ROLE OF RNA PROCESSING MACHINERY AND SIGNALING PATHWAYS IN REGULATING THE DYNAMIC EPIGENETIC LANDSCAPE THROUGH HETEROCHROMATIN ASSEMBLY by Nathan N. Lee A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland October, 2016 © 2016 Nathan N. Lee All Rights Reserved ABSTRACT The regulation of protein coding and noncoding RNAs is linked to nuclear processes including chromatin modifications and gene silencing. Heterochromatin is the major form of chromatin in higher eukaryotes that impacts various chromosomal processes including genomic stability and global gene expression patterns. In the fission yeast Schizosaccharomyces pombe heterochromatin is enriched across large chromosomal domains such as centromeres, telomeres and the mating-type region. In addition, recent work has revealed the existence of small blocks of facultative heterochromatin across the genome. Importantly, the assembly of facultative heterochromatin is mediated by RNA-based mechanisms and dynamically regulated in response to environmental and developmental cues. However, the mechanisms that distinguish RNAs for assembly of heterochromatin at different regions of the genome and how signals trigger changes at the chromatin are poorly understood. I describe the discovery of a nuclear RNA processing network in fission yeast with a core module comprising the Mtr4-like protein named Mtl1 and the zinc finger protein, Red1. The Mtl1-Red1 core (MTREC) promotes degradation of mRNAs and noncoding RNAs, and associates with different proteins to assemble heterochromatin via distinct mechanisms. Mtl1 also forms Red1-independent interactions with evolutionarily conserved proteins named Nrl1 and Ctr1, which associate with splicing factors. Whereas Nrl1 targets transcripts with cryptic introns to form heterochromatin at developmental genes and retrotransposons, Ctr1 functions in processing intron-containing telomerase RNA. Together with our discovery of widespread cryptic introns, including in noncoding RNAs, these findings reveal unique cellular strategies for recognizing regulatory RNAs. ii Furthermore, I have found that Tor2, the yeast homolog of mTOR, functionally connects environmental and developmental signaling cues with remodeling of facultative heterochromatin by regulating cellular protein levels of the MTREC-associated factor Pir1. This process involves Cul4, an E3 ubiquitin ligase in the ClrC complex that also includes the methyltransferase Clr4, Pyp1, a tyrosine phosphatase implicated in TOR pathway, and Swi6/HP1, the heterochromatin binding protein. The mechanism that regulates Pir1 also provides a feedback loop for maintaining the proper level of facultative heterochromatin. These findings reveal signaling pathways and mechanisms that are involved in the dynamic regulation of facultative heterochromatin in response to environmental and developmental signals. Name of Readers/Advisors: Shiv Grewal, Ph.D. (Thesis Advisor) Karen Beemon, Ph.D. Xin Chen, Ph.D. Michael Lichten, Ph.D. iii PREFACE All of the work presented henceforth was conducted in the Chromosome Biology Section of the Laboratory of Biochemistry and Molecular Biology at the Center for Cancer Research of the National Cancer Institute. A version of Chapters 1, 2, and 3 has been published (Lee NN, Chalamcharla VR, Reyes-Turcu F, Mehta S, Zofall M, Balachandran V, Dhakshnamoorthy J, Taneja N, Yamanaka S, Zhou M, Grewal SI. Mtr4-like protein coordinates nuclear RNA processing for heterochromatin assembly and for telomere maintenance. Cell. 2013 Nov 21;155(5):1061-74). I was the lead author of the published work, responsible for areas of hypothesis, data collection, analysis, and manuscript composition while there were a number of co-authors who contributed significantly to the published work. Venkata R. Chalamcharla was involved in purification of proteins and small RNA sequencing. Francisca Reyes-Turcu was involved in the initial concept formation and experiments including protein purification, co-IP, and ChIP-chip. Sameet Mehta helped with bioinformatics analysis. Martin Zofall contributed to some protein purifications, RNA sequencing, and Northern blot analysis. Vanivilasini Balachandran and Jothy Dhakshnamoorthy helped in generating strains. Nitika Taneja contributed to immunofluorescence experiments. Soichiro Yamanaka contributed to one ChIP-chip experiment. Ming Zhou helped with mass-spectrometry analysis. Shiv I.S. Grewal was the supervisory author on this project and was involved throughout the project in concept formation and manuscript composition. A version of Chapters 4, 5, and 6 is being prepared for publication. I am the lead author of the work, responsible for areas of hypothesis, initial concept formation, data iv collection, analysis, and manuscript composition while a number of co-authors also contributed to the project. Gobi Thillainadesan was involved in bioinformatics analysis. David Wang and Avindra Nath provided help with human neural stem cell culture. Shiv Grewal is the supervisory author on this project and was involved throughout the project in concept formation and manuscript editing. v ACKNOWLEDGEMENT I thank everyone who has worked and exchanged ideas with me over this project. I thank every member of the Shiv Grewal laboratory (Chromosome Biology Section of the Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, National Cancer Institute) during my time for valuable contributions and help. I thank my thesis advisor, Shiv Grewal, for support and guidance. And I thank my thesis committee members, Karen Beemon, Xin Chen, and Michael Lichten for advice and support. I thank Peter FitzGerald for intron analysis, Robin Allshire for the cwf10-1 mutant, Masayuki Yamamoto for the tor2-ts6 mutant, Jemima Barrowman for her valuable help in editing the manuscripts, and Michael Lichten for comments. This research was supported by the Intramural Research Program of the National Institutes of Health (NIH), National Cancer Institute, and utilized the Helix Systems and the Biowulf Linux cluster at the NIH. vi TABLE OF CONTENTS Page ABSTRACT ........................................................................................................................ ii PREFACE .......................................................................................................................... iv ACKNOWLEDGEMENT ................................................................................................. vi TABLE OF CONTENTS .................................................................................................. vii LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES .............................................................................................................x INTRODUCTION ...............................................................................................................1 RESULTS ..........................................................................................................................11 Chapter 1. Purification of Red1 and Identification of Its Associated Proteins ......11 1.1. Red1 Interacts with Various Factors ....................................................11 1.2. Red1 and Mtl1 Form a Core Module that Interacts with Different Nuclear Proteins ...................................................................................14 1.3. Red1- and Mtl1-Associated Factors Differentially Affect Heterochromatin Domains ...................................................................18 Chapter 2. MTREC Regulates Gene Expression and Targets Noncoding RNAs and Pre-mRNAs Degraded by the Exosome ..........................................................29 2.1. Mtl1 Regulates Expression of Genes Involved in Sexual Differentiation, Stress Response and Membrane Transport .............................................29 2.2. MTREC Targets Regulatory Noncoding RNA and Pre-mRNA Degraded by the Exosome .......................................................................................31 2.3. Noncoding RNA Regulates Gene Expression in Response to Environmental Changes ..........................................................................33 Chapter 3. Mtl1 Forms Red1-Independent Interactions with Nrl1 and Ctr1 for Regulation of Splicing Associated with Heterochromatin Assembly and Telomere Maintenance ...........................................................................................................38 3.1. Mtl1 Associates with Nrl1 and Ctr1 without Red1 .................................38 3.2. Nrl1 Promotes Assembly of HOODs at Genes and Retrotransposons ...40 3.3. Nrl1 Interacts with Splicing Factors to Assemble HOODs via Cryptic Introns .....................................................................................................42 3.4. Noncoding RNAs and Read-through Transcripts Contain Introns .........51 3.5. Mtl1 and Ctr1 Promote Telomerase RNA Biogenesis and Telomere Maintenance ............................................................................................54 vii Chapter 4. TOR Signaling Pathway Regulates Facultative Heterochromatin .......58 4.1. TOR Signaling Pathway Regulates Heterochromatin Islands ................58 4.2. Tor2 Regulates MTREC-dependent Heterochromatin through Pir1 ......62 4.3. Tor2 and Pir1 Regulate Environmentally-sensitive and Disease- associated Genes .....................................................................................67