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Maize RNA Polymerase IV Complexes and Their Control of Gene Function

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Maize RNA Polymerase IV Complexes and Their Control of Gene Function Maize RNA polymerase IV complexes and their control of gene function by Joy-El Renee Barbour Talbot A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Molecular and Cell Biology in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Jay B. Hollick, Co-chair Professor Jasper D. Rine, Co-chair Professor Michael B. Eisen Professor Michael R. Freeling Summer 2015 Maize RNA polymerase IV complexes and their control of gene function Copyright 2015 by Joy-El Renee Barbour Talbot 1 Abstract Maize RNA polymerase IV complexes and their control of gene function by Joy-El Renee Barbour Talbot Doctor of Philosophy in Molecular and Cell Biology University of California, Berkeley Professor Jay B. Hollick, Co-chair Professor Jasper D. Rine, Co-chair Plants have acquired and maintained an expanded suite of DNA-dependent RNA poly- merases (RNAPs) compared to other eukaryotes. Although their exact roles remain unclear, plant-specific RNAPs (Pol IV and Pol V) are involved in epigenetic silencing of transposable elements (TEs). Zea mays (maize) Pol IV is required for proper plant development as well as the establishment and maintenance of paramutations, which are trans-homolog interactions that facilitate heritable gene silencing. Maize has duplications of Pol IV catalytic subunits, which define multiple Pol IV subtypes, and accessory proteins associated with these subtypes define distinct Pol IV complexes. Understanding the roles of Pol IV will require identify- ing the composition and function of these Pol IV subtypes and complexes. In exploring interactions between the genes encoding a Pol IV catalytic subunit and a putative accessory protein, I identified two new alleles of the Pol IV subunit and a family of potential Pol IV accessory proteins conserved in multicellular plants. Together, these Pol IV complexes are proposed to function through two, potentially overlapping, general mechanisms to control of gene function: direct competition with Pol II and/or generation of 24-nucleotide RNAs that guide de novo cytosine methylation. I analyzed nascent transcriptome data of seedlings lacking Pol IV and their wild-type siblings, which identified a global effect of Pol IV on gene boundary transcription. Pol IV-affected loci serve as molecular and phenotypic models for dissecting the involvement of various Pol IV subtype and complex components. Such analysis implicated a Pol IV-affected allele as being controlled by Pol IV competition with Pol II. Through expanded sequencing of a paramutation participating allele (Pl1-Rhoades), I identified a new Pol IV target of five tandem repeats that may serve as an enhancer element. I used the Pl1-Rhoades allele in particular to compare and contrast the effects of Pol IV loss with that of Pol IV accessory proteins. Together, the work presented here explores the involvement of an uncharacterized protein family of putative Pol IV accessory proteins in Pol IV complexes, expands the roles of Pol IV complexes in controlling maize gene function, and identifies new Pol IV-affected loci for future study. i To Paul and Sheila Barbour For always believing, supporting and listening–even when I make up horrible analogies between my research and videogame plots. To Jared Talbot For helping me get to the other side and being my collaborator in our best genetic experiment yet. Finally to the Yeti–my mostly silent companion in writing this dissertation. I can’t wait to meet your wonderful self. ii Contents Contents ii List of Figures iv List of Tables vi Acknowledgements vii 1 Introduction 1 1.1 Evolutionary origin of plant RNA polymerase structural diversity ...... 2 1.2 Transcriptional activity of Pol IV . ..... 6 1.3 FunctionsofPolIVinplants. ... 8 1.4 Examples of genome regulation by Pol IV . ... 9 1.5 Remainingquestions ............................... 12 1.6 Figures....................................... 14 1.7 References..................................... 15 2 Genetic interactions between Pol IV components 21 2.1 Introduction.................................... 21 2.2 MaterialsandMethods............................. 23 2.3 Results....................................... 24 2.4 Discussion..................................... 31 2.5 Tables ....................................... 34 2.6 Figures....................................... 37 2.7 References..................................... 45 3 Pol IV affects nascent transcription 50 3.1 Introduction.................................... 50 3.2 MaterialsandMethods............................. 52 3.3 Results....................................... 59 3.4 Discussion..................................... 68 3.5 Tables ....................................... 74 3.6 Figures....................................... 91 iii 3.7 References..................................... 108 4 Putative Pl1-Rhoades paramutation element 116 4.1 Introduction.................................... 116 4.2 MaterialsandMethods............................. 118 4.3 Results....................................... 122 4.4 Discussion..................................... 128 4.5 Tables ....................................... 132 4.6 Figures....................................... 134 4.7 References..................................... 145 5 Perspectives 150 5.1 Composition of Pol IV complexes . 150 5.2 Mechanistic models of Pol IV function . 152 5.3 PolIVeffectsongenefunction. 153 5.4 Pol IV effects on paramutable alleles . 153 5.5 Conclusions .................................... 155 5.6 References..................................... 156 Appendices 159 A Complementation cross pedigrees 159 B Small RNA analysis scripts 164 B.1 Awk commands for processing SAM alignment files . 164 B.2 Python function to add total alignment count to SAM file . 166 iv List of Figures 1.1 RNAPholoenzymecomposition . 14 2.1 Conserved carboxy-terminal motifs in the RMR2 family . ........... 37 2.2 Phylogeny of RMR2 family proteins . ..... 38 2.3 Expression of rmr2 anditsparalogsinmaize . 39 2.4 The rp(d/e)2a genemodel.............................. 39 2.5 Pedigree of rmr7-1 and rmr7-5 lineages ...................... 40 2.6 Sectored tassels and anthers from rmr2, rmr7 complementation crosses . 41 2.7 Pedigree of rmr2-1 lineages ............................. 42 2.8 Reciprocal complementation crosses between rmr2-1 and rmr7-5 ........ 44 3.1 GRO-seq reads are similarly distributed in WT and rpd1 mutant libraries . 91 3.2 GRO-seq reads align to both exonic and intronic sequences . ........... 92 3.3 Most TE-like sequences can align to genes . ..... 92 3.4 Genic reads are highly enriched in GRO-seq mappable reads ........... 93 3.5 Nongenic GRO-seq reads are enriched near genes . ....... 93 3.6 Distribution of uniquely mapping WT GRO-seq reads around genes ....... 94 3.7 ATG enrichment at the start of maize gene models . ....... 95 3.8 Alternative TSS definition has little effect on coverage profiles .......... 96 3.9 Comparison of GRO-seq peak and TSS annotations . ...... 97 3.10 Strong antisense coverage upstream of GRMZM2G061206 . ......... 98 3.11 Pol IV loss alters global transcription profiles at gene boundaries. 99 3.12 Uniquely mapping GRO-seq coverage across gene bodies . ......... 100 3.13 Fold change between WT and rpd1 mutant antisense GRO-seq read coverage . 101 3.14 Coverage of uniquely mapping 24mers near gene boundaries ........... 102 3.15 Specific alleles are susceptible to Pol IV-induced changes in gene expression . 103 3.16 qRT-PCR analysis of ocl2 in the absence of RPD1 and DCL3 . 105 3.17 Pol IV loss affects both entire TE families and individual elements . 106 4.1 Pl1-Rhoades shares structural regions with pl1-B73 ................ 134 4.2 Alignment of a Pl1-Rhoades and pl1-B73 repeatunit . 135 4.3 Southern blot of Pl1-Rh-containingBAC . 136 v 4.4 Pl1-Rhoades derivatives have similar structures around the pentarepeats . 137 4.5 Structural diversity of pl1 alleles at the unique subrepeat . 138 4.6 The unique subrepeat is expressed in Pl-Rh florets................. 139 4.7 Expression patterns of pl1 andtheuniquesubrepeat . 139 4.8 Pol IV-dependent 24-nt RNAs align to the unique subrepeat . ........ 140 4.9 Unique subrepeat 24-nt RNAs are made only from Pl1-Rh ............ 141 4.10 Gametophytes contribute to the 24-nt RNA pools of immaturecobs . 142 4.11 Cis-genetic variation does not alter 24-nt RNA profiles in hybrids ........ 143 4.12 Affects of maternal loss of RMR1 on 24-nt RNAs . 144 A.1 rmr7-1 and rmr7-5 pedigree with family identifiers . 160 A.2 rmr2-1 pedigreewithfamilyidentifiers . 162 vi List of Tables 2.1 Primersused ..................................... 34 2.2 Initial ems072087 complementationtests. 35 2.3 Complementation tests between ems051081 and rmr7 alleles . 35 2.4 Complementation tests between ems072087 and rmr7 alleles . 35 2.5 Complementation tests between rmr2-1 and rmr7 alleles . 36 2.6 Complementation tests between rmr2-mum and rmr7 alleles . 36 3.1 Sources of genomic feature annotations . ......... 74 3.2 Primers used for RT-PCR and qRT-PCR analysis . 74 3.3 Genes with increased sense orientation transcription in rpd1 mutants . 75 3.4 Genes with decreased sense orientation transcription in rpd1 mutants . 78 3.5 Genes with increased antisense orientation transcription in rpd1 mutants . 84 3.6 Genes with decreased antisense orientation transcription in rpd1 mutants . 86 3.7 Transposons with increased sense orientation transcription in rpd1 mutants . 87 3.8 Transposons with decreased sense orientation transcription in rpd1 mutants
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