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Investigation of a causal role of transposable element activation in vertebrate aging

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Investigation of a causal role of transposable element activation in vertebrate aging

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Content Investigation of a causal role of transposable element activation
in vertebrate aging.
By
Tu Tang
A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR MEDICINE)
May 2024
Copyright 2024 Tu Tang

ii
Acknowledgements
I would like to thank my principal investigator, Dr. Bérénice Benayoun, for her invaluable
guidance, and continuous support, throughout my master's research. Her expertise and insight
were crucial to the successful completion of this work.
I am also deeply grateful to all the members of our lab for creating such a supportive and
stimulating environment. Special thanks to Dr. Bryan Teefy for mentoring me and giving me
encouragement during the whole project.
Lastly, I would like to thank my family and friends for their endless support and encouragement
during this journey. Thank you all.

iii
Table of contents
Acknowledgements......................................................................................................................... ii
List of Figures................................................................................................................................ iv
Abstract........................................................................................................................................... v
Introduction..................................................................................................................................... 1
Chapter 1: Impact of SUV39H1 on H3K9me3 levels .................................................................... 6
Chapter 2: Activity of human and killifish SUV39H1 in vitro....................................................... 8
Chapter 3: Transposable element activity in SUV39H1 overexpressed cells............................... 10
Chapter 4: Repression of LINE-1 elements in SUV39H1 overexpressed cells............................ 13
Conclusion and future directions.................................................................................................. 15
References..................................................................................................................................... 16
Appendix....................................................................................................................................... 18

iv
List of Figures
Figure 1:Loss of heterochromatin is associated with dysregulation of TEs. Created with
BioRender.com................................................................................................................................ 3
Figure 2: SUV39H1 overexpression alters H3K9me3 levels in HEK293T cells.
(a), SUV39H1 RNA level fold changes 2-day post-transfection. P-value in Wilcoxon test. (b),
Immunoblot staining with anti-FLAG and anti-H3K9me3 antibodies. Human SUV39H1
increases H3K9me3 levels whereas killifish SUV39H1 seemingly decreased global H3K9me3
levels. (c), Quantification of H3K9me3 expression level change by ImageJ. Normalized by
vinvulin expression level. ............................................................................................................... 7
Figure 3: HMTase activity quantification of human and killifish SUV39H1. Recombinant
FLAG-SUV39H1 was purified by immunoprecipitation then tested by color-based assay.
Human and killifish SUV39H1 activity was plotted on HMTase activity standard curve. ............ 9
Figure 4: Transposable element transcriptional activity in SUV39H1 overexpressed cells.
Cells are harvested 1 day or 2 day after transfection and assayed by bulk RNA-seq. Regulation
of TEs are plotted by heatmap. .................................................................................................... 11
Figure 5: GO enrichment analysis of SUV39H1 overexpressed cells. Transcriptionally related
gene sets are downregulated. ........................................................................................................ 12
Figure 6: Impact of SUV39H1 on LINE1 element transcription levels. RNA was extracted
then amplified by RT-qPCR to detect expression levels. P-values in Wilcoxon test.................... 14

v
Abstract
Transposable elements (TEs) are mobile DNA sequences capable of replicating themselves found
in most eukaryotic genomes. TE activation is believed to be directly deleterious to longevity by
promoting genome instability. Drosophila Su(var)3-9 specifically methylates histone H3K9,
which has been proven important in gene repression and chromatin maintenance. Scientists have
shown that overexpressing Su(var)3-9 in Drosophila leads to TE repression and increased lifespan.
However, whether a vertebrate homolog of Su(var)3-9 has a similar effect on both TE expression
and longevity is still unclear. African turquoise killifish (Nothobranchius furzeri) is the shortestlive vertebrate that can be bred in capacity, thus allowing high repeatability and feasibility for
experiments. In this report, we verified the HMTase activity of human SUV39H1 (hSuv) and
killifish SUV39H1 (kSuv), which are homologs of Drosophila Su(var)3-9 in HEK293T cells and
killifish fibroblasts. TE activities are suppressed in both hSuv and kSuv overexpressed cells. We
also found that LINE1 expression was repressed in a human cellular model upon human and
killifish SUV39H1 overexpression.

1
Introduction
The process of progressive organismal and cellular decline known as aging remains one of
the most challenging problems in biology. During the past few decades, scientists have revealed
some major components of aging: genomic instability, telomere attrition, epigenetic changes,
proteostasis, nutrient sensing, mitochondrial dysfunction, etc1
. Among all these contributors,
genomic instability is the most studied.
Genomic instability refers to the accumulation of DNA damage and chromosomal
abnormalities, which can lead to cellular dysfunction, senescence, or apoptosis. It is a consequence
of errors made by the enzymes responsible for chromosome segregation, DNA replication, or DNA
damage repair.
The early development of an organism has many errors because the high number of cell
divisions. However, selection keeps mutation loads under control. As an organism heads into
adulthood and throughout the aging process, the inherent inaccuracy of genome maintenance, such
as accumulation of DNA damage, deficiency of mutation detections, changes in epigenome and
mitochondrial DNA mutations guarantees a steady accumulation of alterations that are no longer
kept in check by selection2
.
There has been a lot of interest in the scientific community into understanding the
relationship between the efficiency of genome maintenance and both interspecies and intraspecies
variation in lifespan. If a genetic factor specifies the potential of an organism to reach old age by
affecting genome stability, then genomic instability can be considered a causal role in aging
process. However, no conclusive evidence shows this is the case. In contrast, accumulating
evidence shows that genome maintenance is involved in intraspecies lifespan variations. For

2
instance, Werner’s syndrome is caused by defect in WRN, which facilitates polymerization by
DNA polymerase across telomeric and other GC-rich sequences in vitro. Other progeroid
syndromes, like Hutchinson-Gilford progeria, also support the conclusion.
Transposable elements (TEs) are mobile DNA sequences capable of replicating themselves
within genomes independently of the host cell DNA. TEs were once thought to be "junk DNA"
with no functional significance, but recent research recognizes them as contributors to genomic
innovations as well as genome instability across a wide variety of species. There are two main
divisions of transposable elements: retrotransposons, which use an RNA intermediate for
transposition, and DNA transposons, which use a DNA intermediate.
TEs affect genomes in two major ways: via a mobilization event or after insertion3
. The
impacts of mobilization are simpler and local; the extent of which is dependent upon the location
of the TE insertion site within the genome. For example, TEs can insert in exons, disrupt coding
sequences and make their way into a mature mRNA; TE insertion into introns can be spliced into
mRNA; TE insertion in 3’ UTR results alternative 3’UTR including TEs. However, mobile TEs
can have global impacts on genome structure and chromosome dynamics. For example, B1 and
AluY elements are actively retrotransposing Short Interspersed Nuclear Elements [SINEs] in mice,
they both function in axial element binding as the anchoring point for the synaptonemal complex.
Other biological aspects, such as genomic and epigenomic landscape alterations, can also be
impacted by TE activity.
Heterochromatin is condensed region in genome with highly repetitive sequences. There
are two kinds of heterochromatin: constitutive heterochromatin and facultative heterochromatin,
classified by amount of methylation on lysine-9 in histone H3. Heterochromatin with H3K9me3
is considered constitutive, otherwise it is facultative. Constitutive heterochromatin makes up over

3
20% of mammalian genome. It is rich in repetitive elements, including satellite DNA and
transposable elements. It is hypothesized that TEs play a crucial role in stability of constitutive
heterochromatin as TEs provide docking sites for protein H3K9 methylation4
. Recent study also
reveals accumulated LINE-1 RNA resulted in loss of heterochromatin5
, which indicates TE
dysregulation may disrupt heterochromatin hemostasis thus affect genome stability.
Figure 1:Loss of heterochromatin is associated with dysregulation of TEs. Created with BioRender.com
Expression of TEs is elevated with age in multiple organisms like mice, nematodes, flies
and human6–9
. TE expression also increases during in vitro senescence10. It has been hypothesized
that TE activation, by promoting genomic instability, may lead to shortened lifespan. Here, we set
out to test the hypothesis that TE activation is a sufficient to promote aging and limit lifespan in a
vertebrate model. Indeed, previous research showed that suppressing age-associated TE activation
extends lifespan in Drosophila7
. In general, aging has been linked to loss of repressive
heterochromatin structure and loss of silencing in constitutive heterochromatin regions (which are
enriched for TE sequences)11. Transcriptomic analysis revealed that many sequences native to
heterochromatic regions, including TEs, increased with age in fly heads and fat bodies. In this

4
context, several genes known to affect heterochromatin maintenance were tested for their impact
on lifespan in flies. By overexpressing Sir2, Su(var)3–9, and Dicer-2, age-related expression of
TEs was mitigated and increased lifespan was observed. Conversely, overexpression of Adar led
to increased age-related expression of TEs and shorter lifespan. Suppressor of Variegation 3-9
(SUV39) is a heterochromatin-associated H3K9 trimethyltransferase. By specifically promoting
H3K9me3 deposition, Su(var)3–9 is a key component of heterochromatin-induced gene silencing.
In Su(var)3-9 transgenic fly lines, Su(var)3-9 was overexpressed 160%-180% above control and
TE activity was suppressed by ~92%, leading to a 36% median lifespan extension. Thus, Su(var)3-
9 is a good candidate modifier to test the impact of suppressing TE activity on aging in a Vertebrate
model.
While the effect of TEs on lifespan in Drosophila has been investigated, the causal role of
TEs on lifespan in Vertebrates remains inconclusive. The African turquoise killifish
(Nothobranchius furzeri) is a small annual fish occupying a seasonal habitat, the ephemeral ponds
of water in southeast Africa, primarily in Zimbabwe and Mozambique12. This species has a fastest
sexual maturity and is the shortest-lived vertebrate that can be breed in captivity. Indeed, the GRZ
lab strain usually lives 4-6 months. All these features make turquoise killifish an attractive model
system for aging and disease research.
A vertebrate homolog of Su(var)3-9, SUV39H1, was the first protein lysine
methyltransferase founded. The SET domain in SUV39H1 (residue 243-366) binds methyl group
donor S- adenosyl-L-methionine (Adomet) and brings it to target lysine residue to methylate it. Cterminally truncated SUV39H1 does not keep its enzymatic activity because of the loss of SET
domain. In this research, we propose to overexpress SUV39H1 in killifish fibroblasts and
HEK293T cells to investigate the role of TE regulation in the regulation of vertebrate aging.

5
The Long Interspersed Nuclear Element 1 [LINE1] is non-LTR retrotransposon that
compromises approximately 17% of human genome. LINE1 expression leads to higher level of
DNA double strand breaks and erodes genomic integrity13. Recent study shows LINE1 also
promotes aging related inflammation8
. Here, we evaluated LINE1 RNA levels to detect the impact
of SUV39H1 overexpression on LINE1 as a proof-of-principle.

6
Chapter 1: Impact of SUV39H1 on H3K9me3 levels
SUV39H1, like its homolog Su(var)3-9, specifically methylates histone H3K914. To investigate the
feasibility of killifish SUV39H1 overexpression in killifish embryos as well as confirm the H3K9
trimethyltransferase activity of killifish SUV39H1 in a cellular context, we tested the impact of
both human and killifish FLAG-tagged SUV39H1 overexpression in the HEK293T cell line. This
cell line was chosen as an easy-to-culture and transfect cell system to investigate the relative
activity of these proteins.
We obtained mammalian overexpression vectors for human and killifish FLAG-tagged
SUV39H1 in the pcDNA3.1 vector from Genscript and transiently transfected them via calcium
phosphate method, using eGFP as negative overexpression control, into HEK293T cells.
Cells were harvested 48 hours after transfection to probe for activity to transfer methyl
groups to histone H3K9. We first performed RT-qPCR to confirm that the vectors were
overexpressed in transfected cells (Fig. 2a). Next, we used polyacrylamide gel electrophoresis with
SDS (SDS-PAGE) followed by Western Blotting to estimate H3K9me3 levels in overexpressing
cells (Fig. 2b, c). Our results confirm that human and killifish SUV39H1 were overexpressed at
RNA level and indicate human SUV39H1 overexpression in HEK293T cells increased methylation
of H3K9. In contrast, killifish SUV39H1 overexpression seemingly led to overall decreased
H3K9me3 levels.

7
Figure 2: SUV39H1 overexpression alters H3K9me3 levels in HEK293T cells. (a), SUV39H1 RNA level fold changes 2-day posttransfection. P-value in Wilcoxon test. (b), Immunoblot staining with anti-FLAG and anti-H3K9me3 antibodies. Human SUV39H1
increases H3K9me3 levels whereas killifish SUV39H1 seemingly decreased global H3K9me3 levels. (c), Quantification of
H3K9me3 expression level change by ImageJ. Normalized by vinvulin expression level.

8
Chapter 2: Activity of human and killifish SUV39H1 in vitro
Because of the different trends in H3K9me3 levels in human and killifish transfected cells, we
surmised that killifish SUV39H1 may have low to inexistent methyltransferase activity. To test our
hypothesis, we ran immunoprecipitation to purify FLAG-tagged SUV39H1 protein and carried out
a histone methyltransferase H3K9 activity quantification assay to compare HMTase activity of
human SUV39H1 with killifish SUV39H1 and quantify the activity with HMT standards using an
ELISA based approach (Fig. 3). Standards for H3K9 methylation (HMT) are pre-methylated
peptides, without enzyme addition during incubation. Unfortunately, despite repeated attempts to
make this assay work, there is some unexpected data even in the standard curve, including lower
than blank absorbance for the standards at lower concentration. However, the HMT standard group
shows good linear increases, so the data may still be interpretable in a preliminary manner. It is
possible that the substrate added in sample, blank and positive control is somehow methylated
before or during the incubation leading to this “lower than blank” result. If the assay can be trusted
despite those negative values, my results suggest that human and killifish SUV39H1 have similar
HMTase activity.

9
Figure 3: HMTase activity quantification of human and killifish SUV39H1. Recombinant FLAG-SUV39H1 was purified by
immunoprecipitation then tested by color-based assay. Human and killifish SUV39H1 activity was plotted on HMTase activity
standard curve.

10
Chapter 3: Transposable element activity in SUV39H1 overexpressed cells
Transposable elements(TEs) are some sequences that can change their position within the genome.
One notable feature of TEs is their transposition activity increases with age in somatic tissues. In
Drosophila, Su(var)3-9 overexpression leads to suppression of TE activity and extended lifespan.
We performed bulk mRNA-seq to assay the expression levels of TEs from human and
killifish SUV39H1 HEK293T overexpressing cells. Sequencing reads were mapped to the human
reference genome (GCA_000001405.15) using STAR, and expression genes and TE sequences
was determined using TEtranscript v2.2.115. Differentially expressed sequences at FDR <5% were
determined using DESeq2 v1.38.316. Interestingly, both human and killifish overexpression cells
showed higher TE activity one-day post-transfection, which is contrary to the results in Drosophila.
However, two-day post-transfection group showed the opposite (and expected) result:
overexpression of human SUV39H1 caused repression of TE activity, and killifish SUV39H1
overexpression showed a similar pattern except upregulation of a few Alu elements (Fig. 4). Thus,
it is likely that our 1 day post transfection results may reflect a transient “crisis” state that is then
resolved at 2 days post transfection.

11
We also screened gene regulation changes by GO enrichment analysis. As expected,
transcriptionally related gene sets such as transcription regulator activity are down-regulated in
both human and killifish SUV39H1 overexpression cells, and human SUV39H1 overexpression
experienced stronger down-regulation than the killifish group as we saw more transcriptionally
related sets like DNA templated transcription and transcription by RNA polymerase II with higher
fold change towards the negative direction. We also found gene sets related to translation like
ribosome, rRNA processing and cytoplasmic translation are up-regulated in killifish group (Fig.
5).
Figure 4: Transposable element transcriptional activity in SUV39H1 overexpressed cells. Cells are harvested 1 day or 2 day after
transfection and assayed by bulk RNA-seq. Regulation of TEs are plotted by heatmap.

12
Figure 5: GO enrichment analysis of SUV39H1 overexpressed cells. Transcriptionally related gene sets are downregulated.

13
Chapter 4: Repression of LINE-1 elements in SUV39H1 overexpressed cells
LINE1 elements are found across vertebrate genomes, although they are particularly abundant in
primate genomes. Indeed, LINE1 transposons compromise 17% of human genomes17. LINE1
activation can induce IFN activation in human senescent cells and promote age-associated
inflammation8
. We directly tested the impact of human and killifish SUV39H1 overexpression on
LINE1 elementslevels in HEK293T cells using RT-qPCR at 48h post transfection. Here we created
cDNA with extracted total RNA, dNTPs and primer d(T)23VN to select mRNA adjacent to 3’
poly(A) tail. Then we used primer sets designed to amplify human L1 sequences, since we
performed experiments in HEK293T human cells8
. Quantification by green fluorescence. We
observed repression of intact L1HS, non-intact L1HS and L1PA2 in human SUV39H1
overexpression group and intact L1HS, L1PA2 in killifish SUV39H1 overexpression group (Fig.
6). This suggests that both human and killifish SUV39H1 may have TE-repressing properties,
although further investigation will be required in vivo.

14
Figure 6: Impact of SUV39H1 on LINE1 element transcription levels. RNA was extracted then amplified by RT-qPCR to detect
expression levels. P-values in Wilcoxon test.

15
Conclusion and future directions
Here we showed that in HEK293T cells, human SUV39H1 overexpression increases H3K9me3
levels while killifish SUV39H1 overexpression seemingly decreases H3K9me3 levels, possibly
because structure difference makes killifish SUV39H1 incompatible in human cells, since they
showed similar activity levels in my preliminary in vitro assay. And both human and killifish
SUV39H1 overexpression repress TE activities, supported by differentially expressed genes and
specific LINE1 element expression pattern. Future study will focus on TE activities in vivo,
which requires microinjection to knock-in and knock-out SUV39H1 in killifish embryos. Effects
on lifespan will also be monitored to reveal the relation between TE activities and lifespan in
killifish.

16
References
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Aging and Cancer. Cell Metab. 2023, 35 (1), 12–35.
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(3) Klein, S. J.; O’Neill, R. J. Transposable Elements: Genome Innovation, Chromosome
Diversity, and Centromere Conflict. Chromosome Res. 2018, 26 (1), 5–23.
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(4) Trono, D. Transposable Elements, Polydactyl Proteins and the Genesis of Human-Specific
Transcription Networks. Cold Spring Harb. Symp. Quant. Biol. 2015, 80, 281–288.
https://doi.org/10.1101/sqb.2015.80.027573.
(5) LINE-1 RNA causes heterochromatin erosion and is a target for amelioration of senescent
phenotypes in progeroid syndromes | Science Translational Medicine. https://www-scienceorg.libproxy2.usc.edu/doi/full/10.1126/scitranslmed.abl6057 (accessed 2024-05-29).
(6) LaRocca, T. J.; Cavalier, A. N.; Wahl, D. Repetitive Elements as a Transcriptomic Marker of
Aging: Evidence in Multiple Datasets and Models. Aging Cell 2020, 19 (7), e13167.
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Hartnett, D. A.; Burhenn, L.; Neretti, N.; Helfand, S. L. Chromatin-Modifying Genetic
Interventions Suppress Age-Associated Transposable Element Activation and Extend Life
Span in Drosophila. Proc. Natl. Acad. Sci. 2016, 113 (40), 11277–11282.
https://doi.org/10.1073/pnas.1604621113.
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A.; Brocculi, G.; Adney, E. M.; Boeke, J. D.; Le, O.; Beauséjour, C.; Ambati, J.; Ambati, K.;
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Sedivy, J. M. L1 Drives IFN in Senescent Cells and Promotes Age-Associated Inflammation.
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(9) Pabis, K.; Barardo, D.; Sirbu, O.; Selvarajoo, K.; Gruber, J.; Kennedy, B. K. A Concerted
Increase in Readthrough and Intron Retention Drives Transposon Expression during Aging
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Appendix
Material and Methods
Cell culture: To generate primary cell line in vitro study on killifish cells , we collected killifish
tail fin tissue and minced it into small pieces. Minced tissue was incubated in sterile tubes at 28°C
with gentle agitation. After incubation, the tissue is resuspended with complete culture medium
(L-15 GlutaMAX (31415029, Gibco), 20% FBS (12103C, Sigma), 1% penicillin/streptomycin
(30-002-CI, Corning), 1% non-essential amino acids, pH=7.4, osmolality=300 mOsm/kg) with 1%
normocin (NOL-41-06, InvivoGene). To make a stock, we mixed the cells with 90% 1x PBS and
10% DMSO in a cryotube and freeze in liquid nitrogen. To revive cells, we slightly warmed up the
cryotube and dissolve in complete culture medium, transfer it to a 10cm dish. For transfection by
electroporation, we replaced media with one without antibiotics (L-15, 20% FBS) and transfered
cells in a filtered flask.
Transfection: To get SUV39H1 overexpressed cells, we performed transient transfection of
human HEK293T using the calcium phosphate method. EGFP is used for negative overexpression
control, with overexpression of a neutral protein. Cells were passaged 1 days before and is changed
media 1-2 hours before transfection. Plasmids (EGFP, human SUV39H1, killifish SUV39H1),
CaCl2 (C4901, Sigma) and 1xTE buffer (786-752, G-Biosciences) were combined for exactly 1min
and added to cells. After one- or two-days incubation at 37°C, we washed transfected cells with
D-PBS (21-031-CV, Corning) and trypsinized with 0.05% Trypsin (25-052-CI, Corning) for
detachment. Then we collected harvested cells into falcon tubes and pelleted by centrifugation.
Cell pellets were frozen and preserved at -80°C. The experiment was performed in biological
triplicates to assess phenotypic robustness.

19
For killifish fibroblasts, we did transfection by electroporation using Neon transfection system
(MPK5000, Invitrogen). 500 to 1500 ng of pcDNA-eGFP plasmid DNA were transfected into
1*105 killifish fibroblast and optimized the condition for electroporation by adjusting amount of
plasmids, voltage, pulse width and pulse number. Cell viability and transfection efficiency were
measured by flow cytometry (MacsQuant 10). We stained the cells by propidium iodide (P1034MP,
Invitrogen). After gating for live single cells (FSC and SSC), cells were sub-gated by fluorescence
emission in FITC channel and propidium iodide channel to measure cell viability and transfection
efficiency. Flow data was exported and analyzed by FlowLogic version 8.7.
Western Blot: We did Gel electrophoresis followed by western blotting to quantify protein
intensity. We extracted Proteins from stored cell pellets by resuspension in RIPA buffer (R0278,
Sigma) with 1x Halt protease inhibitor cocktail (78430, Fisher Scientific). Resuspension buffer
was sonicated for 30 seconds twice, 1 minute apart each time on ice and then centrifuged at
10,000xg, for 20 minutes. The supernatants were stored in -80°C. Protein concentration in each
sample was assessed by using the Pierce BCA protein assay (23227, Thermo Scientific). Then we
divided samples into three groups: eGFP overexpressed, human SUV39H1 overexpressed and
killifish SUV39H1 overexpressed, each included 3 biological replicates. 15 ul of protein samples
were loaded into each well of 4–20% Mini-PROTEAN® TGX™ Precast Protein Gel (4561094,
Bio-Rad) and separated by SDS-Polyacrylamide Gel Electrophoresis [SDS-PAGE] using MiniPROTEAN® Tetra Vertical Electrophoresis system (1658004, Bio-Rad). The running condition
was constant at 120V for 80 min. Then we transferred separated proteins to a PVDF membrane
(1620177, Bio-rad) and blocked with a 2.5% milk solution in PBS-T. Cut the membrane into three
strips, then we incubated separately with anti-vinculin antibody (ab91459, Abcam) as loading
control, anti-FLAG M2 antibody (F1804, Sigma) and anti-H3K9me3 antibody (39161, Active

20
motif) overnight at 4°C. After that, we incubated the strips with secondary antibodies: Goat AntiRabbit IgG (ab205718, Abcam) or Rabbit Anti-Mouse IgG (ab6728, Abcam) at room temperature
for 1 hour. Finally, the signal was detected using Clarity Western ECL Substrate (1705061, Biorad) and captured by Azure 300 Chemiluminescent Western Blot Imager (AZI300-01, Azure
Biosystems).
RT-qPCR: Human SUV39H1, killifish SUV39H1 and eGFP overexpressing cells at 2 days post
transfection were used for RNA extraction, cDNA synthesis, and quantitative PCR. We transferred
each cell pellet into 1.5 mL a microcentrifuge tube with 1 mL TRIzol reagent (15596018,
Invitrogen) and homogenized by pipetting. Then we purified dissolved RNA using Direct-zol RNA
Miniprep Kit (R2052, ZYMO Research). The cDNA was synthesized using the the Thermo
Scientific maximaTM H Minus cDNA Synthesis Master Mix (MAN0016392, Thermo Scientific)
in the Thermal cycler C1000 (1851196, Bio-Rad). We designed specific primers for genes of
interest (human SUV39H1 and killifish SUV39H1) using Primer3 web tools. Then we performed
qPCR reactions using the SensiFAST SYBR® No-ROX Kit (Bioline, BIO-98020) in the Magnetic
Induction Cycler (MIC) machine (Bio Molecular Systems, MIC-2). The running condition is 95°C,
2min (Hold)—>(95°C, 5sec—>60°C, 10sec—>72°C, 10sec) x40—>72-95°C at 0.3°C/sec. We
analyzed the data using the comparative Ct method and compared to the gene expression levels of
GFP transfected cells. Ct values were obtained using the micPCR v2.12.7 software, with a dynamic
threshold setting.
For RT-qPCR on LINE1 elements total we performed RNA extraction in the same way above. The
cDNA was synthesized by mixing oligo d(T)23VN primer (S1327S, NEB), dNTPs (N0447L, NEB),
total RNA and nuclease-free water (SH30538.01, HyClone), then added Induro Reverse
Transcriptase (M0681L, NEB) and RNAse inhibitor (M0314, NEB). Incubated the tubes with

21
condition: 50°C, 10 min—>95°C, 1min. After that, qPCR reactions were performed in the same
way above. Six different LINE1 elements (L1HS intact, L1HS non-intact, L1PA2, L1PA3, L1PA4,
L1PA5)8 were tested. SDHA, ACTB, UDB were used to normalize LINE1 element expression
levels. Running condition is the same as above. The data was analyzed using the comparative Ct
method.
RNA sequencing: We prepared two groups of samples for RNA-seq. One group consisted of
eGFP, human SUV39H1 and killifish SUV39H1 tranfected HEK293T cells harvested 1 day posttransfection. Another group was harvested 2 day post-transfection. Total RNA was isolated by
Trizol and purified by Direct-zol RNA Miniprep Kit from human SUV39H1, killifish SUV39H1
and EGFP overexpressed cells. Then we tested RNA samples on the 4200 TapeStation system
(G2991A, Agilent Technologies) with a High Sensitivity RNA ScreenTape (5067-5579, Agilent
Technologies) to measure RNA quality and concentration. The constructed bulk mRNA-seq
libraries were sequenced on an Illumina Novaseq 6000 generating 150 bp paired-end reads at
Novogene USA. Raw sequencing reads were aligned to the human reference genome (hg38) using
the splice-aware STAR aligner version 2.7.0e with parameters “--outFilterMultimapNmax 200; --
outFilterIntronMotifs RemoveNoncanonicalUnannotated; --alignEndsProtrude 10 ConcordantPair;
--limitGenomeGenerateRAM 60000000000 --outSAMtype BAM SortedByCoordinate”. BAM
files are summarized by TEtranscript version 2.2.1 with parameter “--sortByPos”. DESeq2 version
1.38.3 was used to analyze differential gene expression analysis. We analyzed expression levels of
transposable elements in response to SUV39H1 overexpression.
GO analysis: The results from the differential gene expression were used as input for
ClusterProfiler v4.6.2 to run Gene Ontology enrichment using gene set enrichment analysis

22
(GSEA). Human Gene Ontology terms were obtained from annotation package
org.Hs.eg.db_3.16.0. We used minimum gene set size of 10 and maximum gene set size of 5000
to compute enrichment.
Immunoprecipitation: We purified FLAG tagged SUV39H1 by Anti-DYKDDDDK Magnetic
Agarose (A36797, Thermo Fisher). Lysed cell pellets in binding buffer (25 mM Tris-HCl pH 7.4
(15567-027, Invitrogen), 150 mM NaCl (V4221, Progema), 1 mM EDTA (BDH7830-1, VWR), 1%
NP-40 (25263700, Roche), 5% glycerol (BDH1172-4LP, VWR)) then mixed with magnetic beads
for 20 minutes. Beads were washed with 1x DPBS and purified water, and eluted by 0.1 M glycine,
pH 2.8. The eluted target was neutralized by neutralization buffer (1x Tris, pH 8.5) and saved.
Then we collected the flowthrough during washing. The original lysate, the flowthrough, washes,
and eluted targets were tested by SDS-PAGE to determine purification efficiency.

Abstract (if available)

Abstract Transposable elements (TEs) are mobile DNA sequences capable of replicating themselves found in most eukaryotic genomes. TE activation is believed to be directly deleterious to longevity by promoting genome instability. Drosophila Su(var)3-9 specifically methylates histone H3K9, which has been proven important in gene repression and chromatin maintenance. Scientists have shown that overexpressing Su(var)3-9 in Drosophila leads to TE repression and increased lifespan. However, whether a vertebrate homolog of Su(var)3-9 has a similar effect on both TE expression and longevity is still unclear. African turquoise killifish (Nothobranchius furzeri) is the shortest-live vertebrate that can be bred in capacity, thus allowing high repeatability and feasibility for experiments. In this report, we verified the HMTase activity of human SUV39H1 (hSuv) and killifish SUV39H1 (kSuv), which are homologs of Drosophila Su(var)3-9 in HEK293T cells and killifish fibroblasts. TE activities are suppressed in both hSuv and kSuv overexpressed cells. We also found that LINE1 expression was repressed in a human cellular model upon human and killifish SUV39H1 overexpression.

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Asset Metadata

Creator Tang, Tu (author)

Core Title Investigation of a causal role of transposable element activation in vertebrate aging

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Medicine

Degree Conferral Date 2024-05

Publication Date 06/13/2024

Defense Date 05/23/2024

Publisher Los Angeles, California (original), University of Southern California (original), University of Southern California. Libraries (digital)

Tag aging,OAI-PMH Harvest,Suv39h1,transposable elements

Format theses (aat)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Benayoun, Bérénice (committee chair), Rice, Judd (committee member), Vermulst, Marc (committee member)

Creator Email tangtu0709@gmail.com,tutang@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC1139963R5

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Legacy Identifier etd-TangTu-13097

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Source 20240614-usctheses-batch-1169 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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