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By Submitted in Partial Satisfaction of the Requirements for Degree of in In by Submitted in partial satisfaction of the requirements for degree of in in the GRADUATE DIVISION of the UNIVERSITY OF CALIFORNIA, SAN FRANCISCO Approved: ______________________________________________________________________________ Chair ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ Committee Members ii Acknowledgements I am first and foremost extremely lucky and grateful to have family that is supportive of my academic endeavors throughout my life. As the first of my family to graduate college, I’ve learned the true value of hard-work, determination, and love through my parents. They continue to serve as inspiration for me to grow and become a better version of myself every day. I am incredibly grateful to have had the opportunity to learn and grow as a scientist in the laboratory of Dr. Daniel Amos Lim. Dr. Lim has an unparalleled work-ethic that is motivating and inspiring for his students and mentees. I can truly say that I’ve never worked with a mentor that is so meticulously involved with the growth and success of his mentees. These past 5 years in Dr. Lim’s lab will remain with me forever, and I will never forget the lessons I’ve learned about how to be a good scientist, and a good person. Without a doubt, the academic environment within Dr. Lim’s lab is mentally stimulating – where research spans from fine-grain detail of molecular biology, to broader impact health-care tools. I’d like to thank the lab members for all the help and invigorating discussions we’ve had. In particular, David Wu, a talented MSTP student in the lab was directly involved with my thesis project in the molecular dissection of LOC646329. Outside of the lab, Dr. Aaron Diaz, Soren Muller, and Lin Wang were absolutely instrumental in my thesis project. Their knowledge and approaches from a computational biology standpoint really helped answer questions that elevated our findings and broadened the perspective of our work. I’d like to thank my thesis committee members, which was a team of extraordinary scientists whom I look up to. Dr. Robert Blelloch, Dr. Joe Costello, and Dr. Barbara Panning are all extremely accomplished scientists who gave invaluable guidance throughout my years at UCSF. iii Prior to my graduate education, there are a number of academic mentors that have inspired and instilled the confidence I needed to become a graduate student. I’m extremely appreciative to have had the opportunity to learn from Dr. Stacey Harmer, and Dr. Peggy Farnham at UC Davis. As an undergraduate researcher in Dr. Harmer’s and Dr. Farnham’s lab, I was introduced to the world of academic science. This inspired me to continue working in academic science after my undergraduate education, where I continued to learn under Dr. Abby Dernburg at UC Berkeley. With Dr. Dernburg, I learned about what it meant to be a full-time scientist. This experience launched me into one of the most transformative experiences I’ve had since graduating from UC Davis. I worked with Dr. Benoit Bruneau at the Gladstone Institutes, where I was treated more like a graduate student with intellectual freedom, rather than a staff employee. It was in Dr. Bruneau’s lab where I was truly inspired to become a graduate student myself. Without every single one of these people, and every single one of these events, I would not be where I am today – for that, I am truly grateful. iv miRNA-independent function of lnc-pri-miRNA loci Daniel He Abstract Among the large, diverse set of mammalian long noncoding RNAs (lncRNAs), long noncoding primary microRNAs (lnc-pri-miRNAs) are those that host miRNAs. Whether lnc-pri- miRNA loci have important biological function independent of their cognate miRNAs is poorly understood. From a genome-scale lncRNA screen, lnc-pri-miRNA loci were enriched for function in cell proliferation, and in glioblastoma (GBM) cells with DGCR8 or DROSHA knockdown, lnc-pri-miRNA screen hits still regulated cell growth. To molecularly dissect the function of a lnc-pri-miRNA locus, we studied LOC646329 (a.k.a. MIR29HG), which hosts the miR-29a/b1 cluster. In GBM cells, LOC646329 knockdown reduced miR-29a/b1 levels, and these cells exhibited decreased growth. However, genetic deletion of the miR-29a/b1 cluster (LOC646329-miR29D) did not decrease cell growth, while knockdown of LOC646329-miR29D transcripts reduced cell proliferation. The miR-29a/b1-independent activity of LOC646329 corresponded to enhancer-like activation of a neighboring oncogene (MKLN1), regulating cell propagation. The LOC646329 locus interacts with the MKLN1 promoter, and antisense oligonucleotide knockdown of the lncRNA disrupts these interactions and reduces the enhancer- like activity. More broadly, analysis of genome-wide data from multiple human cell types showed that lnc-pri-miRNA loci are significantly enriched for DNA looping interactions with gene promoters as well as genomic and epigenetic characteristics of transcriptional enhancers. Functional studies of additional lnc-pri-miRNA loci demonstrated cognate miRNA-independent, v enhancer-like activity. Together, these data demonstrate that lnc-pri-miRNA loci can regulate cell biology via both miRNA-dependent and miRNA-independent mechanisms. vi Table of Contents Chapter 1: Introduction ................................................................................................................1 Summary ...................................................................................................................................1 Background ..............................................................................................................................1 Sno-lncRNAs .......................................................................................................................3 Circular lncRNAs.................................................................................................................4 Concluding Remarks ...............................................................................................................6 Chapter 2: lnc-pri-miRNA are at elevated levels in human glioblastoma and enriched for function ...........................................................................................................................................8 Summary ...................................................................................................................................8 Introduction ..............................................................................................................................8 Results .....................................................................................................................................10 lnc-pri-miRNA are enriched in high grade glioma ............................................................10 Discussion................................................................................................................................11 Figures and Tables .................................................................................................................12 Experimental Procedures ......................................................................................................75 Chapter 3: lnc-pri-miRNA loci with miRNA-independent function .......................................77 Summary .................................................................................................................................77 Introduction ............................................................................................................................78 Results .....................................................................................................................................79 vii lnc-pri-miRNA loci regulate cell proliferation in cells with Microprocessor knockdown .........................................................................................................................79 LOC646329 knockdown increases apoptosis and reduces proliferation of GBM cells.....80 Deletion of miR-29a/b1 does not decrease cell growth .....................................................81 LOC646329 has cellular function independent of its cognate miRNAs ............................83 The LOC646329 locus contains transcriptional enhancer activity ....................................85 lnc-pri-miRNA loci are enriched for physical interactions with gene promoters and enhancer characteristics .....................................................................................................87 lnc-pri-miRNAs can regulate local genes independent of its cognate miRNAs ................87 Discussion................................................................................................................................88 Figures and Tables .................................................................................................................96 Experimental Procedures ....................................................................................................127 References ...................................................................................................................................144 viii List of Figures Chapter 1: Introduction ................................................................................................................1
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