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Intron Retention of JARID2 Regulates Gene Expression During Temperature-Dependent Sex Determination

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Intron Retention of JARID2 Regulates Gene Expression During Temperature-Dependent Sex Determination University of North Dakota UND Scholarly Commons Theses and Dissertations Theses, Dissertations, and Senior Projects January 2021 Intron Retention Of JARID2 Regulates Gene Expression During Temperature-Dependent Sex Determination Jacob Robert Bierstedt Follow this and additional works at: https://commons.und.edu/theses Recommended Citation Bierstedt, Jacob Robert, "Intron Retention Of JARID2 Regulates Gene Expression During Temperature- Dependent Sex Determination" (2021). Theses and Dissertations. 3914. https://commons.und.edu/theses/3914 This Thesis is brought to you for free and open access by the Theses, Dissertations, and Senior Projects at UND Scholarly Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UND Scholarly Commons. For more information, please contact [email protected]. INTRON RETENTION OF JARID2 REGULATES GENE EXPRESSION DURING TEMPERATURE-DEPENDENT SEX DETERMINATION by Jacob Robert Bierstedt Bachelor of Science, University of North Dakota, 2017 A Thesis Submitted to the Graduate Faculty of the University of North Dakota in partial fulfillment of the requirements for the degree of Master of Science Grand Forks, North Dakota May 2021 Copyright 2021 Jacob Bierstedt ii Jacob Bierstedt Name: Degree: Master of Science This document, submitted in partial fulfillment of the requirements for the degree from the University of North Dakota, has been read by the Faculty Advisory Committee under whom the work has been done and is hereby approved. ____________________________________ Turk Rhen ____________________________________ Tristan Darland ____________________________________ Manu Manu ____________________________________ ____________________________________ ____________________________________ This document is being submitted by the appointed advisory committee as having met all the requirements of the School of Graduate Studies at the University of North Dakota and is hereby approved. ____________________________________ Chris Nelson Dean of the School of Graduate Studies ____________________________________ Date iii PERMISSION Title Intron Retention of JARID2 Regulates Gene Expression During Temperature-dependent Sex Determination Department Biology Degree Master of Science In presenting this thesis in partial fulfillment of the requirements for a graduate degree from the University of North Dakota, I agree that the library of this University shall make it freely available for inspection. I further agree that permission for extensive copying for scholarly purposes may be granted by the professor who supervised my thesis work or, in his absence, by the Chairperson of the department or the dean of the School of Graduate Studies. It is understood that any copying or publication or other use of this thesis or part thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of North Dakota in any scholarly use which may be made of any material in my thesis. Jacob Bierstedt 05/04/2021 iv TABLE OF CONTENTS LIST OF FIGURES ...................................................................................................................... vii LIST OF TABLES .......................................................................................................................viii ACKNOWLEDGEMENTS .......................................................................................................... ix ABSTRACT ....................................................................................................................................x CHAPTER I ................................................................................................................................... 1 MECHANISMS OF TRANSCRIPTIONAL REGULATION DURING SEX DETERMINATION ...................................................................................................................... 1 Introduction ....................................................................................................................... 1 Sex Determining Mechanisms in Vertebrates ................................................................... 2 Epigenetic Regulation of Gene Expression and Cell Fate Decisions ................................ 6 Specification of Gonadal Progenitor Cells ........................................................................ 8 Differentiation of Steroidogenic Lineages in the Gonad ................................................. 10 Differentiation of Supporting Cell Lineages in the Gonad .............................................. 11 JARID2 as a Candidate Regulator of Differentiation During TSD ................................. 16 Conclusion ....................................................................................................................... 20 References ........................................................................................................................ 22 CHAPTER II ................................................................................................................................ 29 JARID2 INTRON-RETENTION REGULATES GENE EXPRESSION IN TSD ..................... 29 Introduction ...................................................................................................................... 29 Materials and Methods ..................................................................................................... 32 Nanopore Direct cDNA Sequencing .................................................................... 32 Quantification of JARID2 Splicing with qRT-PCR ............................................ 33 JARID2 Structure and Function .......................................................................... 34 Molecular Cloning of JARID2 ............................................................................ 34 Fluorescence Microscopy .................................................................................... 35 In vitro Overexpression of JARID2 Isoforms ..................................................... 36 Expression Data Analysis .................................................................................... 38 Results .............................................................................................................................. 41 JARID2 mRNA is Spliced into Three Main Transcripts ..................................... 41 Confirmation of JARID2 Intron Retention via qPCR .......................................... 43 Structure of JARID2 Isoforms ............................................................................. 43 JARID2 Evolution ............................................................................................... 44 JARID2 Isoforms are Localized to the Nuclei of Gonadal Cells in Culture ....... 46 Temperature Effects on Gene Expression in Gonadal Cells Cultured in vitro .... 47 Overexpression of JARID2-1231-mCherry in Embryonic Gonadal Cells in vitro ......................................................................................................... 53 Overexpression of JARID2-1077-mCherry in Embryonic Gonadal Cells in vitro ......................................................................................................... 55 v Overexpression of JARID2-318-mCherry in Embryonic Gonadal Cells in vitro .................................................................................................................. 59 Overexpression of JARID2 Isoforms Have Common and Unique Effects on Gene Expression ................................................................................. 62 Discussion ........................................................................................................................ 65 JARID2 Exists in Three Distinct Variants in TSD .............................................. 65 Subcellular Localization of JARID2 Isoforms .................................................... 67 Validation of in vitro Model of TSD ................................................................... 67 JARID2-1231 Regulates Genes Involved in Differentiation ............................... 68 Overexpression of JARID2-1077 Represses Transcription of Genes Involved in Steroidogenesis ................................................................................................ 69 JARID2-318 Regulates Expression of Genes Associated with Pluripotency and Wnt Signaling ...................................................................................................... 72 Conclusion ....................................................................................................................... 73 vi LIST OF FIGURES Figure 1: JARID2 occurs in three distinct transcript variants during the TSP .............................40 Figure 2: Quantification of JARID2 splicing with qPCR ............................................................42 Figure 3: JARID2 Functional Domains and Orthogroup Tree .....................................................45 Figure 4: JARID2 isoforms are localized to the nucleus ..............................................................46 Figure 5: Experimental design and validation ..............................................................................48 Figure 6: Genes upregulated at FPT .............................................................................................49 Figure 7: Genes upregulated at MPT ...........................................................................................52
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