Exploring Transposable Elements Regulated by ZNF649 By: Joshua Gu April 2nd 2018 Senior Thesis Project Bachelor of Science in Bioengineering Advisor: Dr. David Haussler University of California, Santa Cruz Abstract KRAB Zinc Fingers Proteins (KZNFs) are the largest family of human transcription factors. Many have evolved to battle against transposable elements (TEs) and protect the genome. When TEs are not repressed, they can disrupt genes and cause diseases. Throughout evolution, different KZNFs have emerged to battle invading TEs, leading to primate specific KZNF-TE interactions. In my thesis project, I investigated different families of transposable elements that were regulated by a specific KZNF, ZNF649, which is expressed in heart, skeletal muscle, and brain tissues of human adults. By analyzing ChIP-seq data of KZNFs and transposable elements, nucleotide KZNF recognition motifs were calculated and validated experimentally. With a binding reporter assay, I discovered that ZNF649 represses the TE L1PA4. Additionally, I showed how specific nucleotide mutations in the calculated ZNF649 DNA recognition motif are vital for ZNF649 to repress L1PA4. The TATA box was also found to be essential for repression of L1PA by ZNF649. To study other TEs that ZNF649 may have regulated, I tested to see if ZNF649 repressed the TE SVA, since it bound the VNTR region of SVA in the ChIP-seq data. Data from my binding assay in mouse embryonic stem cells (mESCs) revealed that ZNF649 does not repress certain SVA elements. However, computational analysis has shown that the tested SVA elements do not contain the consensus sequence defined by SVA elements that bind ZNF649. Furthermore, I used CRISPRi to knockdown ZNFs and study the change in expression levels of the ZNF and the TE it regulates. I determined the molecular requirements for ZNF649 to repress TEs, which includes the required nucleotides of the recognition motif and finger protein domains that recognize the motif, leading to the repression of the TE by ZNF649. Finally, I created a large KZNF library, which established a powerful platform to study how KZNF-TE interactions have influenced human-specific biology. My study will help the Haussler lab identify new KZNF-TE interactions and elucidate the relationships between ZNF649 and the TEs it regulates. Acknowledgments I would like to thank my advisor David Haussler for his time, support, and opportunity to work in the Haussler wet lab since my sophomore year. Next, I want to give special thanks to my mentor Dr. Jason Fernandes for his guidance, insight, and support throughout my time in the Haussler lab. I would also like to thank my mentor Kristof Tigyi, Sofie Salama, and other members of the Haussler lab for answering my questions and teaching me new concepts. Additionally, I would like to thank Bari Nazario from the Institute for the Biology of Stem Cells (IBSC) at UCSC and Ben Abrams from the UCSC Life Science Microscopy Center. Finally, I would like to thank my parents who have supported me throughout my undergraduate career. 1 Contents 1 Introduction 4 2 Background 6 2.1 Transposable Elements . 6 2.2 KRAB-Zinc Finger Proteins . 7 2.3 Transposable Elements in Humans . 8 2.4 Previous Studies . 10 3 Which transposable element(s) does ZNF649 repress? 11 3.1 Mutations commonly seen in unbound L1PA elements . 11 3.2 Which zinc finger domains of ZNF649 are required to repress L1PA4? . 13 3.3 Does ZNF649 repress SVA? . 14 3.4 Luciferase assay method . 15 3.5 Results . 16 4 Knockdown ZNFs with CRISPRi 18 4.1 Lipofection . 19 4.2 Doxycycline induction . 19 4.3 RNA harvest/isolation . 19 4.4 Results . 20 5 ORFeome KZNF collection 22 5.1 Generating a KZNF Library to test on Transposable Elements Overview . 22 5.2 DNA Isolation and Purification . 23 5.3 Gateway Cloning . 25 5.4 Transfection . 27 5.5 Western Blot . 28 5.6 Results . 28 References 32 Appendix A: Miniprep protocol for KZNFs 34 Appendix B: Gateway cloning protocol 37 Appendix C: FLAG tagged KZNF Western Blot Protocol 39 2 Appendix D: CRISPRi Gen1C iPSC Protocol 44 Appendix E: RNA isolation for iPSCs on matrigel Protocol 46 Appendix F: Luciferase Assay Protocol 48 Appendix G: QIAfilter Plasmid Maxi prep protocol 50 List of Figures 1 Peppered moth during industrial revolution . 6 2 Kap1 recruitment . 7 3 Crystal structure of ZNF bound to DNA . 8 4 Primate-specific KZNF timeline . 9 5 Luciferase assay for ZNFs and SVA . 10 6 ChIP-seq data for ZNF649, ZNF93, and Kap1 in L1PA elements . 11 7 ZNF649-L1PA core motif . 12 8 TATA box deletion . 12 9 Mutant L1PA4 constructs for luciferase assay . 13 10 ChIP-seq data for ZNF649 and Kap1 in SVA elements . 14 11 SVA constructs for ZNF649 . 15 12 Mutations for L1PA4 to escape repression from ZNF649 . 16 13 Current results show ZNF649 does not repress SVA . 17 14 CRISPRi Gen1C outline . 19 15 CRISPRi Gen1C +/- Dox image . 20 16 qPCR data of ZNF649 knockdown . 21 17 Knockdown of ZNF649 and ZNF93 setup . 22 18 Miniprepping steps . 24 19 Gateway reaction diagram . 25 20 PID construct design . 26 21 Transfection of KZNFs in PID constructs . 29 22 Western blot of KZNFs . 30 23 Hughes KZNF in PKZ construct restriction digest . 31 3 1 Introduction First discovered in the 1940s by Barbara McClintock, transposable elements are genetic elements that duplicate and "transpose" or in other words, move throughout the genome. These transposable elements have the ability to generate genomic instability and reconfigure gene expression (Reilly et al., 2013). When transposable elements spread within the genome, they can disrupt genes and cause diseases. A certain class of transposable elements, retrotransposons, require transcription in order for them to insert into a new site within the genome. Therefore, repressing their transcription is an effective way to limit their retrotransposi- tion. KRAB-Zinc Finger Proteins (KZNFs), the largest family of human transcription factors, have evolved to battle against transposable elements and protect the genome. Many KZNFs take part in cardiac develop- ment, cardiovascular diseases, differentiation of several tissues, tumorigenesis, or neurodegeneration diseases (Jen and Wang, 2016). Waves of primate-specific transposable element insertions have been accompanied by new primate KZNFs (Thomas and Schneider, 2011). One of the functions of KZNFs includes using their zinc finger protein domains to recognize transposable element DNA and silencing them by recruiting KRAB-associated protein-1 (Kap1) to repress transposable element transcription (Wolf and Goff, 2007; Wolf and Goff, 2009). Understanding the way KZNFs regulate transposable elements is key to revealing how the genome protects itself from invading transposable elements that influence human evolution. Our lab recently demonstrated that two primate-specific KZNFs, ZNF93 and ZNF91 evolved to repress specific subclasses of transposable elements, Long Interspersed Nuclear Element (LINE) 1 and SINE-VNTR-Alu (SVA), respec- tively (Jacobs et al., 2014). Furthermore, repression of transposable elements can lead to repression of genes nearby, which can lead to species-specific evolution. (Jacobs et al., 2014). While KZNFs evolve to recognize and regulate new transposable elements, transposable elements evolve and mutate recognition sites to escape the recognition of KZNFs. This selective pressure between KZNFs and transposable elements has resulted in an \evolutionary" arms race, which has led to primate-specific KZNF-transposable element pairs. However, many questions still remain unknown. How many KZNFs are sufficient to repress an active transposable element? Are all the zinc finger domains of a KZNF needed to repress a transposable element? What binds to the youngest L1 transposable element in humans? Chromatin immunoprecipitation sequencing (ChIP-seq) data can illuminate the unknown surrounding these questions by identifying ZNF recognition motifs of transposable elements. ChIP-seq is a method that studies the interaction between protein and DNA. DNA-bound protein are immunoprecipitated using a specific antibody, then coprecipitated, purified, and sequenced. To predict DNA binding sites from ChIP-seq read count data, peak calling is used, which identifies areas within a genome where protein interacts with DNA. Analysis of previously published ChIP-seq data revealed that ZNF649 as well as ZNF93 recognized L1PA, a subclass of the L1 transposable elements (Imbeault et al., 2017; Schmitges et al., 2016). Additionally, evolutionary analyses of the L1PA elements and KZNFs known to bind it, ZNF93 and ZNF649, revealed nucleotide 4 mutations that support the arms race model. ZNF649 is located on the human chromosome 19q13.41 according to NCBI and contains 5 exons. It has been found in adults that ZNF649 is expressed predominantly in heart, skeletal muscle, and brain tissues, but weaker in other tissues (Yang et al., 2005). Data from ChIP-seq helped determine where the ZNF bound to on the transposable elements. Analyz- ing the ChIP-seq data from previous studies revealed ZNF649 bound to certain L1PA and SVA elements (Imbeault et al., 2017; Schmitges et al., 2016). While ZNF649 did not bind to the most recent L1PA el- ement, L1HS (human specific), it bound to L1PA4, which arose around 18 million years ago. The larger number after the L1PA element indicates an older element. My goal was to determine the molecular requirements for ZNF649, and other KZNFs, to repress transposable elements. The molecular requirements include the required nucleotides of the recognition motif and finger protein domains that recognize the motif, leading to the repression of the transposable element by ZNF649. To test this, I utilized a binding reporter assay to determine if ZNF649 caused expression change in the transposable element. I showed that ZNF649 repressed L1PA4 and nucleotides in the recognition motif that allow L1PA4 to escape repression. This was achieved by introducing single nucleotide mutations into the recognition motif on L1PA4 reporter constructs. Our lab previously showed that ZNFs can gain zinc fingers to regulate transposable elements (Jacobs et al., 2014), which led to the question if ZNF649 required all 10 of its zinc finger domains to repress L1PA4.