REGULATION OF CHROMATIN BY FACT AND NUA3 by Adam Tadeusz Chruscicki B.Sc.Hon., The University of British Columbia, 2008 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUTATE STUDIES (Biochemistry and Molecular Biology) THE UNIVERISTY OF BRITISH COLUMBIA (Vancouver) April 2013 © Adam Tadeusz Chruscicki, 2013 Abstract The presence of nucleosomes over vast regions of genome negatively influences transcription creating a need for temporal and structural regulation of chromatin. The default transcription-repressive state can be countered by addition of post-translational modifications by chromatin modifiers or chromatin alteration by histone chaperones. Chaperones alter chromatin structure before the RNAPII passage and restore it afterwards. How modifiers and chaperones function within chromatin is an area of intense research. Here we show how two complexes, the yeast FACT and NuA3 contribute to chromatin function. Yeast Facilitates Chromatin Transactions (yFACT) is a histone chaperone that maintains chromatin structure. The model of yFACT function in vivo is a subject of much debate. We provide evidence that yFACT acts by stably binding and altering nucleosomes. We also present the EM structure of yFACT associated with nucleosomes. We find that yFACT-associated nucleosomes are hyper-acetylated and show evidence for it being an effect of a direct interaction between yFACT and NuA3. At the same time, acetylation of the H3K56 residue by the histone acetyltransferase Rtt109, acts to recruit yFACT to chromatin through a nucleosome-dependent mechanism. To determine the distribution of yFACT-associated nucleosomes we constructed a map of yFACT-nucleosome localization at single-nucleosome resolution. We show that while yFACT-bound nucleosomes are distributed thought the genome they are also positioned over the canonical Nucleosome Depleted Regions (NDR). The yFACT-bound nucleosomes are positioned around TATA-elements and Nhp6-target sequences genome-wide. Deletion of NHP6A/B leads to loss of chromatin at these loci. Our work suggests the first ever sequence-dependent mechanism of histone chaperone action in Saccharomyces cerevisiae. We also examined NuA3 recruitment to chromatin and showed that Yng1, a subunit of NuA3 with a known affinity for H3K4me3 is a bivalent protein. While as previously shown, the C-terminal PHD finer of Yng1 binds to H3K4me3, the N-terminus of Yng1 can also bind to unmodified chromatin. Although these motifs can bind independently, together they increase the apparent association of Yng1 with chromatin. Yng1 binding to chromatin is regulated by the HDA1 complex. ii Preface Chapter 1: “yFACT binds to nucleosomes in vivo” is based on a first author manuscript currently in preparation. All experiments were designed by myself and Dr.LeAnn Howe. I conducted the majority of experiments, while the supplementary work was done by Nancy Fang, Dr. Calvin Yip and Nicolas Coutin. While I prepared the samples, Nancy Fang performed the MS. The EM images were taken with Dr. Calvin Yip. Nicolas Coutine performed the bioinformatic analysis of EM images. Chapter 2: “yFACT-bound nucleosomes are organized by Nhp6 binding sequences” is based on a first author manuscript in preparation. All experiments were designed by myself and Dr. LeAnn Howe. I conducted the majority of experiments, while Benjamin Martin, Nancy Fang, Dr. Julie Brind’Amour and Dr. Mark Hills performed supplementary work and provided help with data analysis. Benjamin Martin helped me to establish the bioinformatics platform for sequence analysis and provided intellectual input to the project. Nancy Fang provided experimental expertise. Dr. Julie Brind’Amour constructed the libraries for sequencing and Dr. Mark Hills helped with the processing of fastq files. The draft of the manuscript was written by myself, and then revised with help of Benjamin Martin and Dr. LeAnn Howe. A version of Chapter 3:” Critical Determinants for Chromatin Binding by Saccharomyces cerevisiae Yng1 Exist Outside of the Plant Homeodomain Finger” was published. Chruscicki AT, MacDonald VE, Young BP, Loewen CJR, and Howe LJ.(2010). Critical iii Determinants for Chromatin Binding by Saccharomyces cerevisiae Yng1 Exist Outside of the Plant Homeodomain Finger. Genetics. 185(2):469-77. DOI: 10.1534/genetics.110.116285. I conducted the majority of experiments for the project and wrote the first version of the manuscript that was later revised by Dr. Vicki Maltby (MacDonald) and Dr. LeAnn Howe. My exact contributions to this work were Figures 4.1 b, c; 4.3a, b, c; 4.5a, b; 4.6a, I also performed, analyzed and validated the high- throughput Synthetic Dosage Lethality (SDL) screen. The remainder of the Figures was a shared contribution between Dr. Vicki Maltby (MacDonald) and Dr. LeAnn Howe. In addition, Dr. Vicki Maltby (MacDonald) constructed the plasmids used in this study. iv Table of Contents Abstract ............................................................................................................................ii Preface ............................................................................................................................ iii Table of Contents ............................................................................................................ v List of Tables ...................................................................................................................ix List of Figures .................................................................................................................. x List of Abbreviations ....................................................................................................... xii Acknowledgments ......................................................................................................... xvi Dedication ................................................................................................................... xviii 1 Chapter: Introduction ................................................................................................ 1 1.1 Chromatin .................................................................................................................... 1 1.2 Histone Post-Translational Modifications ..................................................................... 1 1.2.1 Histone acetylation ............................................................................................... 2 1.2.2 Histone methylation .............................................................................................. 4 1.3 Chromatin Modifying Complexes ................................................................................. 6 1.3.1 Readers ................................................................................................................ 6 1.3.2 Lysine-acetyltransferases ..................................................................................... 8 1.3.3 Lysine-methyltransferases .................................................................................... 9 1.4 The Histone Chaperones ............................................................................................10 1.4.1 FACT ...................................................................................................................13 1.5 Nucleosome Positioning..............................................................................................17 2 Chapter: yFACT Binds to Nucleosomes In Vivo ..................................................... 19 2.1 Introduction .................................................................................................................19 2.2 Methods ......................................................................................................................21 v 2.2.1 Yeast cell culture .................................................................................................21 2.2.2 Antibodies ............................................................................................................22 2.2.3 Chromatin ImmunoPrecipitation (ChIP) ................................................................22 2.2.4 Pob3TAP based FACT-purification and MNase digestion .......................................23 2.2.5 FACT fractionation ...............................................................................................24 2.2.6 Electron microscopy .............................................................................................24 2.2.7 Mass spectrometry...............................................................................................25 2.3 Results ........................................................................................................................29 2.3.1 yFACT stably binds nucleosomes in vivo .............................................................29 2.3.2 yFACT-associated nucleosome particles display altered structure .......................34 2.3.3 yFACT binds to hyper-acetylated nucleosomes in vivo ........................................38 2.3.4 yFACT is recruited to chromatin by H3K56ac.......................................................43 2.3.5 The effect of the cell cycle on yFACT-associated chromatin ................................49 2.3.6 The yFACT-bound nucleosomes are directly acetylated by NuA3 ........................52 2.4 Discussion ..................................................................................................................55 3 Chapter: