Identification and Characterisation of Murine Metastable Epialleles Conferred by Endogenous Retroviruses
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Identification and characterisation of murine metastable epialleles conferred by endogenous retroviruses Anastasiya Kazachenka Department of Genetics Darwin College University of Cambridge September 2017 This dissertation is submitted for the degree of Doctor of Philosophy The research in this dissertation was carried out in the Department of Genetics, University of Cambridge, under the supervision of Professor Anne Ferguson-Smith. This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except specified in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution except specified in the text. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University or similar institution except specified in the text It does not exceed the prescribed word limit of 60,000 words. 1 Summary Anastasiya Kazachenka Identification and characterisation of murine metastable epialleles conferred by endogenous retroviruses Repetitive sequences, including transposable elements, represent approximately half of the mammalian genome. Epigenetic mechanisms evolved to repress these potentially deleterious mobile elements. However, such elements can be variably silenced between individuals – so called ‘metastable epialleles’. The best known example is the Avy locus where an endogenous retrovirus (ERV) of the intracisternal A-particle (IAP) class was spontaneously inserted upstream of the agouti coat colour gene, resulting in variable IAP promoter DNA methylation, variable expressivity of coat phenotype, and environmentally modulated transgenerational epigenetic inheritance within genetically identical individuals. It is not known whether the behaviour exhibited by the ERV at Avy represents a common occurrence throughout the genome or is unusual. Taking a genetic approach in purified cell populations, I have conducted a systematic genome-wide screen of murine metastable epialleles. I have identified over 100 murine IAPs with properties of metastable epialleles. Like Avy, each exhibits a stable epigenetic state within an individual but epigenetic variability between individuals. Methylation levels are locus-specific within an individual, suggesting cis-acting control. The same screening strategy was applied for identification of metastable epialleles associated with other types of LTR-retroelements. However, many of identified candidates showed no inter-individual methylation variation upon experimental validation. These results suggest that IAPs are the dominant class of ERVs capable of acquiring epigenetic states that are variable between genetically identical individuals. I have conducted an analysis of IAP induced initiation and termination of transcription events using de novo assembled transcriptomes generated for B and T cells. 142 IAPs have been identified to overlap de novo assembled transcripts. 32 IAPs are metastable epialleles. Several of them show an inverse correlation between LTR promoter methylation and adjacent gene expression. In addition, I have shown that metastable epialleles have a characteristic pattern of histone modification and are flanked by the 2 methylation sensitive binding factor CTCF, providing testable hypotheses concerning the establishment and/or maintenance of the variable methylation state. My findings indicate that metastability is, in general, specific to the IAP class of ERVs, that only around 1% of these elements have this unusual epigenetic property and that the ability to impact transcription, such as at agouti in Avy, is not a ubiquitous feature of these loci. 3 Contents Summary 2 Contents 4 List of figures 6 List of tables 9 Abbreviations 10 Chapter 1 - Introduction 12 Genome-wide epigenetic reprogramming 15 Epigenetics of repetitive genome 16 Impact of retroelements on transcriptome 18 Epialleles 19 Metastable epialleles 20 Metastable epialleles are sensitive to environmental exposures 22 Metastable epialleles as a model for transgenerational epigenetic inheritance 23 Transgenerational effects at metastable epialleles 26 Identification of novel metastable epialleles 27 Blueprint project 28 B and T cell models 29 Research questions and experimental aims 31 Part 1. Validation of BLUEPRINT datasets and manual curation of cell type specific differentially methylated regions (DMRs). 31 Part 2. Genome-wide identification of novel metastable epialleles. 32 Part 3. Characterization of IAP impact on transcription 33 Chapter 2 - Materials and Methods 35 Chapter 3 - Validation of BLUEPRINT datasets 43 Validation of BLUEPRINT datasets for WGBS and RNA-seq 43 Is there selective absence of imprinting in hematopoietic cell types? 49 Identification of cell type specific differentially methylated regions (DMRs) 52 Summary and discussion 59 Chapter 4 - Genome-wide identification and characterization of murine metastable epialleles 62 Introduction 62 Genome-wide identification of IAP-derived metastable epialleles 63 Characterization of metastable epiallele candidates 74 Relationship between metastability and IAP sequence, chromatin, and genetic background. 79 Strain-specific behaviour of conserved integrations. 86 4 Genome-wide identification of non-IAP-derived metastable epialleles 88 Metastable IAP methylation dynamic during male and female germline development 90 Summary and discussion 95 Chapter 5 - Impact of IAPs on transcription 99 Introduction 99 Strategy design 100 Characterization of IAP-driven initiation and termination events 102 Characterization of splicing events within IAPs 117 Summary and discussion 118 Chapter 6 - General conclusion and perspectives 120 Acknowledgments 127 References 128 5 List of figures Figure 1.1 Project outline. 34 Figure 2.1 Simplified schematic representation example of the 41 genome-wide screening strategy to identify variably methylated ERVs. Figure 2.2 Schematic representation of the identification of transcripts 42 initiated or terminated within IAP and transcripts spliced within IAP. Figure 3.1 Validation of imprinting regions in C57BL/6J. 44 Figure 3.2. Validation of genomic regions differentially methylated 45-46 between B and T cell. Figure 3.3. Validation of imprinting regions in CAST/EiJ. 47 Figure 3.4. Loss of Dlk1 imprinting in B and T cells. 51 Figure 3.5. Distribution of DMRs 55 Figure 3.6. Analysis of manually curated DMRs in B and T cells. 56 Figure 3.7. Relation between DMRs and expression of host genes. 58 Figure 4.1. Summary of the biased screen for metastable epialleles. 65 Figure 4.2. Validation of IAPs identified during biased screen. 68 Figure 4.3. Validation of ERVs identified during biased screen. 69 Figure 4.4. Genome-wide screen for metastable IAPs. 72 Figure 4.5. Summary of genome wide screen 73 Figure 4.6. Validation of interindividual methylation variation at 3’ LTR. 75 Figure 4.7. Relation between 5’ and 3’ LTR methylation. 76 Figure 4.8. Characterization of metastable IAPs. 78 Figure 4.9. Distribution of metastable IAPs across 18 mouse strains 79 6 Figure 4.10. Neighbour-joining tree for IAPLTR1 subtype. 81 Figure 4.11. Methylation variation at closely related IAPs. 82 Figure 4.12. Relative H3K9me3 enrichment profiles of metastable IAP 84 flanking regions. Figure 4.13. Relative CTCF enrichment profiles of metastable IAP 85 flanking regions. Figure 4.14. ChIP-seq profiles of 3 metastable IAPs. 86 Figure 4.15. Strain-specific behaviour of conserved integrations. 87 Figure 4.16. Validation of ERV metastable epiallele candidates. 89 Figure 4.17. IAP methylation dynamics during male germline 93 development. Figure 4.18. Methylation dynamic at metastable (ME) and non-metastable 94 (not ME) IAPs during female germline development. Figure 4.19. Comparison of genome-wide screens for metastable 98 epialleles. Figure 5.1. Screenshots of IAPs impacting transcription 103 Figure 5.2. Characterization of IAPs involved in transcription termination 104 and/or initiation. Figure 5.3. Relation between IAP methylation and its ability to impact 106 transcription initiation and/or termination. Figure 5.4. Validation of Eps8l1 expression. 108 Figure 5.5. Validation of 2610035D17Rik expression. 109 Figure 5.6. Validation of Slc15a2 expression. 110 Figure 5.7. Validation of Bmf and Bub1b expression. 111 Figure 5.8. Validation of Tfpi and Gm13710 expression. 113 7 Figure 5.9. Epigenetic profiles of metastable IAP flanking regions that 115 either overlap (MEs with transcription) or do not overlap (MEs with no transcription) with de novo assembled transcripts. 8 List of tables Table 1. Function and localization of histone marks. 13 Table 2. Murine metastable epialleles. 21 Table 3. Non-genetic inter-/transgenerational effects of parental 25 exposures in rodents Table 4. RNA-seq validation. 48 Table 5. Gene ontology of large 5’end DMRs 57 Table 6. Summary of ERVs present in C57BL/6J and CAST/Eij strains 63 published in Nellaker et al., 2012 Table 7. Summary of identified ERVs 66 Table 8. Summary of potentially metastable IAPs that were not identified 97 in our screen Table 9. Summary of genes potentially regulated by metastable IAPs 114 9 Abbreviations 5mC - 5-methylcytosine 5hmC - 5-hydroxymethylcytosine 5fC - 5-formylcytosine 5caC - 5-carboxylcytosine BER - base excision