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Transcriptional Poising Prior to the Midblastula Transition Underlies Dorsal Cell Fate Specification Yb the Wnt/Beta-Catenin Pathway

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Transcriptional Poising Prior to the Midblastula Transition Underlies Dorsal Cell Fate Specification Yb the Wnt/Beta-Catenin Pathway University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Fall 2009 Transcriptional Poising Prior to the Midblastula Transition Underlies Dorsal Cell Fate Specification yb the Wnt/Beta-Catenin Pathway Shelby A. Blythe University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Developmental Biology Commons Recommended Citation Blythe, Shelby A., "Transcriptional Poising Prior to the Midblastula Transition Underlies Dorsal Cell Fate Specification by the Wnt/Beta-Catenin Pathway" (2009). Publicly Accessible Penn Dissertations. 242. https://repository.upenn.edu/edissertations/242 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/242 For more information, please contact [email protected]. Transcriptional Poising Prior to the Midblastula Transition Underlies Dorsal Cell Fate Specification yb the Wnt/Beta-Catenin Pathway Abstract Following fertilization in many multicellular organisms, zygotic transcription is suppressed for several hours and cell divisions, until a major embryonic transition termed the midblastula transition (MBT). Nevertheless, steps critical for later patterning of the embryo occur during this early stage. To address this question, we have optimized the chromatin immunoprecipitation technique to allow the investigation of pre-MBT chromatin architecture. We find that, in the context of transcriptional quiescence before the MBT in Xenopus, Wnt signaling through β-catenin primes dorsal gene expression by establishing transcriptionally poised chromatin architecture at target promoters. This is later resolved into active gene expression following the large-scale activation of zygotic transcription at the MBT. During pre-MBT dorsal specification, β-catenin interacts with a histone H3 methyltransferase activity that targets arginine 8 (R8). Recruitment of the arginine methyltransferase Prmt2 to β-catenin target promoters is necessary and sufficiento t establish the dorsal developmental program, indicating that Prmt2-mediated histone H3R8 methylation plays a critical role downstream of β-catenin in establishing poised chromatin architecture and marking key organizer genes for later expression. This work demonstrates a mechanism whereby a signal transduction pathway can establish poised chromatin architecture at target genes, which could have implications for the regulation of gene regulatory networks during development. Additionally, our results suggest the possibility that transcriptional poising plays a broader role in maintaining zygotic genome silencing before the MBT. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Peter S. Klein Keywords Wnt Signaling, Embryogenesis, Chromatin, Histone Methylation, Transcriptional Poising Subject Categories Developmental Biology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/242 Acknowledgments I am grateful to Peter Klein for providing an intellectually challenging and stimulating lab, and for allowing me to pursue the idea that became this dissertation. I am also grateful to my thesis committee for being patient and extremely supportive of my project, even at times when things looked difficult. Everyone’s encouragement to follow the work through made it possible for me to get this story together. I am also grateful to the members of the Klein lab for providing a lively and enjoyable place to be a student. And, of course, none of this would have been very easy or enjoyable without the support of my friends and family. I also acknowledge former lab member Dr. Mahmoud Najafi for initially generating the N-β- catenin antibody and Dr. Said Sif of Ohio State University for sharing his anti-H3R8me2s antiserum. During development of the ChIP protocol, I also appreciated the contributions of Christine Reid and Dan Kessler, whose ability to successfully perform the procedure and obtain nice reproducible results gave me confidence that the protocol was good. Finally, I am grateful for the intellectual and financial support of the Cell and Molecular Biology graduate group and the Department of Cell and Developmental Biology. This work was supported by both the C&MB and Dev. Biol. training grants, as well as an NIH grant to Peter Klein. ii ABSTRACT TRANSCRIPTIONAL POISING PRIOR TO THE MIDBLASTULA TRANSITION UNDERLIES DORSAL CELL FATE SPECIFICATION BY THE WNT/β-CATENIN PATHWAY Shelby A. Blythe Peter S. Klein Following fertilization in many multicellular organisms, zygotic transcription is suppressed for several hours and cell divisions, until a major embryonic transition termed the midblastula transition (MBT). Nevertheless, steps critical for later patterning of the embryo occur during this early stage. To address this question, we have optimized the chromatin immunoprecipitation technique to allow the investigation of pre-MBT chromatin architecture. We find that, in the context of transcriptional quiescence before the MBT in Xenopus, Wnt signaling through β- catenin primes dorsal gene expression by establishing transcriptionally poised chromatin architecture at target promoters. This is later resolved into active gene expression following the large-scale activation of zygotic transcription at the MBT. During pre-MBT dorsal specification, β- catenin interacts with a histone H3 methyltransferase activity that targets arginine 8 (R8). Recruitment of the arginine methyltransferase Prmt2 to β-catenin target promoters is necessary and sufficient to establish the dorsal developmental program, indicating that Prmt2-mediated histone H3R8 methylation plays a critical role downstream of β-catenin in establishing poised chromatin architecture and marking key organizer genes for later expression. This work demonstrates a mechanism whereby a signal transduction pathway can establish poised chromatin architecture at target genes, which could have implications for the regulation of gene regulatory networks during development. Additionally, our results suggest the possibility that transcriptional poising plays a broader role in maintaining zygotic genome silencing before the MBT. iii Table of Contents Title......................................................................................................................... i Acknowledgments ................................................................................................. ii Abstract ................................................................................................................ iii List of Figures ....................................................................................................... vi Chapter 1: Introduction ..........................................................................................1 1.1: Summary 1 1.2: The Midblastula Transition 2 1.2.1: Two Developmental Timers 4 1.3: Transcriptional Control Before the MBT 6 1.3.1: Suppression of Basal Transcription in pre-MBT Embryos 8 1.3.2: Counteracting Trans-Activator Function 9 1.3.3: Heritability of Active Chromatin Structure Before the MBT 12 1.4: Transcriptional Poising 14 1.5: Dorsal Specification by the Maternal Wnt/β-catenin Pathway 18 1.6: Research Summary 22 Chapter 2: Chromatin Immunoprecipitation in Early Xenopus laevis Embryos ...24 2.1: Introduction 24 2.2: Results and Discussion 27 2.2.1: Crosslinking Optimization 28 2.2.2: General Sonication Guidelines 32 2.2.3: Shearing Optimization 32 2.2.4: Chromatin Immunoprecipitation in Blastula Stage Embryos 33 2.2.5: Controls for Antibody Specificity 37 2.2.6: Quantitative PCR Analysis 41 2.2.7: Further Considerations 45 2.2.8: Concluding Remarks 49 2.3: Experimental Procedures 49 2.3.1: ChIP Protocol 50 2.3.2: PCR 60 2.3.3: Cloning of Additional Xnr6 Genomic Sequence 64 2.3.4: Western Blotting 64 2.3.5: Antibodies 65 iv Chapter 3: β-catenin Primes Organizer Gene Expression By Recruiting a Histone H3 Arginine Methyltransferase, Prmt2......................................................66 3.1: Introduction 66 3.2: Results 69 3.2.1: β-catenin Establishes Poised Chromatin Architecture at Target Promoters Before the Midblastula Transition 73 3.2.2: β-catenin Associates with a Histone H3 (R8) Methyltransferase Activity in early Xenopus Embryos 79 3.2.3: β-catenin Recruits the Arginine Methyltransferase Prmt2 to Target Loci 89 3.2.4: Directing Prmt2 to β-catenin Target Promoters Is Sufficient to Drive Dorsal Specification in the Absence of β-catenin. 95 3.2.5: Prmt2 Is Necessary for Dorsal Specification and for Conferring Resistance to ΔNTcf3 Before the MBT. 99 3.3: Discussion 106 3.3.1: H3R8 Methylation and the Histone Code 106 3.3.2: Pre-Setting Patterns of Gene Expression in the Embryo 108 3.3.3: Context-Dependent Chromatin Modifying Activities for β-catenin? 110 Chapter 4: Conclusions and Future Directions..................................................112 4.1: Accounting for pre-MBT β-catenin Activity: Prmt2 Function 112 4.1.1: The H3R8me2 “Reader”/ H3K4me3 “Writer” Hypothesis 113 4.1.2: The H3R8me2 “Lock” Hypothesis 115 4.1.3: The H3R8 / H3K9 Competition Hypothesis 119 4.1.4: Concluding Remarks: The Function of H3R8 Methylation at β-catenin Target Genes Before the MBT 124 4.2: Poised Chromatin Architecture 125 4.2.1: β-catenin, H3K4me3, and ISWI: A Potential Regulatory Node in Poised Chromatin Architecture? 128 4.3: Transcriptional Poising and Cell Fate Determination 134 4.3.1: p-TEFb Before the MBT, A Clue From Primordial
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