the techniques used for investigating theactivity of repair pathways in oocytes and preimplantation embryos . 301.3.1.1. Functional studies . 301.3.1.2. Proteomic studies . 311.3.1.3. Gene expression studies . 311.3.1.3.1. Expression of DNA damage sensor genes and cell cyclecheckpoint genes in oocytes and preimplantation embryos . 321.3.1.3.2. Expression of base excision repair genes in gametes andpreimplantation embryos . 331.3.1.3.3. Expression of nucleotide excision repair genes in gametes andpreimplantation embryos . 351.3.1.3.4. Expression of double strand break repair genes in gametesand preimplantation embryos . 391.3.1.3.5.Homologous recombination repair . 401.3.1.3.6.Non-homologous end-joining repair . 421.3.1.3.7. Expression of mismatch repair genes in gametes andpreimplantation embryos . 441.3.1.3.8.1.4.Interstrand crosslink repair . 47Control of gene expression . 501.4.1.Control of gene expression by methylation . 507

Table of Contents1.4.1.1. Methylation status of the zygote and the preimplantation embryos511.4.1.2. Methylation status of the gametes . 531.4.2.Control of gene expression by miRNA regulation . 531.4.2.1. MiRNA biogenesis . 531.4.2.2. Regulation of mRNA expression by miRNAs . 591.4.2.3. MiRNA expression in gametes and preimplantation embryos . 601.4.2.4. Regulation of DNA repair genes by microRNAs expressed ingametes and preimplantation embryos . 61Base excision repair genes and miRNAs expressed in preimplantationembryos. 67Nucleotide excision repair genes and miRNAs expressed in preimplantationembryos. 69Double strand break repair and miRNAs expressed in preimplantationembryos. 71Mismatch repair and miRNAs expressed in preimplantation embryos . 741.5.Aims and hypothesis . 761)The expression level of miRNAs and their target repair transcriptsin human oocytes and preimplantation embryos . 762)Differential gene expression in preimplantation embryos andpotential relation with differential methylation . 773)Functional assay development for mismatch repair . 782. Materials and Methods . 802.1.General laboratory methods and standards . 812.2. Potential correlation analysis between miRNA and their target repairtranscripts in human oocytes and preimplantation embryos . 842.2.1.Sample collection . 852.2.2.Target selection . 862.2.3.Sample processing and analysis . 882.2.3.1. Reverse transcription for mRNA and miRNA expression . 888

Table of Contents2.2.3.2. Real time PCR for mRNA and miRNA. 892.2.3.3. Statistical analysis . 902.3. Differential gene expression in preimplantation embryos and potentialrelation with differential methylation . 912.3.1.Sample collection . 922.3.1.1. Sample selection . 922.3.2.Sample processing and analysis . 932.3.2.1. DNA extraction from whole blood samples. 932.3.2.2. Lymphocyte separation using the ficoll-paque plus method . 942.3.2.3. Primer design . 942.3.2.4. Polymerase chain reaction (PCR) . 952.3.2.5. Agarose gel electrophoresis . 962.3.2.6. Sequencing . 97Purification of amplified samples . 97Cycle sequencing reaction . 97Purifying the extension product . 98Analysis of sequencing products . 982.3.2.7. DNA and RNA extraction from embryos . 982.3.2.8. Assessment of nucleic acids by Nanodrop and Bioanalyzer . 992.3.2.9. Reverse transcription of RNA obtained from embryos . 1002.3.2.10. Mini-sequencing (SNaPshot ) . 1002.3.2.11.SNaPshot assay sensitivity for semi-quantitative analysis . 1022.3.2.11.1. Real time PCR validation . 1022.3.2.11.2. Chromosome copy number determination by PCR . 1032.3.2.11.3. Aneuploidy screening by aCGH . 104Whole genome amplification . 1049

Table of ContentsLabelling of the test and re

This study investigated microRNA and mRNA expression and protein function associated with DNA repair in human oocytes and embryos. MicroRNAs have been shown to down-regulate and in some cases to stabilise the expression of several genes including repair genes. The first aim of this study was to analyse the differences in the expression of microRNAs and their target mRNAs involved in repair .