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Functional Investigations of Duf1220 Protein Domains

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Functional Investigations of Duf1220 Protein Domains FUNCTIONAL INVESTIGATIONS OF DUF1220 PROTEIN DOMAINS by JONATHON G. KEENEY B.A., University of Colorado Boulder, 2006 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Neuroscience Program 2014 ii This thesis for the Doctor of Philosophy degree by Jonathon G. Keeney has been approved for the Neuroscience Program by S. Rock Levinson, Chair Thomas Finger David Pollock Diego Restrepo Matthew Taylor James Sikela, Advisor Date __5/9/14__ iii Keeney, Jonathon G. (Ph.D., Neuroscience) Functional Investigations of DUF1220 Protein Domains Thesis directed by Professor James Sikela ABSTRACT Copy number expansion of genetic content is a powerful way in which species adapt to environments, and a powerful mechanism by which novel genes are generated. DUF1220 domains were previously identified as showing the largest human lineage- specific increase in copy number of any protein coding sequence with the human genome. While the function of DUF1220 domains remains obscure, the Sikela lab has identified a strong correlation between increasing DUF1220 copy number and increases in both brain size and cortical neuron number within great apes. In addition, a significant association has been reported between reduction and gain of DUF1220 copy number and brain size in microcephaly and macrocephaly, respectively . This thesis investigates the function of DUF1220 domains by further investigating the correlation between DUF1220 copy number and several physical brain measurements within anthropoid primates (monkey, ape and human). The spatiotemporal expression pattern in human fetal brain tissue was then evaluated, and suggested a role in neural progenitor expansion. In vitro cell culture experiments were then used to demonstrate the ability of DUF1220 to promote proliferation of human neural stem cells. Transcriptomics and proteomics studies both suggested a role in down regulating mitochondrial function, and this result was confirmed by live cell imaging using a iv mitochondrial dye. Finally, a functional evaluation of mitochondrial output in DUF1220 over expressing cells was performed, and found to slightly reduce metabolic output. I offer potential explanations linking the ability of DUF1220 to promote proliferation with the apparent reduction in mitochondrial output. The form and content of this abstract are approved. I recommend its publication. Approved: Jim Sikela v DEDICATION I would like to dedicate this thesis to my sisters, Julia Morgan, Josie Keeney and Chelsea Keeney, for all of their steady, incredible support over many difficult years. Through many chaotic and unpredictable events, I have found in them the unconditional love and support I needed to overcome challenges and become a better person. vi ACKNOWLEDGEMENTS I would like to acknowledge Jack Davis for statistical analysis of data pertaining to my cell proliferation experiment (Figure III.7). I would also like to thank Miriam Post for providing human fetal brain tissue, Boye Schnack Nielson for in situ hybridization using LNA probes, and Julie Siegenthaler for her expert advice in analyzing these samples. I’d like to thank David Astling for his analysis of my RNAseq data and for providing me with Figure III. and Table III.1, Table III.2, Table III.3 and Table III.4 based on this data. Additionally, I’d like to thank Karl Vandepoele, Vanessa Andries and Frans van Roy for their DLD1Tr/NBPF1 and DLD1Tr/Mock cell lines, as well as a proteomic analysis of these cells (not included). I would like to thank Pamela Lopert and Manisha Patel for access to the Seahorse XF24 bioanalyzer, and for help in analyzing my results (Figure III.). Thank you very much to Suzana Herculano-Houzel and Mariana Gabi for evaluating the cellular composition of transgenic mice I developed during this project, and for their thoughtful analysis of this data, which was used to generate Figure III.. Finally, I would like to thank the Coleman Institute for Cognitive Disabilities for their generous support over four years of productive research. vii TABLE OF CONTENTS CHAPTER I. BACKGROUND ............................................................................................................ 1 Copy Number Variations ............................................................................................ 1 Developmental Neuroscience in Primates .............................................................. 18 II. SCOPE OF THE PROJECT ........................................................................................... 35 III. ORIGINAL RESEARCH ................................................................................................ 36 Additional Associations ............................................................................................ 36 DUF1220 Domains Are Associated with Increased Neural Stem Cell Proliferation in Simian Primates and Drive Proliferation in Human Neurons .................................................................................................................... 37 DUF1220 Domain Expression Down Regulates Mitochondrial Pathways .................................................................................................................. 53 A Mouse Model of DUF1220 in vivo ........................................................................ 76 IV. SUMMARY, CONCLUSIONS AND FUTURE DIRECTIONS ........................................... 86 Summary .................................................................................................................. 86 Conclusions .............................................................................................................. 90 Future Directions ..................................................................................................... 99 REFERENCES .................................................................................................................... 104 viii LIST OF TABLES TABLE III.1: Pathways associated with genes down regulated in our RNAseq analysis after exogenous expression of NBPF1 in DLD1Tr/NBPF1 cells .................................................. 59 III.2: Pathways associated with genes down regulated in our RNAseq analysis after exogenous expression of NBPF1 in DLD1Tr/Mock cells ................................................... 60 III.3: Pathways associated with genes up regulated in our RNAseq analysis after exogenous expression of NBPF1 in DLD1Tr/NBPF1 cells .................................................. 61 III.4: Pathways associated with genes up regulated in our RNAseq analysis after exogenous expression of NBPF1 in DLD1Tr/Mock cells ................................................... 62 III.5: Raw values generated by the Seahorse XF24 ........................................................... 67 ix LIST OF FIGURES FIGURE I.1: Mechanism of Copy Number Variations ....................................................................... 1 I.2: The Fate of Duplicated Genetic Content ...................................................................... 3 I.3: Array-Based CGH Setup ................................................................................................ 7 I.4: Compilation of Primate Lineage Specific Copy Number Variations ............................. 9 I.5: Graphical Depiction of DUF1220 Organization Within Genes .................................... 12 I.6: Graphical Depiction of the Evolutionary Expansion of DUF1220 Domains ................ 15 I.7: Summary of genomic copy number variations known to be associated with cognitive diseases ............................................................................................................................. 17 I.8: Proposed Mechanism Linking DUF1220, Brain Evolution and Disease. ..................... 19 I.9: Specification of transitory, developmental zones in the neocortex .......................... 23 I.10: The trajectory of cells within a radial unit expands the neocortex .......................... 25 I.11: Graphical Description of the Neuronal Scaling Rules Within the Cerebral Cortex of the Primate and Rodent Lineages ..................................................................................... 33 III.1: Probe Quantification ................................................................................................. 41 III.2: Manual Inspection of Cell Coverage ......................................................................... 43 III.3: In situ hybridization results of human fetal brain tissues throughout development, part I: Stages 6 - 15 ........................................................................................................... 44 III.4: In situ hybridization results of human fetal brain tissues throughout development, Part II: Stages 16 - 25 ........................................................................................................ 45 III.5: In situ hybridization results of human fetal brain tissues throughout development, Part III: Stages 28 - 39 ....................................................................................................... 46 III.6: Timeline of DUF1220 Expression and Human Fetal Brain Development ................. 47 III.7: Exogenous Expression of DUF1220 promotes Neural Progenitor Cell Number ....... 50 III.8: Histogram of False Discovery Rates .......................................................................... 62 III.9: Induction of NBPF1 over expression decreases
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