The Effects of Overexpression of DNA Methyltransferase 3-like (DNMT3L) and Transmission Through the Murine Male and Female Germlines A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science Department of Human Genetics at McGill University Montreal, Quebec, Canada July 2014 © Patrick Boisvert, 2014 1 TABLE OF CONTENTS Table of Contents 2 Abstract 6 Résumé 7 List of Figures 9 List of Tables 10 List of Abbreviations 11 Acknowledgements 13 Contribution of Authors 17 Introduction 18 o Epigenetics 18 DNA methylation 18 o Biochemistry 18 o Genomic distribution of CpGs 19 o CpG islands 19 o Non-CpG methylation 20 o Role of DNA methylation in transcriptional regulation 20 o Genomic imprinting 21 o X-inactivation 21 o Disorders linked to abnormal methylation 22 o DNA methylating enzymes 22 o DNMT1 22 o DNMT2 23 o DNMT3 23 o DNMT3L 24 o Interaction between DNMT3L and DNMT3a2 24 Developmental dynamics of DNA methylation 25 o Disorders due to Dnmt deficiency 25 o DNA methylation dynamics in embryogenesis 25 2 o Spermatogenesis 26 o DNA methylation in male gametogenesis 28 o DNA methylation analysis using RRBS 30 o Recent DNA methylation studies using bisulfite sequencing 31 o Dnmt3L in male gametogenesis 31 o Studies of germline modulation of Dnmt3L expression 32 Project Outline 34 Materials and Methods 37 Mice 37 Characterization of transgene transmission to F1, F2 and F3 37 o Determination of transgene copy number 37 o Propagation of transgene from founder (F0) to F1, F2, and F3 37 o Measurement of reproductive organ weights 38 o Sperm collection 38 o Assessment of the quality of seminiferous tubules 38 o Sperm counts 38 o Assessment of transgene expression in testis cross-sections by fluorescence microscopy 39 Sperm DNA methylation analysis using RRBS 39 o RRBS library preparation 39 o RRBS library sequencing and methylation data analysis 40 Dnmt3L (-/-) infertility rescue experiment using the EF1α-Dnmt3L transgene 40 o Generation of EF1α-Dnmt3L transgenic, Dnmt3L (-/-) males and females 40 Statistical Analysis 41 Results 42 Outcomes of Dnmt3L transgene overexpression, transmission through the male germline 42 o Matings produced F1, F2 and F3 animals in male germline transgene transmission study 42 o Transgene expression in male germ cells localized near basal lamina of 3 seminiferous tubules 42 o Rates of male germline transgene transmission to F1 and F2 were mostly normal in all lineages 42 o Significant decrease in average litter size from F1 to F2 in high copy lineage 43 o No difference in reproductive organ weights between transgenic and non- transgenic males 43 o No difference in sperm counts between F2 transgenic and non-transgenic males 44 o Decline in quality of seminiferous tubules in testis cross-sections of F1 males 44 o Decline in quality of seminiferous tubules in testes of high-copy transgenics from F1 to F2 45 Genome-wide DNA methylation analysis of transgenic, non-transgenic sperm in F1 and F2 45 o DNA methylation levels similar overall among transgenic, non-transgenic and control samples 45 o Predominance of differently methylated tiles found in intergenic, intronic, and exonic regions 46 o Methylation in DMRs found in exons higher in transgenics vs controls, and F2 vs F1 transgenics 46 o Methylation in DMRs in exons up in F2 non-transgenics vs controls, F2 vs F1 non-transgenics 47 Attempt to rescue infertility and methylation abnormalities in Dnmt3L deficient males 47 o Generation of TG+, Dnmt3L (-/-) and TG-, Dnmt3L (-/-) males for infertility rescue 47 o Infertility in low copy control males 48 o Lack of fertility rescue in TG+, Dnmt3L(-/-) males from the low copy lineage 48 4 o Infertility in high copy control males 48 o Lack of fertility rescue in TG+, Dnmt3L (-/-) females from low and high copy lineages 49 o No difference in paired testes weights of TG+, Dnmt3L (-/-) and TG-, Dnmt3L (-/-) males 49 Characterization of the transgene model upon transmission through female germline 50 o Low rate of transgene transmission from female founders to F1 in most lineages 50 o Trend to decrease in average litter size between birth and weaning age 50 Discussion 51 Reproductive effects of EF1α-Dnmt3L transgene transmission through the male germline 51 On the discrepancy between the apparent effect of Dnmt3L overexpression on testicular histology, and the lack of effect on organ weights and sperm counts 53 Effects of EF1α-Dnmt3L transgene overexpression on mature sperm DNA methylation 56 Attempt to correct infertility in Dnmt3L (-/-) males using the EF1α-Dnmt3L transgene 61 Comparison of outcomes upon EF1α-Dnmt3L transgene transmission through male and female germlines 63 References 65 Figures 81 Tables 103 5 ABSTRACT The DNA methyltransferase 3-like (DNMT3L) enzyme is critical to proper de novo DNA methylation acquisition in germ cells and to gametogenesis. In mice, Dnmt3L deficiency or haploinsufficiency leads to reproductive abnormalities and germ cell methylation defects. Dnmt3L heterozygous males sire offspring with an increased incidence of aneuploidy, and Dnmt3L-deficient males and females are both infertile. These abnormal reproductive outcomes correlate with hypomethylation in germ cells of these animals. In terms of DNMT3L dosage then, it is known that lack/reduction of this enzyme in germ cells leads to DNA methylation and phenotypic defects. The reproductive effects of increased DNMT3L dosage are not known, however. Therefore we evaluated the outcome of Dnmt3L overexpression in male and female mouse germ cells, using an EF1α-Dnmt3L transgene previously created in our lab, and the effect of propagating the transgene through the male and female germlines for successive generations. In the female germline transmission experiment, 28.8% of all F1 pups died between birth and weaning, and the rate of transgene transmission to surviving pups was only 22.3%. The low number of transgenic pups in F1 led to termination of 5 of the 7 lineages in F1, and the 2 remaining lineages were terminated in F2. In the male germline transmission experiment, males from the lineage with the highest number of transgene copies displayed a significant decrease in average litter size at weaning from F1 to F2. Transgenic males from this lineage showed significantly more abnormal seminiferous tubules in their testis cross sections than controls, and a significant increase in abnormal tubules from F1 to F2. These findings prompted us to examine the DNA methylation patterns in the sperm of these males, using a genome-wide, sequencing method called reduced representation bisulfite sequencing (RRBS). RRBS analysis revealed that, overall, DNA methylation patterns in transgenic sperm were not different from non-transgenic or control sperm. However, some differentially methylated regions (DMRs) between transgenic and control sperm were found. DMRs located in exons were hypermethylated in transgenic versus control sperm, and hypermethylated in F2 vs F1 transgenic sperm. It therefore appears that transgenic overexpression of Dnmt3L in our model leads to subtle reproductive and sperm DNA methylation defects. Although preliminary, it is possible that the worsening of these defects from F1 to F2 is due to transgenerational epigenetic inheritance. 6 RÉSUMÉ L’enzyme ADN méthyltransférase 3-like (DNMT3L) est essentiel durant l’établissement initial de la méthylation de l’ADN dans les cellules germinales mâles et femelles, ainsi que dans le développement de ces dernières. Chez la souris, une déficience de cet enzyme mène à des troubles au niveau de la reproduction, ainsi qu’à des défaults de méthylation d’ADN dans la lignée germinale. La progéniture de mâles hétérozygotes démontre un taux plus élevé d’aneuploïdies, et les mâles et femelles homozygotes mutants sont infertiles. Ces anomalies reproductives sont liées à une hypométhylation de l’ADN dans la lignée germinale de ces animaux. Donc, en terme de dose de DNMT3L, nous savons qu’une réduction donne lieu à des défauts de méthylation d’ADN ainsi qu’à certains phénotypes. Par contre, nous ne connaissons pas l’effet d’une augmentation de la dose de DNMT3L. Nous avons donc évalué l’issue d’une surexpression de Dnmt3L dans la lignée germinale mâle et femelle chez la souris, en utilisant un transgène, EF1α-Dnmt3L, que nous avons propagé à travers la lignée germinale durant plusieurs générations. Dans l’expérience de transmission du transgène à travers la lignée germinale femelle, 28.8% de tous les descendants à la génération F1 sont morts entre la naissance et l’âge de sevrage, et le taux de transmission du transgène aux descendants qui ont survécu jusqu’au sevrage était de 22.3%. Le manque de descendantes femelles à la génération F1 nous a fait mettre fin à 5 des 7 lignées, tandis que nous avons mis fin aux 2 autres lignées à la génération F2. Dans l’expérience de transmission du transgène à travers la lignée germinale femelle, les mâles issus de la lignée contenant le plus haut nombre de copies du transgène ont démontré une diminution significative du nombre de descendants par portée au sevrage de la génération F1 à F2. Les mâles transgéniques de cette lignée ont aussi démontré un nombre significativement plus élevé de tubes séminifères anormaux dans les coupes transversales de leurs testicules comparativement aux contrôles, ainsi qu’une augmentation significative de tubes séminifères anormaux de la génération F1 à F2. Ces observations nous ont mené à examiner les patrons de méthylation d’ADN dans le sperme de ces mâles, en utilisant le séquençage à l’échelle du génome entier par technique de représentation réduite de séquençage bisulfite (RRBS). Les résultats de l’analyse par RRBS ont révelé que, globalement, les patrons de méthylation d’ADN dans le sperme de mâles transgéniques n’étaient pas différents de ceux du sperme de mâles non- 7 transgéniques ou contrôles. Par contre, certaines régions méthylées différemment (DRMs) ont été découvertes.