A Thesis entitled The Maize TFome 2.0: Genomic Analysis of Transcription Factor Repertoire by Rachael Wasikowski Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master’s Degree in Bioinformatics, Proteomics and Genomics ________________________________________ John Gray, Committee Chair ________________________________________ Scott Leisner, Committee Member _________________________________________ Alexei Fedorov, Committee Member _________________________________________ Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo May 2018 Copyright 2018, Rachael Wasikowski This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of The Maize TFome 2.0: Genomic Analysis of Transcription Factor Repertoire by Rachael Wasikowski Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master’s Degree in Bioinformatics, Proteomics and Genomics The University of Toledo May 2018 The complete transcription factor (TF) repertoire of an organism is a very useful resource in the study of regulomics. A physical clone collection of each TF’s open reading frame (ORF) comprises a TFome library and has the potential to experimentally place transcription factors within a gene regulatory network. The Maize TFome 2.0 is a correction and expansion of the transcription factor repertoire previously identified in Maize TFome 1.0 (Burdo et al., 2014). The predicted gene models for uncloned transcription factors and coregulators were analyzed using RNA-seq data from 42 different tissues and growth conditions. Models were ranked for future cloning efforts based upon the level of read support. In the future, all well-supported TFs and CRs will be cloned and added to the maize TFome 2.0 collection. As part of expanding the maize TFome, the second largest family of maize TFs named the Apetela2/Ethylene Response Element Binding Proteins (AP2/EREBP) family was analyzed using synteny and coexpression data. As a result of a whole genome duplication event in maize, it was found that 54% of the AP2/EREBP genes are duplicated. Synteny data and an HMM scan of the new Maize genome revealed 24 new members of the AP2/EREBP family. iii Members of the AP2/EREBP family were sorted into four subfamilies based on the domains present and the sequence of their DNA binding motif. Some syntelog pairs were highly coexpressed suggesting functional redundancy while others are considered to have diverged in function based on their different levels of expression during plant development. iv Acknowledgements I would like to thank Todd Lenz, Patrick Dusza, and Alex Fazekas for assisting with the gene model rankings and RNA-seq data extraction. Their efforts helped keep this project running smoothly. v Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ..............................................................................................................v Table of Contents ............................................................................................................... vi List of Tables ................................................................................................................... ix List of Figures ......................................................................................................................x List of Abbreviations ......................................................................................................... xi 1 Analysis of Uncloned Transcription Factors and CoRegulators in Maize TFome 1.1 Introduction ........................................................................................................1 1.1.1 The Maize Regulome .......................................................................1 1.1.2 General Utility of TFome Collections .............................................2 1.1.3 TFome Studies in Plants and other Organisms ................................4 1.1.4 Study Objectives ..............................................................................6 1.2 Materials and Methods .......................................................................................7 1.2.1 Collection and Refinement of the Maize TFome RNA-seq Data ....7 1.2.2 Interpretation of RNA-seq Data and Ranking of Gene Model Quality ........................................................................................................8 1.3 Results ..........................................................................................................9 1.3.1 Census of Uncloned TFs and CRs in the Maize Genome ................9 1.3.2 Evaluation of Gene Model Quality for Uncloned TFs and CRs in the maize genome. .....................................................................................13 1.4 Discussion and conclusions .............................................................................19 vi 2 Analysis of the AP2/EREBP Transcription Factor Family in Maize ....................24 2.1 Introduction ......................................................................................................24 2.1.1 Overview of the AP2/EREBP TF Family in Maize .......................24 2.1.2 Known Functions of AP2/EREPB Genes ......................................26 2.1.3 AP2/EREBP Subfamily RAV ........................................................28 2.1.4 The Influence of Whole Genome Duplication (WGD) on maize TF repertoire ....................................................................................................29 2.1.5 The Effect of Redundant Syntenic Genes ......................................30 2.1.5 Study Objectives ............................................................................31 2.2 Materials and Methods .....................................................................................32 2.2.1 Detection of Synteny Amongst Maize AP2/EREBP Family Members ....................................................................................................32 2.2.2 Coexpression Analysis of the AP2/EREBP Family ......................33 2.2.3 Phylogenetic Analysis of the AP2 domain ....................................34 2.3 Results ........................................................................................................35 2.3.1 Survey of EREBP Gene Structures in Maize ................................35 2.3.2 Phylogenetic Analysis of the EREBP family in maize .................36 2.3.3 Detection of Synteny amongst Maize EREBP family members .. 38 2.3.4 Coexpression Analysis of Maize EREBP family members ..........41 2.4 Discussion and conclusions………………………………………………….47 References ..........................................................................................................................53 A Table A1: List of Uncloned TF Genes and Gene Model Rankings .......................62 B Table A2: List of Uncloned CR Genes and Gene Model Rankings ......................82 vii C Table A.3: List of Syntelogs of EREBP genes in Maize .......................................86 D Table A.4: List of EREB Genes with likely deletions of syntelog ........................93 E CIRCOS Ribbon file ..............................................................................................95 F CIRCOS Gene coordinates file ..............................................................................97 G CIRCOS Ideogram file ..........................................................................................99 H CIRCOS Configuration file .................................................................................100 I R script for coexpression analysis ........................................................................102 viii List of Tables 1.1 Tandem Duplications of Uncloned Transcription Factors .....................................10 2.1 List of Maize AP2/EREBP Genes with known association ...................................27 2.2 Summary of Self-Synteny in Maize Genome V4 ..................................................39 2.3 List of Syntenic EREB Gene Pairs in Maize .......................................................40 2.4 List of syntenic EREB family members that are highly coexpressed .................42 2.5 List of syntenic EREB family members that are not coexpressed ......................44 2.6 List of non-syntenic EREB family members that are highly coexpressed ..........46 A.1 List of Uncloned TF Genes and Gene Model Rankings ........................................62 A.2 List of Uncloned CR Genes and Gene Model Rankings .......................................82 A.3 List of Syntelogs of EREBP genes in Maize .........................................................86 A.4 List of EREB Genes with likely deletions of syntelog ..........................................93 ix List of Figures 1-1 Bar graph of the total number of maize transcription factor V4 gene models and the number of previously cloned genes within each gene family. .........................12 1-2 Bar graph of the total number of maize CoRegulator (CR) V4 gene models and the number of previously cloned genes within each gene family. .........................13 1-3 Analysis pipeline for mapping of RNA-Seq reads to maize genome V4.37 .........14 1-4 Ranking System for Maize Gene Model Support based on RNA-Seq Data. ........15 1-5 Summary of Ranked Maize TF and