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Nuclear Organization and the Epigenetic Landscape of the Mus Musculus X-Chromosome Alicia Liu University of Connecticut - Storrs, [email protected]

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Nuclear Organization and the Epigenetic Landscape of the Mus Musculus X-Chromosome Alicia Liu University of Connecticut - Storrs, Alicia.Liu@Uconn.Edu University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 8-9-2019 Nuclear Organization and the Epigenetic Landscape of the Mus musculus X-Chromosome Alicia Liu University of Connecticut - Storrs, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Liu, Alicia, "Nuclear Organization and the Epigenetic Landscape of the Mus musculus X-Chromosome" (2019). Doctoral Dissertations. 2273. https://opencommons.uconn.edu/dissertations/2273 Nuclear Organization and the Epigenetic Landscape of the Mus musculus X-Chromosome Alicia J. Liu, Ph.D. University of Connecticut, 2019 ABSTRACT X-linked imprinted genes have been hypothesized to contribute parent-of-origin influences on social cognition. A cluster of imprinted genes Xlr3b, Xlr4b, and Xlr4c, implicated in cognitive defects, are maternally expressed and paternally silent in the murine brain. These genes defy classic mechanisms of autosomal imprinting, suggesting a novel method of imprinted gene regulation. Using Xlr3b and Xlr4c as bait, this study uses 4C-Seq on neonatal whole brain of a 39,XO mouse model, to provide the first in-depth analysis of chromatin dynamics surrounding an imprinted locus on the X-chromosome. Significant differences in long-range contacts exist be- tween XM and XP monosomic samples. In addition, XM interaction profiles contact a greater number of genes linked to cognitive impairment, abnormality of the nervous system, and abnormality of higher mental function. This is not a pattern that is unique to the imprinted Xlr3/4 locus. Additional Alicia J. Liu - University of Connecticut - 2019 4C-Seq experiments show that other genes on the X-chromosome, implicated in intellectual disability and/or ASD, also produce more maternal contacts to other X-linked genes linked to cognitive impairment. The observation that there are dif- ferences between the maternal and paternal X interactomes is bolstered by potential variation in Atrx binding and H3K27me3 enrichment between XM and XP, sug- gesting that there may be broad-scale differences of the X-chromosome, related to parent-of-origin effects. Taken together, these results provide intriguing insight into the maternal X susceptibility to cognitive and social impairment. Nuclear Organization and the Epigenetic Landscape of the Mus musculus X-Chromosome Alicia J. Liu Bachelor of Science, University of Delaware, 2012 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2019 Copyright by Alicia J. Liu 2019 i APPROVAL PAGE Doctor of Philosophy Dissertation Nuclear Organization and the Epigenetic Landscape of the Mus musculus X-Chromosome Presented by Alicia J. Liu, B.S. Major Advisor Michael O’Neill Associate Advisor Rachel O’Neill Associate Advisor John Malone University of Connecticut 2019 ii To Fat Stanley, protector of the pumpkin patch. iii ACKNOWLEDGEMENTS I wish to express my deepest gratitude to the people in my life whose encourage- ment and support made this dissertation possible. First and foremost, the biggest thanks go to Mike. Thank you for being my advi- sor. Thank you for inspiring me and motivating me to meet every challenge, while also giving me the independence to grow and mature into the scientist I am today. The skills I learned in your lab I will carry with me the rest of my life. Besides my advisor, I extend thanks to the rest of my committee members for their years of support and invaluable advice. Rachel and John, your insights have been an incredible resource to me over the course of this dissertation. Leighton and Bibi, your course work and expertise provided the foundation upon which a lot of this work was built. What I have learned from my committee has been a tremendous help in designing and analyzing experiments, and for that I am incredibly grateful. I would also like to thank my lab mates, both past and present, for their contin- ued support, and for making the M. O’Neill lab a wonderful, fun, and productive environment. Natali, for always being there as a great friend and supportive brain to lean on. Rob, who answered my endless questions and was always willing to lend a helping hand. And to Glenn, Matt, Katelyn, and Tyler, thank you. Special thanks go to Dylan. You are my motivation and my inspiration. Your unwavering faith in me made this possible. Last but not least, I would like to thank my friends and family for their constant encouragement, love, and support throughout my long graduate career. Thank you. iv TABLE OF CONTENTS Introduction : Setting the Problem :::::::::::::::::::::::::::::::::::::::: 1 0.1. Overview . .1 0.2. Genomic Imprinting . .3 0.3. Eutherian X-Chromosome Dosage Compensation . .5 0.4. Turner Syndrome . .6 0.5. Autism Spectrum Disorder . .8 0.6. X-linked Genomic Imprinting . 10 0.7. Mechanisms of Epigenetic and Imprinting Regulation . 12 0.7.1. Differential DNA Methylation . 13 0.7.2. Allele-Specific Histone Modification . 14 0.7.3. Interactome Analyses of Imprinted Domains . 16 Ch. 1 : Nuclear Organization of the Imprinted Xlr3/4 Locus ::::::::::::::::: 18 1.1. Abstract . 18 1.2. Background . 19 1.3. Results . 24 1.3.1. Generating a library of genome-wide chromatin interactions . 24 1.3.2. Long-range interactions at the imprinted Xlr3/4 locus . 25 1.3.3. 4C domains reside in open chromosomal compartments . 30 1.3.4. Short-range comprehensive analysis of looping interactions . 33 1.3.5. 4C domains enriched with genes linked to cognitive impairment . 40 1.4. Discussion . 45 Ch. 2 : Epigenomic Profiling of the Maternal and Paternal X-Chromosomes :: 48 2.1. Abstract . 48 2.2. Background . 49 2.3. Results . 53 2.3.1. Interaction profiles of the maternal and paternal X-chromosomes . 53 2.3.2. XM 4C domains are enriched with genes involved in cognitive function . 55 2.3.3. X-chromosome profiling of H3K27me3 and Atrx . 58 2.4. Discussion . 68 Ch. 3 : Regulation of Xlr3b Gene Expression ::::::::::::::::::::::::::::::: 73 3.1. Background . 73 3.2. Results . 78 3.3. Discussion . 83 Ch. 4 : Synthesis and Future Directions ::::::::::::::::::::::::::::::::::: 86 v Ch. 5 : Materials and Methods :::::::::::::::::::::::::::::::::::::::::::: 93 5.1. Animal Breeding and Tissue Collection . 93 5.2. Xlr3b Expression Analysis . 93 5.3. Chromosome Conformation Capture (3C) . 94 5.3.1. Mouse Neonatal Brain Tissue . 94 5.3.2. Mouse Fibroblast Cultured Cells . 96 5.4. 3C-Carbon Copy (5C-Seq) . 97 5.4.1. 3C Control Library Generation . 97 5.4.2. 5C Primers and Library Generation . 97 5.4.3. Illumina MiSeq Sequencing . 98 5.4.4. Data Analysis . 98 5.5. Circular 3C (4C-Seq) . 99 5.5.1. 4C Primers and Library Generation . 99 5.5.2. Illumina NextSeq500 Sequencing . 100 5.5.3. Data Analysis . 101 5.6. Chromatin Immunoprecipitation (ChIP-Seq) . 102 5.6.1. ChIP Library Generation . 102 5.6.2. Illumina NextSeq500 Sequencing . 103 5.6.3. Data Analysis . 103 5.7. Assay for Transposase-Accessible Chromatin (ATAC-Seq) . 103 App. A : :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 104 A.1. Primers . 104 A.2. BACs............................................................... 118 A.3. Antibodies . 118 App. B : :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 119 B.1. Sequencing Metrics . 119 Bibliography :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 125 vi LIST OF FIGURES 0.4.1 Cognitive defects in individuals with Turner syndrome. .7 0.6.2 Genomic imprinting of Xlr3b, Xlr4b, and Xlr4c......................... 11 0.6.3 Schematic representation of the Xlr3/4/5 locus. 12 0.7.4 Increased Xlr3b expression in 5-aza-2-dC treated cell lines. 15 1.2.1 Overview of 3C-derived methods. 21 1.3.1 Overview of 4C-Seq bait region at the imprinted Xlr3/4 locus. 25 1.3.2 Distribution of 4C-Seq reads. 26 1.3.3 Distribution of 4C interactions. 27 1.3.4 Significant 4C domains in cis and in trans.............................. 28 1.3.5 Long-range cis interactions of Xlr3b and Xlr4c.......................... 29 1.3.6 DNA replication timing of 4C domains. 31 1.3.7 Enrichment of active chromatin signatures around 4C domains. 32 1.3.8 Repetitive elements within 4C domains. 34 1.3.9 Near-bait interactions of Xlr3b and Xlr4c.............................. 35 1.3.10 Near-bait interactions show parent-specific differences within a TAD. 36 1.3.11 Overview of 5C-Seq primer design at the imprinted Xlr3/4 locus. 37 1.3.12 5C interaction profile of Xlr3b........................................ 38 1.3.13 4C interacting regions display characteristics of regulatory elements. 39 1.3.14 Distribution of region-gene associations within 4C domains. 41 1.3.15 Functional annotation of genes within Xlr4c 4C domains. 42 1.3.16 Functional annotation of genes within Xlr3b 4C domains. 44 2.3.1 Significant 4C domains of six viewpoints across the X-chromosome. 54 2.3.2 Number of associated genes per 4C domain. ..
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