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. To test this, I deleted zinc fingers of ZNF649 to use in a binding reporter assay. The output of the ChIP-seq data correlation uncovered an evolutionary arms race and provided a recog- nition motif that I tested. I determined how ZNF93 and ZNF649 recruit Kap1 at each L1PA element and investigated if ZNF93 and ZNF649 worked together to repress L1PA elements as they attempt to escape recognition. To test if ZNF93 and ZNF649 are both needed to repress L1PA elements, I knocked down (silence) one of the KZNFs to determine if the repression of the L1PA element remains the same. I used an inducible CRISPR-interference (CRISPRi) Gen1C human induced pluripotent stem cell (iPSC) line to express guide RNAs and deactivated Cas9-KRAB fusion construct to knock- down genes, then assayed ZNF knockdown and transposable element expression using qPCR. This CRISPRi system was used to inhibit transcription of KZNFs to determine if inhibition of specific KZNFs lead to de-repression of specific transposable elements. The CRISPRi system tests the presence of the recognition motif by using unique primers for the qPCR. In order to determine which primate-specific KZNFs recruits KAP1 to primate-specific transposable elements, I cloned a library of 146 KZNFs in order to determine their activities in stable transposable element cell lines, where the transposable element gene is integrated into the genome. The KZNFs were cloned into a FLAG tagged lentiviral construct before being introduced to 293FT human cells using a lentivirus packaging system. The resulting lentivirus were used to infect mouse embryonic stem cell (mESC) lines engineered with

5 a single instance of a primate-specific transposable element reporter. My work establishes a powerful platform to study how KZNF-transposable element interactions have influenced human-specific biology.

2 Background

2.1 Transposable Elements

Transposable elements, also known as “jumping genes”, are DNA sequences that duplicate and move within the genome. KRAB-Zinc Finger Proteins (KZNFs) regulations are one of the methods of regulating them. Transposable elements can impact the genome through various mechanisms including alternative splicing, insertional mutagenesis, and exon shuffling (Hancks and Kazazian, 2010). There are two types of transposable elements, Class I (retrotransposons) and Class II (DNA transposons). Retrotransposons are transcribed from DNA to RNA, then reverse transcription of the RNA produces DNA, which is inserted back into the genome at a new location. On the other hand, DNA transposons are cut and inserted into a target site in the genome. I studied retrotransposons since they are active in humans. An important challenge of evolutionary biology is discovering the mutational events that help adaptation to environmental change. A common example used in classrooms of visible evolutionary response is the color change of the peppered moth (Biston betularia) during the industrial revolution, as seen in Figure 1. During the industrial revolution, the black (Carbonaria) form of the peppered moth outnumbered the common pale form due to their ability to blend to the soot covered surroundings, as opposed to the common pale moth that could not easily blend in and evade their prey. A recent study revealed that an insertion of a large, tandemly repeated, transposable element into the first intron of the Cortex gene enhanced Cortex expression levels, which underlay adaptive cryptic coloration during the industrial revolution. (van’t Hof et al., 2016).This shows that transposable elements can generate phenotypic changes.

Figure 1: The polymorphic Carbonaria transposable element impacted the color genotype of the peppered moth by inserting into the first intron of the Cortex gene. Source: Chuong et al. Nature Rev Gen 2016

6 Three currently active retrotransposable element classes are short interspersed nuclear element (SINE)- VNTR- Alu (SVA), long interspersed nuclear element (L1), and Alu. SVA transposable elements have been associated with human diseases such as Leukemia (Hancks and Kazazian, 2010). Recently, an SVA insertion in the TAF1 locus locus has shown to trigger aberrant splicing of TAF1 and cause the X-Linked Dystonia- Parkinsonism disease (Aneichyk et al., 2018). An L1 insertion was found to cause Haemophilia A in humans, the first study to display a transposable element causing disease via transposable element insertion in the human genome (Kazazian et al., 1988). L1 transposable elements are estimated to have 500,000 copies per genome and may contribute to tumorigenesis, the formation of tumor(s) (Rodic and Burns, 2013). By studying transposable element expression defects that affect motor neuron diseases such as Amyotrophic Lateral Sclerosis (ALS), a more in-depth understanding of the diseases can be achieved so that a treatment or cure can be provided in the future (Li et al., 2012).

2.2 KRAB-Zinc Finger Proteins

KRAB-Zinc Finger Proteins (KZNFs), the largest family of human regulatory proteins, have evolved to battle against invading transposable elements in the genome. There are around 350 KZNFs encoded in the human genome (Imbeault et al., 2017). While the functions of the majority of KZNFs are unknown, some have shown to repress transposable elements in embryonic stem cells (ESCs) by recruiting transcriptional regulators (Kap1, also known as TRIM28) and associated mediators of histone H3 Lys9 trimethylation (H3K9-me3)-dependent heterochromatin formation and DNA methylation (Imbeault et al., 2017). KZNFs function to silence or regulate transposable elements when they encounter their recognition motifs for the KZNF. The KZNF binds to the transposable element DNA and recruits KRAB-associated protein-1 (Kap1) to repress them (Jacobs, et al., 2014), as seen in Figure 2.

Figure 2: A KZNF binds to the DNA of the transposable element, then recruits Kap1, which transcriptionally represses the transposable element. Kap1 recruits factors that cause a closed chromatin state. By analyzing ChIP-seq data, it is known that ZNF649 binds to L1PA.

7 Zinc finger proteins are small functional domains that use a zinc ion to stabilize the integration of the protein (Laity et al., 2001). The ”finger” is the secondary structures (α-helix and β-sheet), held together by the zinc ion.

Figure 3: Crystal structure (1G2F) of a ZNF bound to DNA illustrates the amino acids involved in base- specific DNA recognition (Provided by Dr. Jason Fernandes and adapted from Wolfe et al., 2001)

2.3 Transposable Elements in Humans

One of the major scientific questions of interest from our lab is “What makes humans different from other primates?” It has been found that the DNA sequences of the chimpanzee and human genomes are almost 96% identical with DNA insertions and deletions taken into account (Waterson et al., 2005). Among the protein coding sequence, the identity between humans and chimpanzees is around 98% (Waterson et al., 2005). Primate-specific transposable elements are one of the factors that distinguish humans from their close relative, chimpanzees. While chimpanzees and humans share similar protein encoding genes due to chimpanzees being a close relative of humans, they are phenotypically (observable characteristics) distinguishable. For example, humans have a larger brain size than chimpanzees. Part of the reason that chimpanzees and humans are phenotypically different is because of transposable element insertions in the genome. Investigating how transposable elements cause primates to “branch off” the phylogenetic tree will provide a better understanding of the evolutionary process and the differences between humans and its relatives. Regulated transposable element expression can lead to adaptive evolution. Analyzing transposable elements that drive population diversity and cause primate-specific phenotypes will ultimately provide a better understanding of the evolutionary process and the differences between humans and other primates.

8 The Red Queen hypothesis is an evolutionary hypothesis that states that organisms must constantly adapt and evolve to “stay in place” and survive. As seen in Figure 4, primate-specific KZNFs arise throughout evolution to battle invading transposable elements. A recent study by Jacobs et al. (2014) uncovered an evolutionary arms race by showing repression of transposable element SVA and L1 retrotransposons by ZNF91 and ZNF93, respectfully. The retrotransposon classes studied in my thesis are the Long Interspersed Nuclear Element (L1) and SINE- VNTR-Alu (SVA).

Figure 4: As transposable elements invade the genome, KZNFs rise to combat against transposable elements. Each primate has a specific set of KZNF-transposable element interactions. ZNF93 and ZNF649 arose to battle the L1PA elements and are specific to the primates after Old World monkeys. L1PA5, L1PA4 and L1PA3 are transposable elements that arose 20 million, 18 million and 16 million years ago respectively. Adapted from: Jacobs, et al., 2014

9 2.4 Previous Studies

Previous studies of KZNFs include the uncovering of the evolutionary arms race between ZNF91/93 with SVA and L1PA transposable elements respectively (Jacobs, et al., 2014), determining the genomic targets of human KZNFs with ChIP-seq on 257 HEK 293FT cell lines (Imbeault et al., 2017), and ChIP-seq analysis of 131 human C2H2-ZF proteins (Schmitges et al., 2016). Our lab studied 33 KZNFs and found 14 to be highly expressed in human embryonic stem cells (Jacobs, et al., 2014). They used a luciferase binding assay, as seen in Figure 5, to measure the relative luciferase activity, determining the level of transposable element repression. The large collection of ChIP-seq studies of KZNFs and transposable element were leveraged to calculate and validate experimentally KZNF recognition motifs at a nucleotide resolution.

Figure 5: A luciferase binding assay was used to determine luciferase activity. A lower bar indicated low luciferase activity, which meant the KZNF repressed the SVA transposable element more. ZNF91 had the lowest relative luciferase activity. Source: Jacobs, et al., 2014

10 3 Which transposable element(s) does ZNF649 repress?

With a vast collection of ChIP-seq data of 246 KZNFs that were recently published (Imbeault et al., 2017; Schmitges et al., 2016) and KAP1 ChIP-seq data in different cell types (Jacobs et al., 2014; Theunissen et al., 2016), I determined which KZNF is responsible for targeting specific transposable elements by recruiting KAP1. Using ChIP-seq data, I determined the binding locations of KZNFs to the transposable elements. However, a binding interaction may not indicate repression. Therefore, I used a luciferase binding assay to determine if the KZNF repressed the transposable element it bound to successfully. When viewing the KAP1 and ZNF ChIP-seq data on the UCSC repeat browser, co-localization of KZNF and KAP1 ChIP-seq peaks suggest recruitment of KAP1. As seen in Figure 6, ZNF649 recruited KAP1 to repress L1PA elements until L1HS. It is still unknown as to what ZNF regulates L1HS since both ZNF649 and ZNF93 do not regulate L1HS.

Figure 6: This UCSC repeat browser data shows ZNF649 and KAP1 ChIP-seq peaks aligned for L1PA6 to L1PA2 elements, indicating the recruitment of KAP1 by ZNF649. The ZNF93 and KAP1 ChIP-seq peaks are aligned for L1PA6 to L1PA3-Long elements, indicating the recruitment of KAP1 by ZNF93 in these L1PA elements. The KAP1 ChIP-seq data was in hESCs while the ZNF649 and ZNF93 ChIP-seq data from Imbeault et al., 2017 were from HEK 293FT cells. The KAP1 ChIP-seq peak that aligns with the ZNF93 peaks in L1PA4 and L1PA3 were higher than the ZNF649’s. The x-axis was the nucleotide position, which included 1000 nucleotides for each L1PA element window.

3.1 Mutations commonly seen in unbound L1PA elements

Using previously published ChIP-seq data, the location of where ZNF649 bound to the transposable element L1PA was revealed. Analysis of L1PA6 to L1HS (human specific) elements provided a core DNA recognition motif GCACACCAGGAGATTATATCCCGC. Motif 1 and motif 2, as seen in Figure 7, had the highest score when testing for motifs with the highest number of bound instances. With thousands of

11 L1PA elements in the human genomes, not all had ZNF649 bound to the motif.

Figure 7: The DNA recognition motif for ZNF649. Motif 1 had the higher score and included the TATA box (shown in blue).

When the 5’ untranslated region (UTR) of the consensus L1PA elements was aligned, it was discovered that 30 million years ago in L1PA7, the TATA box was deleted. The TATA box is a sequence of non-coding DNA which serves as the nucleating site for the RNA-PolII transcription initiation complex. It is located in the promoter region of genes and contains repeating T and A base pairs. The TATA box is found in both archaea and eukaryotes. As seen in Figure 8, a new TATA box was formed in a 200 nucleotide region of L1PA6 through L1HS. To determine whether the TATA sequence contributes to ZNF649 binding, I introduced mutations in the TATA box by replacing the A nucleotide with a C nucleotide. This TC mutation resulted in a TCTC box instead.

Figure 8: Alignment of the 5’ UTRs of the consensus L1PA element to show TATA box deletion in L1PA7 30 milion years ago. A new TATA box was formed in L1PA6.

I used a luciferase assay to determine if mutations common in unbound L1PA elements and TC mutation would hinder the repression of the L1PA element by ZNF649. The setup, as seen in Figure 9, included 4 common mutation that were present in unbound L1PA elements. ZNF649 or pCAG empty vector was co-transfected for each construct in a 24 well plate of mESCs plated at 50,000 cells per cm2. I transfected into mESCs to have a null-primate background, preventing primate factors to effect KZNF-transposable

12 element interaction. The functional assay was normalized to Oct4 Enhancer and pCAG empty vector. The L1PA4-1* was an A to G mutation from L1PA4 to L1PA3. The L1PA4-2* included an additional G to C mutation from L1PA3 to L1PA2. The L1PA4-3* included an additional T to C mutation from L1PA2 to L1HS. And the L1PA4-4* included an additional G to C mutation.

Figure 9: Constructs used in the luciferase assay to determine the level of repression of L1PA mutations by ZNF649. Each construct was co-transfected with either ZNF649 or pCAG empty vector in a 24 well plate of mESCs. Firefly luciferase (FFL) was at the end of the sequence.

3.2 Which zinc finger domains of ZNF649 are required to repress L1PA4?

Zinc fingers gain and lose finger domains throughout evolution. For example, ZNF91 gained finger domains to bind to L1PA and recruit KAP1 to repress SVA. Zinc finger domains each recognize and bind to 3 nucleotides on the transposable element. ZNF649 has 10 finger domains. However, as new finger domains evolve, are the old finger domains still required to bind to a transposable element? I specifically studied ZNF649’s finger domains binding to L1PA. I used truncated mutant ZNF649s to test which finger domains were necessary to bind and repress L1PA. By co-transfecting mutant ZNF649s and L1PA4 into mESCs and testing with a luciferase assay, I observed the level of repression for different combinations of zinc finger domains.

13 3.3 Does ZNF649 repress SVA?

After analyzing published ChIP-seq data of ZNF649, I learned that it also binded to the transposable element SVA in the VNTR region. My goal was to determine if ZNF649 recruited KAP1 to repress SVA. As seen in Figure 10, ZNF649 ChIP-seq peaks do not align with KAP1 ChIP-seq peaks. I hypothesized that this indicated that ZNF649 would recruit KAP1 weakly and repress SVA slightly.

Figure 10: The ChIP-seq data for KAP1 in hESC and ZNF649 ChIP-seq data in HEK 293FT cells from Imbeault et al., 2017. The y-axis was the number of ChIP-seq peaks. The x-axis was the nucleotide position, which included 2000 nucleotides.

A previous study (Jacobs et al., 2014) showed that ZNF91 repressed SVA. I re-transformed the constructs used in the study, which are shown in Figure 11. The constructs I used were the pGL4cp, SINE-R, no VNTR, full length, VNTR with Oct4Enh, and Oct4Enh constructs. The VNTR region consists of 15 repeats, which are GC rich. The constructs were consensus sequences of SVA D, which does not exist in the human genome.

14 Figure 11: SVA constructs co-transfected with ZNF649 or pCAG empty vector into mESCs (null-primate background). The full length construct was pGL4cp-SVA D-SV40 P. The pGL4cp included hex (6) and Alu. Firefly luciferase (FFL) was at the end of the sequence.

3.4 Luciferase assay method

The luciferase assay was performed using to the Promega dual-luciferase kit and protocol in Appendix F. E14 mESCs were plated on 0.1% gelatin-coated-24-well plates at 50,000 cells per cm2. The next day, 100ng of pCAG-ZNF was co-transfected with 20ng of SV40P-luciferase reporter and 2ng of pRL-TK-renilla (a 10:1 firefly to renilla ratio) per 24 wells using Lipofectamine2000 in duplicate wells. Three hours after transfection, the media was changed. After 24 hours, the wells were washed with DPBS twice and harvested using 100µl of 1X Passive Lysis Buffer for 15 minutes on a room temperature rocker. To read as duplicates for each well, 45µl of lysate was transferred twice to a 96-well white opti-plate well. Then, 50µl of Luciferase Assay Reagent (LARII) was added to each well and the opti-plate was read on a Perkin-Elmer luminometer with Wallace Victor Light software counting 1 second per well. Next, 50µl of Stop and Glo reagent was added to each well of the opti-plate to measure the renilla activity for a transfection efficiency control. The opti-plate was then read on the luminometer with the same settings. Data was normalized in Microsoft Excel by dividing firefly by renilla then taking the average of 4 technical replicate measurements. The average was normalized against an SV40P-luciferace control for each KZNF pCAG construct and then again with pCAG empty vector set to 100%. Lipofection is a transfection technique used to introduce DNA into a cell using liposomes, vesicles that easily enter the cell membrane due to the fact that they are both phospholipid bilayers. The purpose of the

15 lipofection was to introduce the ZNF into the mESCs, which do not natively have ZNF649 or ZNF93.

3.5 Results

As seen in Figure 12, L1PA4-0 and L1PA4-1 had about 30% relative luciferase activity percentage, indicating 70% repression. L1PA4-2 had a decrease in repression at about 55% repression. L1PA4-3, L1PA4- 4, and L1PA4-TC were appeared to not have any repression. This data suggests that the first mutation did not alter the level of repression. However, the two mutation L1PA4 construct caused a decrease in repression. After introducing the third mutation, the repression essentially did not occur. The mutations were cumulative, so it is not possible to know if the mutations can perform the same way individually. To solve this question, I will test single mutations in the same setup. The TC mutation also caused ZNF649 not to repress L1PA4, suggesting that the TATA box is an important feature for ZNF649 to repress L1PA4. As mentioned before, the TATA box was deleted in L1PA7 30 million years ago, but a new TATA box was formed in L1PA6 and is still present. It is possible that L1PA4 required the TATA box itself to survive, which is why it was not deleted again.

Figure 12: Data from the luciferase assay of L1PA4 mutants and ZNF649. L1PA4-0 has no mutations while L1PA4-TC is the TC mutation in the TATA box. The relative luciferase activity percentage correlates with the repression percentage. The 4 data sets were averaged and normalized to Oct4 and pCAG empty vector. Data obtained: 2/21/18

16 Initial results for the ZNF649 with SVA luciferase assay indicates that in mESCs, the SVA constructs tested were not repressed much by ZNF649 (shown in Figure 13). As a control, ZNF91 and SVA were also tested. Results from the ZNF91-SVA assay showed a 60% repression for full length. SINE-R was shown to be a strong enhancer. The SVA constructs used were consensus sequences use to test ZNF91. However,the SVA constructs were not the bound ZNF649 element consensus sequence. To further test this, I will use a construct with the consensus sequence of bound ZNF649 elements from the ChIP-seq data. To determine if there is non-specific repressing, I will perform a titration with 50/100/200/400ng of ZNF/EV DNA. Additionally, I will test this assay in Human Embryonic Kidney (HEK) 293FT cells, which were the cells used for the ChIP-seq data. There may be a factor in the human cells that are not present in mouse that could be needed for ZNF649 to repress SVA.

Figure 13: Data from the luciferase assay of ZNF649 and SVA using 100ng of ZNF/EV DNA. The VNTR+Oct4 reporter was normalized to Oct4. Reporters No VNTR and full length were normalized to SINE-R. Data obtained: 2/23/18

17 4 Knockdown ZNFs with CRISPRi

Clustered regularly interspaced short palindromic repeats interference (CRISPRi), in which a doxycycline- inducible catalytically dead Cas9 (dCas9) is fused to a KRAB domain, can specifically and reversibly inhibit gene expression in iPSCs (Mandegar, et al., 2016). A guide RNA (gRNA) is a short RNA that targets the promoter element of the target gene with a scaffold sequence for dCas9 to bind, allowing for epigenetic editing. I used a stable CRISPRi Gen1C human iPSC line to knockdown ZNFs and observe the changes in repression of the transposable element. A ZNF guide RNA was transfected into the Gen1C iPSCs. See Appendix D for more detail about the CRISPRi Gen1C iPSC protocol. When viewing previously published ChIP-seq data of KAP1, ZNF93, and ZNF649 on the UCSC repeat browser for L1PA elements, the ChIP-seq peaks of the ZNFs and KAP1 align with each other, revealing where the ZNF binds to the L1PA element, as seen in Figure 6. Both ZNF93 and ZNF649 bind to L1PA subfamilies L1PA5 to L1PA3-L, but Kap1 recruitment by each KZNFs varies for each L1PA subfamily. Which leads to the question, what causes the KAP1 ChIP-seq peaks of ZNF93 and ZNF649 to fluctuate between L1PA5 and L1PA3? I determined how ZNF93 and ZNF649 recruit Kap1 at each L1PA element and investigated if ZNF93 and ZNF649 worked together to repress L1PA elements as they attempt to escape recognition. To test if ZNF93 and ZNF649 are both needed to repress L1PA elements, I knocked out ZNF649 to observe if it affects Kap1 recruitment to L1PA elements. I used an inducible CRISPRi Gen1C human induced pluripotent stem cell (iPSC) line to express guide RNAs and doxycycline-induce the deactivated Cas9-KRAB fusion construct to knockdown genes, then assayed ZNF knockdown and L1PA expression using qPCR. This CRISPRi system was used to inhibit transcription of KZNFs and transposable elements to determine if inhibition of specific KZNFs lead to de-repression of specific transposable elements. Due to the deletion of the ZNF93 binding site in L1PA3, ZNF93 did not have any ChIP-seq peaks that aligned with the KAP1 ChIP-seq peaks starting from L1PA3-short. The L1PA3-long still included the ZNF93 binding site. The ZNF guide RNA contained a mU6 promoter, the ZNF guide, and green fluorescence protein (GFP) sequence. The construct can be seen in Figure 14. Stably integrated in the Gen1C iPSC was a Tet Response Element (TRE) promoter, KRAB domain fused to a dCas9, and mCherry sequence. The TRE includes 7 repeats of a 19 nucleotide tetracycline operator (tetO) sequence and is recognized by the tetracycline repressor (tetR). When doxycycline, a derivative of tetracycline, is present, tetR will bind to doxycycline instead of the TRE, allowing transcription. With dCas9 transcribed, the ZNF will be knocked down.

18 Figure 14: The ZNF guides were transfected into Gen1C iPSCs, which had the KRAB-dCas9 construct stably integrated. After lipofection, selection with zeocin antibiotic is performed to kill off cells without the gRNA. Doxycycline (dox) induction is then performed for 48 hours. The addition of dox caused the knockdown of the ZNF.

4.1 Lipofection

Lipofection is a transfection technique used to introduce DNA into a cell using liposomes, vesicles that easily enter the cell membrane due to the fact that they are both phospholipid bilayers. The purpose of the lipofection was to introduce the ZNF guide RNA into the human iPS cells. Each ZNF guide RNA had duplicates in separate wells labeled (G1 and G2). Once 24 hours passed after lipofection, I changed the media and included zeocin (zeo) antibiotic to only select the cells that included the ZNF guide, which had zeocin resistant gene.

4.2 Doxycycline induction

The CRISPRi Gen1C was a doxycycline-inducible system. The addition of doxycycline (dox) caused the knockdown of the ZNF. As seen in Figure 14, the addition of dox produced a red fluorescence from the mCherry protein. After selection, the cells were imaged with a live cell imaging microscope to confirm that only cells that accepted the guide RNA, which produced a green fluorescence due to the GFP gene in the guide, were left.

4.3 RNA harvest/isolation

Once the cells were dox induced for 48 hours, the cells were lysed to extract and harvest the RNA. See Appendix E for more detail about the RNA extraction protocol. The isolated RNA was then purified and analyzed using qPCR.

19 4.4 Results

After 48 hours of dox induction, the GenC iPSCs were imaged using the live image microscope with the TRITC and GFP channel. The TRITC channel detects the red fluorescence from the mCherry when Dox was added, as seen in Figure 15. As expected, the TRITC channel for -Dox showed no fluorescence since the mCherry was not transcribed without the Dox. The GFP channel for both +/- Dox showed green fluorescence, indicating that the transfection was successful. A total of 24 ZNF guides were tested with the Gen1C CRISPRi system.

Figure 15: Live image microscope of ZNF93 Guide 1 (G1) and ZNF649 Guide 2 (G2) with +/- Dox. Each ZNF had 2 guides. In the -Dox TRITC channel, nothing is seen since Dox was absent. In the +Dox TRITC channel, red fluorecence from the mCherry can be seen. In the + and -Dox GFP channels, GFP can be seen, indicating a successful transfection. The +Dox images for each ZNF was from the same 2cm2 well while the -Dox images were from a seperate well. Data obtained: 12/8/17

To determine expression levels of the transposable element that is repressed by the ZNF of the guide RNA, qPCR was performed. In the +Dox conditions, the ZNF is knocked down and is expected to have a low expression while the transposable element is expected to have a high expression if other ZNFs do not regulate it. However, in -Dox conditions, the ZNF is expected to have a high expression while the transposable element is expected to have a low expression since the ZNF is present and regulating the transposable element.

20 Figure 16: Expression levels of ZNF649 and L1PA in +Dox and -Dox conditions. Knockdown of ZNF649 was not sufficient to reactive L1PA. The level of expression of endogenous ZNF649 was low in the +Dox condition as expected since it was knocked down. Data provided by Dr. Jason Fernandes.

As observed in the qPCR data (Figure 16) of ZNF649 knockdown, L1PA expression was not high when ZNF649 was knocked down. However, ZNF93 also represses L1PA and may have still be regulating L1PA since it was not knocked down. To determine if L1PA could be reactivated, I designed my next experiment to knockdown both ZNF649 and ZNF93. I used the previous Gen1C iPSCs with ZNF649 guide transfected and transfected ZNF93 guide. The setup can be seen in Figure 17.

21 Figure 17: The ZNF93 guide was transfected into Gen1C iPSCs that had the ZNF649 guide already trans- fected. GFP and Zeocin resistance gene was already integrated from the ZNF649 guide. The expected qPCR results show ZNF93 with a higher expression in the +Dox condition than ZNF649 because not all the cells had ZNF93 since selection for them could not be performed due to all the cells having the zeocin resistance from the ZNF649 guide. With both ZNF649 and ZNF93 knocked down in the +Dox condition, L1PA expression is expected to be high, unless another ZNF is also regulating L1PA.

5 ORFeome KZNF collection

5.1 Generating a KZNF Library to test on Transposable Elements Overview

I generated a lentiviral KRAB-Zinc Fingers (KZNF) library of various KZNFs to test on different trans- posable elements, which allowed for the study of the interactions between transposable elements and KZNFs. I purified and isolated the KZNFs by miniprepping the plasmid DNA in the inoculated KZNF clones from the ORFeome and Dr. Tim Hughes, then cloned into engineered DNA constructs using an LR Gateway reaction. To confirm the success of the DNA constructs, I transfected the plasmids into human embryonic kidney (HEK) 293FT cells and performed a western blot, a technique used to detect proteins expressed by the plasmid DNA. Gateway cloning is a method to insert fragments into a plasmid. KZNFs were inserted into pDONR221 destination vector that included sequences for a FLAG tag (polypeptide protein tag), barcode for identifi- cation, Internal ribosome entry site (IRES), tdTomato (red fluorescent protein), and EF1α promoter. The screening process determined if there were any functional interactions by detecting the fluorescence produced when a KZNF interacts with a transposable element. ChIP-seq was then performed to study the interaction

22 between the protein and DNA and determine if KZNFs physically bonded to the DNA sequence. The data from the screening system will later be analyzed by Dr. David Haussler’s Lab at UC Santa Cruz to help understand the significance of each KZNF-transposable element pair.

5.2 DNA Isolation and Purification

I inoculated the glycerol stocks, which contained E. coli cultures harboring the desired plasmid construct with glycerol added to allow the plasmids to be safely frozen and stored for years, of various KZNFs purchased before isolating and purifying them with a Qiagen miniprep kit. Using a micropipette, I added 2µl of the glycerol stock into 5ml of Luria Broth (LB) media that also contained Carbenicillin (Carb) antibiotic and placed it into a 30◦C shaker at 200 rpm for 24 hours. Due to being cloned into a lentival vector, the inoculation occurred at 30◦C instead of 37◦C, the typical temperature for growing E. coli. The lentivirus vector has long terminal repeat (LTR) sequences that tend to recombine. Bacteria tend to remove viral sequences with recombination, which is less efficient at 30◦C. Reducing the inoculation temperature decreases the chance for bacteria from removing the viral sequence (including the KZNF). The KZNFs were purchased from the ORFeome (http://horfdb.dfci.harvard.edu/index.php?page=home). The inoculation process is used to grow bacteria. The resulting culture was spun down to create a pellet of cells from which the plasmid DNA was purified. I resuspend the pellet by adding the P1 buffer and then used the P2 buffer to chemically lyse, break open the cells, as seen in Figure 18. I used N3 buffer, a neutralization buffer, to neutralize the lysis buffer. Next, the solution was loaded on a spin column and bound to the column by spinning the solution through the column. Then, I used 500µl of PB and two 750µl PE washes, binding and washing buffer, to ensure that the DNA bound to the spin column while the debris flowed through. Lastly, I used 105µl of EB buffer to elute the DNA off the spin column. See Appendix A for more details about the DNA Isolation and Purification protocol.

23 Figure 18: The steps for inoculating and miniprepping KZNF plasmids. In step A, 5µl of the glycerol stock was added using a p-20 tip to 5ml of the LB media with antibiotics and placed in a 30◦C shaker at 200 rpm for 24 hours. After 24 hours, the tube was spun down in a bucket centrifuge at 3000 rpm for 5 minutes and the supernatant was poured out. In step B, the supernatant was resuspended with the P1 buffer and the cells were lysed with the P2 and N3 buffers. In step C, the DNA was transferred to a spin column before using binding and washing buffers (PB and PE buffer) on the spin columns. In step D, the spin column was spun for 5 minutes at 15,000 rpm to dry the membrane in the column and then EB buffer was used to elute the DNA off the column.

24 5.3 Gateway Cloning

Gateway cloning is a method based on lambda recombinase from Invitrogen, a company that manufactures biotechnology products, that is used to transfer DNA fragments between plasmids efficiently using proprietary Gateway att sites and enzyme mixes LR Clonase and BP Clonase. The two types of reactions for Gateway cloning are LR and BP. After mixing the LR or BP Clonase with the plasmids, the reaction is incubated at 25◦C for an hour. For an LR reaction, entry clones and destination vectors are mixed with LR Clonase to produce expression clones and donor vectors as seen in Figure 19. The BP reaction is the opposite of the LR reaction. By mixing the expression clones, donor vectors, and BP Clonase, entry clones and destination vectors are produced in the BP reaction. I used an LR reaction to clone the KZNF fragment from the entry clone into the destination vector, pDONR221. The KZNF library included N terminal FLAG tags and C terminal FLAG tags, which are polypeptide protein tags. If the KZNF had a stop codon at the end, I Gateway cloned, with an LR reaction, the KZNF into an N terminal FLAG construct while the KZNFs without a stop codon at the end were Gateway cloned into a C terminal FLAG construct as seen in Figure 20.

Figure 19: Gateway reaction diagram for an LR reaction. Mixing the entry clone and destination vector with the LR Clonase resulted in expression clones and donor vectors as the product. The LR Clonase is incubated at 25◦C for an hour before transforming the DNA into competent cells.

25 As seen in Figure 20, the KZNF fragment in the entry clone switches places with the ccdB fragment—a lethal marker gene for bacterial cells, in the destination vector to create the expression clone when the LR Clonase is mixed with the plasmids. The ccdB fragment caused the donor vector to be killed because it is lethal to bacterial cells without resistance towards the ccdB marker. The donor vector is killed by both the ccdB and the Carbenicillin (Carb) in the LB on the plate, resulting in only expression clones on the plate that I could pick and inoculate. See Appendix B for more details about the LR reaction protocol. I used an EF1α promoter in the FLAG constructs, which is also present in normal human genes, for its resistance to silencing by the cells. The two different kinds of lentiviral FLAG tagged constructs were PID constructs and PKZ constructs. The PID constructs, as seen in Figure 20, included a barcode while the PKZ constructs did not. The barcode is a 15-nucleotide sequence, TCTAGAC–C–C–, with 6 nucleotides that are different for each plasmid so that they can be easily identified without sequencing entire sequence. I used the 3X FLAG tag in the DNA construct so that the primary anti-FLAG antibodies could recognize the expressed protein during the western blot detection of protein process. Between the FLAG tag and KZNF sequence is a 2X Glycine-Glycine-Serine (GGS) sequence that allows the FLAG tag to bend without affecting the KZNF structure. The KZNFs were flanked by attB sites, which were used for Gateway cloning. At the end is a tdTomato sequence, which provided a red fluorescence that I used for the confirmation of a successful transformation when the Axiovert 200M live imaging scope was used. Instead of using another promoter before the tdTomato, I used an Internal Ribosome Entry Site (IRES) to include tdTomato in the same mRNA. If a promoter was used instead of the IRES, the tdTomato would become a separate mRNA strand from the mRNA strand before the promoter.

Figure 20: Design for both N terminal and C terminal PID FLAG constructs. The EF1α promoter was used for its resistance to silencing by the cell. The barcode after the EF1α promoter is a unique sequence I used for each construct and recorded for identification purposes. The 3X FLAG tag was recognized by the antibodies during the western blot detection of protein process. The 2X Glycine-Glycine-Serine (GGS) sequence allowed the FLAG tag to bend without affecting the KZNF sequence. The KZNFs were flanked by attB sites, which were used for Gateway cloning. At the end was an Internal Ribosome Entry Site (IRES) that allowed the entire construct to be read as a single mRNA strand instead of 2 strands. The end had a tdTomato sequence that provided the red fluorescence used to confirm a successful transfection.

26 To study more KZNFs, I received KZNF clones from Dr. Tim Hughes that were used in ChIP-seq analysis (Schmitges et al., 2016). The KZNF samples from Dr. Hughes were in entry or destination clones and arrived in Polymerase Chain Reaction (PCR) tubes. I centrifuged the PCR tubes to ensure that the samples were at the bottom of the tube. I then nanodropped to decide if I would need to transform the KZNF samples into Top 10 competent cells. The KZNF samples arrived as either donor (spectinomycin resistant) or expression (ampicillin resistant) clones. If the nanodrop result was lower than 50ng or lower, I transformed the sample into Top 10 competent cells. For samples with nanodrop results above 50ng, I performed a BP Gateway reaction for expression vectors and LR Gateway reactions for donor vectors. The BP Gateway reaction included the expression vector and pDONR221 while the LR Gateway reaction included the donor vector and PKZ NF0, which included a FLAG tag. Once the BP Gateway reactions were complete, I performed the LR Gateway reaction with PKZ NF0. The samples were then sequences with EF1α Forward primer to confirm the KZNF sample and if the vector was in the correct frame. To confirm the success of the Gateway reaction, I created a python program to compare the DNA sequence of the expression clone to a library of KZNF sequences. The program matched the input sequence to a FASTA file, a file format that can include multiple nucleotide sequences, of KZNF’s in the KZNF library after analyzing the input sequence. Before matching the input sequence to the KZNF library, I wrote a method that removed the last 300 nucleotides of the input sequence if they were 55% or more ambiguous nucleotides, which were labeled as “N’s”. This was because the input sequences were either sequenced with a forward or reverse primer, which would have “N’s” at the end, making it difficult to get an accurate match. The next method split the sequence into blocks of 147 nucleotide bases and placed them into a list and match against the library of KZNF sequences. The overall score of the match was then sorted and the overall match with the highest score would be the match. I used the program to confirm the identity of the input sequence, find the specific barcode in the input sequence, and find the GC content percentage. I used the GC content percentage to decide if a dGTP protocol, a protocol used for GC rich sequences, would be used when sequencing the sample.

5.4 Transfection

Transfection is a process that introduces plasmids into cells for protein expression. The purpose of the transfection was to deliver the PID construct DNA into cells to express the protein. I grew a 1x10cm petri dish of HEK 293FT cells 2 days before the transfection process. Once the cells were about 70% confluent, I plated them into 6 well dishes with 300,000 cells per well. For this project, I used a lipid reagent, Lipofectamine, to form a complex with the plasmid DNA to efficiently deliver the plasmid into the cell. I mixed the DNA with Opti-MEM, a reduced serum media for cell growth, and Lipofectamine before being introduced to the cells. I then transfected the DNA into the cells by adding 250µl of the DNA and reagents mixed earlier for

27 each well. Each of the 6 wells contained 2ml of a solution that included Dulbecco’s Modified Eagle’s Medium (DMEM) + 10% Fetal Bovine Serum (FBS) media and 300,000 cells. As a positive control, pMax Green Fluorescent Protein (GFP) was transfected into the HEK 293FT cells in a separate well. When imaging the 6 well plates, the GFP well were expected to have a green fluorescent glow due to the GFP transfected into the cells while the wells with PID constructs transfected into the cells would have a red glow due to the tdTomato in the constructs. See Appendix C for more details about the western blot/transfection protocol.

5.5 Western Blot

A Western Blot is a technique used to detect the protein expressed of the DNA transfected. To begin, I lysed the HEK 293FT cells used in the transfection with a RIPA buffer to break open the cells. The RIPA buffer composition can be seen in the protocol in Appendix C. The protein lysate was run on a 3-8% tris-acetate gel that was then transferred to a nitrocellulose membrane using the iBlot 2 dry transfer system. After the transfer, I soaked the membrane in a milk blocking solution to prevent the antibodies from binding to the nitrocellulose membrane nonspecifically. After each solution change, the membrane was washed with Phosphate Buffered Saline Tween (PBST). For the detection of the protein, I used anti-FLAG mouse primary antibodies to detect the protein since the PID constructs had the FLAG tag for the anti-FLAG antibody to bind to. Then, I used goat-anti-mouse horseradish peroxidase(HRP) as the secondary antibody to locate the primary antibody. Before imaging the nitrocellulose membrane, I added a Chemiluminescent substrate solution to illuminate the secondary antibody so that I could visualize the bands on the nitrocellulose membrane. See Appendix C for more details about the western blot/transfection protocol.

5.6 Results

While performing the Western Blots, I was not able to detect the protein bands on the nitrocellulose membrane. To troubleshoot the problem, I optimized the transfer method and lysing method by increasing the transfer time from 7 to 13 minutes and using a stronger lysis buffer to further break open the cell. When I imaged the transfection, the wells had the correct fluorescent color as seen in Figure 21, proving that the transfection was successful. Since the positive control, Green Fluorescence Protein (GFP), was seen, the problem was not with the antibodies or blocking process. The problem was narrowed down to the transfer and lysing methods since the positive control was not a KZNF. KZNFs are longer, which may have required a longer transfer time from the gel to the nitrocellulose membrane. I concluded that a stronger lysis buffer was required since when running both the supernatant and pellet of the protein lysate, the pellet was bright while the supernatant lane was empty, supporting the theory that the protein was in the pellet. After using the RIPA buffer, which had 25mM Tris HCl, 150mM NaCl, 1% NP-40, 1% Sodium deoxycholate, and 0.1%Sodium dodecyl sulfate (SDS), the cell was broken up even more and the protein was detected on the

28 Western Blot bands, as seen in Figure 22 and 23.

Figure 21: The Axiovert 200M live imaging scope was used to visualize the Green Fluorescence Protein (GFP) with a green fluorescent glow as expected and the PIDs with a red glow from the tdTomato as expected. The top left corner well with GFP was the positive control for transfection success.

29 Figure 22: The Western Blot result from 12/9/2016 shows the KZNF91A, KZNF91B, KZNF93A, KZNF93B, and C2C4B band at the expected length. KZNF91s were expected around 137kDa, KZNF93s were expected around 71kDa, and C2C4B were expected around 38kDa. The bands were slightly lower than expected due to the FLAG tag’s charge. C2C4B was the positive control. There were 2 samples for each KZNF since each had a different barcode in the DNA construct. The A’s and B’s of each KZNF had the same band length, which indicated that the barcode did not affect the overall length.

30 Figure 23: This restriction digest using EcoR1-HF and Xba1 enzymes shows that the Gateway cloning process successfully inserted the KZNF fragment into the PKZ construct. The positive control, KZNF91, was used. The lanes labeled 4A and 4B were ZNF214. The lanes labeled 5A and 5B were ZNF250. The lanes labeled 6A and 6B were ZNF257. The lanes labeled 7A and 7B were ZNF273. The top bands around 10kb were the backbone of the construct while the bands below were the KZNF fragments since the enzymes cut the KZNF fragment out of the PKZ construct. The multiple bands were caused by EcoR1-HF cutting within the KZNF fragment.

While some KZNFs from the ORFeome collection were Gateway cloned successfully, others had long untranslated regions (UTRs), causing the cloning process to fail. To solve this issue, I had to use Polymerase Chain reactions (PCR) to remove the UTRs. Due to the complicated issue of designing primers for each KZNF, we worked with the 55 KZNF samples from the Hughes Lab from the University of Toronto. After receiving 55 KZNF samples from the Hughes Lab, I Gateway cloned them into the them into the PID constructs. However, problems occurred after Gateway cloning. To troubleshoot what caused the problem, I tested the same KZNF sample Gateway cloned into a PID construct and a PKZ construct, which did not include the barcode. The result was that the PKZ constructs resulted in around a 90% success rate while the PID constructs resulted in a 10% success rate. A possible explanation for why the barcode in the PID constructs caused an issue was that the ligation of the barcode into the PKZ construct was not efficient.

31 References

[1] Aneichyk, T., Hendriks, W., Yadav, R., Shin, D., Gao, D., Vaine, C. A., ... and Hanscom, C. (2018, Febru- ary 22). Dissecting the Causal Mechanism of X-Linked Dystonia-Parkinsonism by Integrating Genome and Transcriptome Assembly. Cell, 172(5) 897-909.

[2] Chuong, E. B., Elde, N. C., and Feschot, C. (2016, November 21). Regulatory activities of transposable elements: from conflicts to benefits. Nature Reviews Genetics.

[3] Hancks, D. C., and Kazazian, H. H. (2010, August). SVA retrotransposons: Evolution and genetic instability. Seminars in cancer biology (Vol. 20, No. 4, pp. 234-245). Academic Press.

[4] Imbeault, M., Helleboid, P. Y., and Trono, D. (2017). KRAB zinc-finger proteins contribute to the evolution of gene regulatory networks. Nature, 543(7646), 550.

[5] Jacobs, F. M., Greenberg, D., Nguyen, N., Haeussler, M., Ewing, A. D., ...and Haussler, D. (2014). An evolutionary arms race between KRAB zinc-finger genes ZNF91/93 and SVA/L1 retrotransposons. Nature, 516(7530), 242-245.

[6] Jen, J., and Wang, Y. C. (2016). Zinc finger proteins in cancer progression. Journal of biomedical science, 23(1), 53.

[7] Kazazian Jr, H. H., Wong, C., Youssoufian, H., Scott, A. F., Phillips, D. G., and Antonarakis, S. E. (1988). Haemophilia A resulting from de novo insertion of L1 sequences represents a novel mechanism for mutation in man. Nature, 332(6160), 164.

[8] Khan, H., Smit, A., and Boissinot, S. (2006). Molecular evolution and tempo of amplification of human LINE-1 retrotransposons since the origin of primates. Genome research, 16(1), 78-87.

[9] Laity, J. H., Lee, B. M., and Wright, P. E. (2001). Zinc finger proteins: new insights into structural and functional diversity. Current opinion in structural biology, 11(1), 39-46.

[10] Li, W., Jin, Y., Prazak, L., Hammell, M., and Dubnau, J. (2012). Transposable elements in TDP-43- mediated neurodegenerative disorders. PloS one, 7(9), e44099.

[11] Mandegar, M. A., Huebsch, N., Frolov, E. B., Shin, E., Truong, A., Olvera, M. P., ... and Judge, L. M. (2016). CRISPR interference efficiently induces specific and reversible gene silencing in human iPSCs. Cell Stem Cell, 18 (4), 541-553.

[12] Reilly, M. T., Faulkner, G. J., Dubnau, J., Ponomarev, I., and Gage, F. H. (2013). The role of trans- posable elements in health and diseases of the central nervous system. Journal of Neuroscience, 33(45), 17577-17586.

32 [13] Rodi´c,N., and Burns, K. H. (2013). Long interspersed element–1 (LINE-1): passenger or driver in human neoplasms?. PLoS Genet, 9(3), e1003402.

[14] Schmitges, F. W., Radovani, E., Najafabadi, H. S., Barazandeh, M., Campitelli, L. F., Yin, Y., ... and Dai, W. F. (2016). Multiparameter functional diversity of human C2H2 zinc finger proteins. Genome research, 26(12), 1742-1752.

[15] Theunissen, T. W., Friedli, M., He, Y., Planet, E., O’Neil, R. C., Markoulaki, S.,...and Duc, J. (2016). Molecular Criteria for Defining the Naive Human Pluripotent State. Cell Stem Cell.

[16] Thomas, J. H., and Schneider, S. (2011). Coevolution of retroelements and tandem zinc finger genes. Genome research, 21(11), 1800-1812.

[17] van’t Hof, A. E., Campagne, P., Rigden, D. J., Yung, C. J., Lingley, J., Quail, M. A., ... and Saccheri, I. J. (2016). The industrial melanism mutation in British peppered moths is a transposable element. Nature, 534(7605), 102.

[18] Waterson, R. H., Lander, E. S., and Wilson, R. K. (2005). Initial sequence of the chimpanzee genome and comparison with the human genome. Nature, 437(7055), 69.

[19] Wolf, D., and Goff, S. P. (2007). TRIM28 mediates primer binding site-targeted silencing of murine leukemia virus in embryonic cells. Cell, 131(1), 46-57.

[20] Wolf, D., and Goff, S. P. (2009). Embryonic stem cells use ZFP809 to silence retroviral DNAs. Nature, 458(7242), 1201.

[21] Yang, H., Yuan, W., Wang, Y., Zhu, C., Liu, B., Wang, Y., ... and Liu, M. (2005). ZNF649, a novel Kruppel type zinc-finger protein, functions as a transcriptional suppressor. Biochemical and biophysical research communications, 333(1), 206-215.

33 Appendix A: Miniprep protocol for KZNFs

Purpose: This protocol provides detailed instructions to miniprep plasmid DNA from bacterial cultures. The process purifies the DNA using the membrane in the spin columns of the QIAGEN Miniprep kit.

Overview: The P1/P2 Buffers resuspend and lyse the DNA while the N3 neutralization buffer neutralizes the spin column. The PB and PE washes bind the DNA to the column and washes away the debris. The silica membrane in the spin column of the QIAGEN Miniprep kit binds the denatured DNA. The elution step is when the DNA is removed from the spin column. This protocol requires 48 hours.

Materials:

• Luria Broth (LB) media with appropriate antibiotic (5 ml/culture)

• 5ml culture tubes

• 5µl of glycerol stock of each sample

• QIAGEN Miniprep kit (Catalog # 27106)

• P-20, P-200, and P-1000 micropipettes

• Bucket centrifuge

• Microcentrifuge

• 30◦C shaker

• 1.5 ml Eppendorf tubes

• 2 ml Eppendorf tubes

• Nanodrop device

If LB media does not have antibiotic included, take antibiotic stock (from antibiotic stocks box in freezer; carb is in its own box) and dilute in LB media. Add antibiotic stock to the LB so that the concentration is 100µg/ml. Methods: Day 1:

(a) Take out 5 µl of a glycerol stock and pipette into culture tube using p-20 micropipette. Label the sample name, date, and your name on the tubes with tape on the tube (NOT the cap).

34 (b) Grow cultures in shaker at 30◦C, 200 rpm overnight.

Day 2:

(a) Make sure centrifuge is balanced. Spin culture tubes at 3000 rpm for 5 min in the bucket centrifuge. If samples do not form a solid pellet and clear supernatant, centrifuge again for 5 additional minutes.

(b) Discard supernatant into sink (run the sink to wash away LB) and dry tubes by inverting on paper towel.

(c) Remove Buffer P1 from refrigerator. If it is an old bottle, make sure RNAse A was added. If making new bottle, add RNAse A from kit into the P1 bottle. Resuspend each pellet in 250 ul P1 and transfer to labeled 2 ml Eppendorf spin tube.

(d) Add 250 µl P2 to each tube (solution will turn blue because of the LyseBlue in the P1 buffer). Don’t let tubes sit for more than 5 minutes before performing next step.

(e) Add 350 µl N3 to each tube; close and invert. If you have a lot of tubes do in sets of 4-6. Don’t let tubes sit unmixed for too long. If LyseBlue was added tube should turn white. You should see some cell debris at this point.

(f) Spin at max for 10 min in the microfuge. Make sure it is balanced. Orient tubes all the same way so that you know where the pellet will be.

(g) Label spin columns (on the column) with an ethanol resistant pen.

(h) Apply the supernatant to the appropriate spin column. Don’t disturb the pellet. Discard the 2 ml tube, which should have nothing but pellet in it.

(i) Spin the columns with supernatant at max speed for 30 sec and discard flow through.

(j) Add 500 µl PB buffer to each column, spin max 30 seconds and discard flow through.

(k) Add 750 µl PE (make sure ethanol was added to bottle!) to each column, spin at max speed for 30 seconds and discard flow through.

(l) Repeat previous step (k) exactly.

(m) Spin column at max speed, 2-5 min to dry membrane.

(n) Transfer spin column to clean 1.5 ml ep tube (label as appropriate).

(o) Pippete 105 µl EB to center of spin column membrane, let sit for 5 min.

35 (p) Spin at 10000 rpm for 30 seconds; properly label tube if not already done.

(q) Nanodrop 1.2 µl of each sample and record concentration.

(r) Create labels for each tube and store in the 20◦C refrigerator.

Data Analysis: The expected result is that after eluting the sample (Day 2 step p), about 100 µl of purified DNA should be present. The concentrations of DNA from the nanodrop are expected to be between 60 and 200 ng/µl.

Source: QIAprep Miniprep Handbook 4th Edition

36 Appendix B: Gateway cloning protocol

LR reaction Transferring your gene from a Gateway entry clone to destination vector (1 hour reaction). See below for an overview of the set-up. 1. Add the following components to a 1.5 ml tube at room temperature and mix:

(a) Entry clone (50-150 ng) 1–7 µl

(b) Destination vector (150 ng/µl) 1 µl

(c) TE buffer, pH 8.0 to 8 µl

2. Thaw on ice the LR Clonase II enzyme mix for about 2 minutes. Vortex the LR Clonase II enzyme mix briefly twice (2 seconds each time). 3. To each sample (Step 1, above), add 2 µl of LR Clonase II enzyme mix to the reaction and mix well by vortexing briefly twice. Microcentrifuge briefly. 4. Return LR Clonase II enzyme mix to -20◦C storage. 5. Incubate reactions at 25◦C for 1 hour, shaking at 300 rpm. 6. Add 1 µl of the Proteinase K solution to each sample to terminate the reaction. Vortex briefly. Incubate samples at 37◦C for 10 minutes.

BP reaction Creating a Gateway entry clone from an attB-flanked PCR product (1 hour reaction). See below for an overview of the set-up. 1. Add the following components to a 1.5 ml tube at room temperature and mix:

(a) attB-PCR product (=10 ng/µl; final amount 15–150 ng) 1–7 µl

(b) Donor vector (150 ng/µl) 1 µl

(c) TE buffer, pH 8.0 to 8 µl

2. Thaw on ice the BP Clonase II enzyme mix for about 2 minutes. Vortex the BP Clonase II enzyme mix briefly twice (2 seconds each time). 3. To each sample (Step 1, above), add 2 µl of BP Clonase II enzyme mix to the reaction and mix well by vortexing briefly twice. Microcentrifuge briefly. 4. Return BP Clonase II enzyme mix to –20◦C storage. 5. Incubate reactions at 25◦C for 1 hour, shaking at 300 rpm.

37 6. Add 1 µl of the Proteinase K solution to each sample to terminate the reaction. Vortex briefly. Incubate samples at 37◦C for 10 minutes.

Transformation Use 1µl of either the LR or BP reaction, depending on the type of reaction that is being performed, and add to 25µl of thawed Top 10 competent cells. 1. Incubate on ice for 15-30 minutes. 2. Heat shock at 42◦C for 30 seconds. 3. Incubate on ice 2-5 minutes. 4. Add 250µl of SOC media. 5. Recover in 30◦C and shake at 300 rpm for 1 hour. 6. Warm up LB plates with appropriate antibiotics. 7. Plate 100µl of each transformation tube on the LB plates using beads. 8. Place in 30◦C incubator for 24 hours.

Source: ThermoFisher Scientific Gateway Protocol

38 Appendix C: FLAG tagged KZNF Western Blot Protocol

Joshua Gu Draft 2: 1/9/17

Goal: Visualize FLAG tagged KZNF protein expression on the Western Blot after transfection into 293FT cells. Strategy: Day 1, 1x10cm dish of confluent 293FT (p11-30) cells were plated at 300,000 cells/well in a 6 well dish. Day 2, the wells were transfected.

Reagent A (130µl/sample) Reagent B (130µl/sample) Opti-MEM + 10 µl Lipofectamine Opti-MEM + DNA

Mix reagents A and B (20 mins). Then add 250µl into each well.

well plasmid 1 2 3 4 5 6

Buffers/Reagents: 50ml RIPA buffer 25 mM Tris HCl (pH 8) 1.25ml of 1M 150 mM NaCl 1.88ml of 4M 1% NP-40 5ml of 10% 1% sodium deoxycholate 5ml of 10% 0.1% SDS 0.5ml of 10% Complete Protease Inhib. 1 tab

Remainder = H2O (37.37ml)

PBST (0.05% Tween)

• 1L of 1xPBS

39 • 500µl of Tween20

TA running buffer

• 475ml H2O • 25ml 20x NuPAGE Tris-Acetate SDS running buffer

About the antibodies: cat# ZNF Antigen Vendor Concentration Ab source Predicted size

Methods: Cell Lysate: 1. Aspirate media. Rinse 1x PBS (2ml/well). Resuspend with cold 250µl RIPA buffer in each well. Place in cold room rocker for 15 mins. 2. Transfer contents into 1.5µl Eppendorf tubes while plate is on ice or in cold room. 3. (Spin protein lysate 10 mins. max speed in cold room.) *optional 4. Remove the supernatant containing the proteins of interest to a new tube and leave the debris. 5. Store in -4◦C fridge.

Western Blot: Denaturing samples for gel: resuspend in sample buffer + reducing reagent, heat at 95◦ for 10min, spin 2 seconds.

• 10ml of sample, 10ml 4x LDS buffer, 4ml of Reducing reagent 10X reducing agent, 16 ml H2O

1. Assemble HLife Technologies Mini Gel Tank: NuPAGE 12 well 3-8% tris-acetate 1mm gel in TA running buffer. 2. Remove the comb carefully. Then load 20µl of sample into each well. (use buffer on ends to prevent samples with curved ends)

Transfer: 1. Once gel has finished running, turn on iBlot 2 system and select KZNF saved method (13 min P0 transfer). 2. Open iBlot 2 Mini Transfer Stack and remove separator below Top Stack. 3. Run at 200V for about 40 minutes (or when the bottom band reaches 1-1.5cm above the bottom).

40 Top Stack of the Mini Transfer Stack. Source: iBlot 2 Dry Blotting System User Guide Revision C.0

4. Use putty knife to crack open the gel case. Cut off wells and end slot/foot and rinse with mill-Q water. 5. Optional: soak gel with back casing in 20% ethanol for 10 mins to improve transfer efficiency. 6. *Carefully* slide the gel onto the Bottom Stack of the Mini Transfer Stack without folds. 7. Place bottom stack onto the iBlot 2 dry blotting system.

Placing of the bottom stack onto the iBlot 2 dry blotting system. Source: iBlot 2 Dry Blotting System User Guide Revision C.0

7. Place pre-soaked iBlot Filter Paper onto gel then Top Stack above it and remove any air bubbles. 8. Place iBlot 2 Absorbent Pad on top of Mini Transfer Stack with electrical contacts aligned.

41 Placement of the absorbent pad. Source: iBlot 2 Dry Blotting System User Guide Revision C.0

9. Close lid of iBlot 2 system and press start. 10. Make block buffer: 5% non-fat dry milk in PBS (1.65g non-fat dry milk/33 ml PBST). Place nitrocellu- lose membrane in block buffer overnight, rocking slow, 4◦C room.

Next day: After blocking overnight in 5% Non-Fat Milk in PBS, using a tuperware rocking in 4◦C: Do a quick PBST wash. 1. Primary Ab (anti-FLAG) mouse M2 1:2000 a. 1:2000 is relative to the 1mg/ml for primary antibody incubation. b. In 15ml conical (12ml PBST+6µl Ab) 1.5-3hrs in PBST (0.05% Tween: 500µl Tween20 in 1L PBS) room temp roll. 2. Save primary antibody at 4◦C Fridge in PBST. 3. Wash 3x PBST 5 min in cleaned pipette box. 4. Secondary antibody: SCBT goat anti-mouse-HRP 1:10,000 a. [200µg/0.5ml] 2µl/20ml = 1:10K b. 45 minutes 1:10K (2µl/20ml) Room temp. 5. Wash 3x PBST 5 min in cleaned pipette box. 6. Cut a 3-ring binder page protector in half horizontally and the bottom and side without 3-ring holes so that the page protector can be opened like a book. 7. Substrate: pico super signal 1ml each a 2ml total in 15ml conical and pipette to mix. 8. Transfer 1 ml of pico super signal to the inside of half of the page protector. Place nitrocellulose membrane onto pico super signal. 9. Move around membrane with tweezers. Add 1ml of pico super signal next to the membrane. Use tweezers to carefully flip the membrane onto the 1ml just placed onto the page protector. 10. Move around membrane with tweezers. Close page protector and cover membrane from light with pipette box for 5 mins. 11. Use BIO-RAD ChemiDoc Chemi-Hi Sensitive filter to image 30-180secs with 6 images. Export image as

42 a tiff file and save image. 12. Store membrane in PBST in cold room. 13. Overlap the figures in Powerpoint. Place figure in notebook. 14. Analyze the results.

Materials:

Catalog # Item Vendor IB23002 iBlot DNA Transfer LifeTech Stack LA0041 NuPAGE Tris-Acetate Thermo SDS Running Buffer (20X) EA03752BOX NuPAGE Novex 3-8% Thermo LOT:16100470 Tris-Acetate Protein Gels, 1.0 mm, 12-well LC5699 HiMark Pre-stained Thermo Protein Standard 11668019 Lipofectamine 2000 Lifetech F1084-5mg Anti-Flag antibody Sigma

Source: iBlot 2 Dry Blotting System User Guide Revision C.0

43 Appendix D: CRISPRi Gen1C iPSC Protocol

Joshua Gu Draft 2: 1/12/18 Necessary Items:

• mTESR 1, Stem cell Technologies, 85850 • Corning matrigel, growth factor reduced, 356230 • Pen/Strep, LifeTech, 11668-027 • Lipofectamine 2000, LifeTech, 11668-027 • Accutase, LifeTech, A1110501 • ROCK inhibitor, ATCC, ACS-3030, 10uM (1:1000) • 0.5mM EDTA

Gen1C dCas9-KRAB cell line protocol: EDTA Passaging:

• To maintain cells, we use EDTA to passage colonies. • 0.5µlM EDTA is used. • Coat plates with diluted matrigel for 30 minutes at room temp. • 3mL of matrigel per 6cm plate. 1.5mL per 6 well.

1. Aspirate off old media and wash 1x with dPBS. 2. Add 2mL of EDTA to 6cm plate (1mL to 6 well). 3. 5 minutes at 37◦C 4. Aspirate off EDTA. Some colonies may come off and that is ok. 5. Wash colonies off with 4mLs of media 6. Add 1mL from this mixture to new 6cm plate with 4mLs of mTESR. 1:4 splits, essentially.

Lipofection protocol:

• Plate 30k cell/cm2 on matrigel the day before transfection. (300k cells/well) • Aspirate, wash, aspirate, add 1.5mL of accutase, 10 minutes at 37◦C. • Add 4mL media, triturate, spin 300xG for 5 minutes. • Apirate media, resuspend in 2mL, count cells. • Use ROCK inhbitor when splitting to spingle cell. (1µl/mL media) • 1ug of DNA to 5µl of lipofectamine for each well of 6 well plates • 250µl reaction volume. 130µl optimem/DNA + 130µl optimem/Lipo

44 • Pre-load 1ug of DNA into 1.5mL eppendorf • Take 15mL conical and add 1690µl of optimem (13 well x 130µl) and add 65µl of lipofectamine 2000 (13 well x 5µl) and wait 5 min.

1. Add 130µl of optimem to each DNA tube. 2. Add 130µl of optimem/Lipo to this tube. 3. Wait 20 minutes. 4. Add 250µl to each corresponding well. 5. Change media after 3 hours. 6. 2mLs of media, mTESR, NO ZEO OR DOX. 7. Begin selection with zeocin antibiotic 24 hrs later. 8. Dox induce for 48 hours when about 60-70% confuent.

45 Appendix E: RNA isolation for iPSCs on matrigel Protocol

Joshua Gu 1/12/18

For 1 well of a 6-well:

1. Aspirate media and scrape cells with cell scraper into 1mL PBS (scale volume up for larger vessels). 2. Transfer to 15mL Falcon and spin 300xg. 3. Aspirate PBS and repeat wash with 1mL PBS. 4. In a fume hood: Add 300µl Trizol to cell pellet (scale to 600µl if greater than 106 cells). Add 300µl (equal volume) 100% EtOH to lysed cells; transfer to clearly labeled eppendorf tube and store in -80◦C or proceed with Direct-Zol isolation.

RNA purification: Centrifuge at 15,000g for 30 seconds 1. In a hood: Transfer mixture into Zymo-Spin IIICG Column in a Collection Tube and centrifuge. Transfer column into new collection tube and discard flow-through. 2. At RNA-free bench: Add 400µl Direct-Zol RNA PreWash to column at centrifuge. Discard flow-through and repeat this step. 3. Add 700µl RNA Wash Buffer to the column and centrifuge for 2 minutes at 15,000g to remove wash buffer. Transfer column carefully into new RNase-free 1.5 epp tube. 4. To elute RNA, add 45µl of DNase/RNase-Free Water directly to the column matrix and centrifuge at 11,000g for 30 seconds.

DNase I treatment: DNase I in -20◦C chest 1. Make master mix of DNase I and DNA digestion Buffer. 5µl each per sample. 2. Each sample: 40µl of RNA sample, 5µl DNase I, 5µl DNA Digestion Buffer. Mix gently in new RNase- Free epp tube. 3. Incubate at room temperature (25◦C) for 45 minutes.

RNA purification: 1. Add 2 volumes RNA Binding Buffer to each sample and mix. (100µl buffer and 50µl sample) 2. Add and equal volume of ethanol (200 proof) and mix (Add 150µl ethanol).

46 3. Transfer sample to Zymo-Spin IC Column in a Collection Tube and centrifuge for 30 seconds. Discard flow-through. 4. Add 400µl RNA Prep Buffer to column, centrifuge for 30 seconds, discard flow through. 5. Add 700µl RNA Wash Buffer to column, centrifuge for 30 seconds, discard flow through. 6. Add 400µl RNA Wash Buffer to column, centrifuge for 2 minutes. Transfer column carefully into new RNase-Free epp tube. 7. Add 16.5µl DNase/RNase-Free Water directly to column matric and centrifuge for 30 seconds at 11,000g. 8. Use nanodrop to determine concentration. 9. Store at -70◦C

Adapted from: Zymo RNA purification protocol

47 Appendix F: Luciferase Assay Protocol

Joshua Gu Draft 3: 1/12/18 1x 24-well plate with duplicates

Goal: Measure luciferase activity with luminometer to determine repression level of the transposable element L1PA under certain conditions.

• 100ng : 20ng : 2ng, Rescue : Reporter : Renilla • mESC media +LIF • 24 well plate set up • Gelatin coat plates, TrypLE split, 50k cells/cm2, lipofect the next day. • Rescue normalized to 250ng/uL • Reporter normalized to 50ng/uL • pRL-TK control to 50ng/uL

Day 0:

• Pull media out to warm, gelatinize plates (including maintenance plate) • Aspirate media from 6cm dish, wash, and add 0.5mL trypsin (break up with 1mL pipette tip) • After 4 min, wash media with 2mL warmed media, collect • Spin 5min, 300x G • Resuspend in 2mL mESC media+LIF, count cells • Calculate cell dilution for 50,000 cells/cm2 (24well area is 2 cm2) • Make the cell dilution according to your calculations. Seed 100K cells onto the maintenance plate with 4mL of media

Day 1:

• Pre-setup the eppendorf tubes with appropriate amount of DNA’s. 100ng/20ng/2ng ratios per well of the rescue, reporter, renilla. When doing duplicates, calculate for 3 sets. Multiply by 3 for each tube, resulting in 300ng of either ZNF or pCAG empty vector and 60ng of the reporter. • In TC, take two 15mL eppendorf tubes and label them Opti+Renilla and Opti+Lipo. • For renilla, we want 2ng per well. We have 24 total wells. Calculate for 40 wells (duplicates). 80ng of Renilla into the 15mL conical labeled renilla. • Calculate for 3 wells/tube and 100µl each so for 40 wells, we will need 4mL total (Calculating for 40 wells). Divide this in 2, 2mL of Opti into the Optimem+Lipo tube and 2mL of Opti into the Optimem+renilla tube.

48 • 100µl of lipofectimine into the appropriate conical, swirl, wait 5 min. • During this wait, aliquot 155µl of Optimem+renilla into each DNA tube (if doing triplicates) • After 5min lipo wait, combine 155µl of this Optimem+lipo into the Optimem+DNA tubes. • Wait 20 minutes. • Add 100µl transfection into each well. • Change media 3 hours later.

Day 2:

• Take out luciferase reagents from -20◦C and let come up to room temp (not the supplements). • Make 1x passive lysis buffer from the supplied 5x stock (10mLs). • Aspirate, wash, aspirate wash and then add 100µl passive lysis buffer and rock for 15 minutes. • Take 45µl of the lysate and add to optiplate. • Make the firefly(FF) reagent by adding 10mLs buffer to the lyophilized reagent. • Use a reagent trough and multichannel. • 50µl of this reagent to each well and read 1s on luminometer. This is your FF value. • Add 200µl stop and glow reagent to the appropriate buffer. • 50µl of this reagent to each well and read 1s on luminometer. This is your renilla value

Source: Promega dual-luciferase assay system manual

49 Appendix G: QIAfilter Plasmid Maxi prep protocol

Joshua Gu Date: 1/19/18

Cat. Nos. 12262 and 12263 Notes:

• Add RNase A solution to Buffer P1 mix, and store at 2-8◦C • Optional: Add LyseBlue reagent to Buffer P1 at a ratio of 1:1000. • Prechill Buffer P3 at 4◦C. Check Buffer P2 for SDS precipitation. • Isopropanol and 70% ethanol are required.

100µl starter culture + 100mL LB media with antibiotic 1. Harvest bacterial culture after 12-16 hrs. of growth by centrifuging at 6000 x g (RCF) for 15 minutes at 4◦C. 2. Discard/pour out supernatant. 3. Completely resuspend pellet in 10 mL Buffer P1. 4. Add 10mL Buffer P2, mix/invert 4-6 times, incubate at room temperature for up to 5 minutes. If using LyseBlue reagent, solution will turn blue. 5. During incubation, screw the cap onto outlet nozzle of the QIAfilter Cartridge (w/ plunger) and place onto QIArack. 6. Add 10mL prechilled Buffer P3 to lysate, mix/invert 4-6 times. If using LyseBlue reagent, mix until solution is colorless. 7. Pour lysate into barrel of QIAfilter Cartridge (label!). Incubate at room temp for up to 10 minutes. Do not insert plunger! 8. Equilibrate QIAGEN-tip by applying 10mL Buffer QBT, and allow the column to empty by gravity flow. Label QIAGEN-tip! 9. Remove cap from QIAfilter Cartridge outlet nozzle. Gently insert plunger into QIAfilter Cartridge barrel. Filter cell lysate into equilibrated QIAGEN-tip. Allow lysate to enter resin by gravity flow. 10. Wash QIAGEN-tip with 30mL Buffer QC (X2) 11. Elute DNA with 15mL Buffer QF into labeled 50mL conicals. 12. Precipitate DNA by adding 10.5mL room-temp isopropanol, mix, centrifuge at 15,000 x g (RCF) for 30 minutes at 4◦C. 13. Aspirate supernatant CAREFULLY! 14. Wash DNA pellet with 1.5mL room temp 70% ethanol using borehole pipette tip and transfer to 2mL

50 Eppendorf tube. Centrifuge at 15,000 x g(RCF) for 10 minutes. Carefully remove supernatant with pipette. 15. Air dry pellet for 5-10 minutes in hood. 16. Redissolve DNA with 200µl water.

Source: QIAfilter Plasmid Purification Handbook 3rd Edition

51