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Identification of target genes and protein partners of ZNF711 in glioblastoma cells
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Identification of target genes and protein partners of ZNF711 in glioblastoma cells
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Content
IDENTIFICATION OF TARGET GENES AND PROTEIN PARTNERS OF ZNF711 IN
GLIOBLASTOMA CELLS
by
Jemima Pangemanan
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)
August 2024
ii
ACKNOWLEDGEMENTS
I would like to express my gratitude to my professor and chair of my committee, Dr.
Peggy Farnham, for her support and invaluable patience and feedback during my time as a
student in her lab. I also would like to thank my committee members; Dr. Judd Rice and Dr.
Oliver Bell who generously provided knowledge and expertise.
I also could not have undertaken this journey without the help from fellow lab members;
Charles and Emily also previous lab members; Shannon, Yao, and Katie who patiently guide
me through experiments and helped me with my questions. Thank you for sharing all your
knowledge and experience with me.
Lastly I would like to thank my parents, brothers, partner, and friends for giving me
motivation and endless support through my study. Their belief in me has kept my spirits and
motivation high during this process. I would also like to thank Lembaga Pengelola Dana
Pendidikan (LPDP) Indonesia for funding my study and supporting me.
iii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS.......................................................................................................ii
LIST OF TABLES....................................................................................................................iv
LIST OF FIGURES ...................................................................................................................v
ABSTRACT..............................................................................................................................vi
CHAPTER I. Introduction .........................................................................................................1
CHAPTER II. Methods..............................................................................................................4
Overall design study ..............................................................................................................4
Plasmid cloning......................................................................................................................5
Cell culture.............................................................................................................................6
Transient transfections...........................................................................................................7
Pre-experimental controls......................................................................................................7
RNA sequencing (RNA-seq) .................................................................................................8
Chromatin Immunoprecipitation Sequencing (ChIP-seq) .....................................................8
Turbo-ID ................................................................................................................................9
CHAPTER III. .........................................................................................................................10
Functional Characterization of ZNF711 ..................................................................................10
Effects on the transcriptome after ZNF711 transfection......................................................10
ZNF711 has different effects on the transcriptome than ZFX and ZFY..............................15
ZNF711 binds downstream of the TSS and binds to the same promoters as does ZFX......17
ZNF711 directly regulates genes involved in neurogenesis ................................................18
CHAPTER IV. .........................................................................................................................22
Identification of ZNF711 Interaction Complexes....................................................................22
CHAPTER V. ..........................................................................................................................25
ZFX Family Member Domain Swapping ................................................................................25
CHAPTER VI. Discussion.......................................................................................................26
REFERENCES ........................................................................................................................28
iv
LIST OF TABLES
Table 2.1. Primers used for cloning of ZNF711 N-terminus ZFX miniTurbo plasmid ….5
Table 2.2. Primers used for RT-qPCR and ChIP-qPCR ……………………………….....7
v
LIST OF FIGURES
Figure 1.1 ZFX family members………………………………………………………..... 1
Figure 1.2 ZFX family gene structures………………………………………………….... 2
Figure 2.1 ZNF711-miniTurbo fusion protein………………………………………….... 4
Figure 2.2 ZNF711-Nterminus_ZFX_miniTurbo fusion protein……………………........ 4
Figure 2.3 ZFX-Nterminus_ZNF711_miniTurbo fusion protein……………………........ 5
Figure 2.4 Gel visualization of PCR product…………………………………………….. 6
Figure 3.1 Experimental controls for ZNF711 RNA-seq in LN-229…………………… 11
Figure 3.2 ZNF711 effects on the transcriptome……………………………………….. 13
Figure 3.3 Gene Ontology (GO) biological process analysis of ZNF711
up-regulated and down-regulated genes ranked by FDR values……………………...... 14
Figure 3.4 Comparison of ZNF711, ZFX, and ZFY effects on transcriptome…………. 15
Figure 3.5 Gene Ontology (GO) biological process analysis…………………………... 16
Figure 3.6 ZNF711 genomic binding………………………………………………..… 18
Figure 3.4 Comparing ZNF711 and ZFX direct target genes…………………………... 20
Figure 4.1 ZNF711 interaction map…………………………………………………..... 23
Figure 4.2 Overlap between ZFX and ZNF711 interacting proteins………………….... 24
Figure 5.1 ZFX family N-terminus amino acids alignment…………………………….. 25
Figure 5.2 Experiment design for N-terminus swapping……………………………….. 25
vi
ABSTRACT
The ZNF711 gene is a member of the ZFX family of C2H2 zinc finger proteins. It is
known that ZFX family members act as transcriptional activators and bind to CpG islands
promoter. ZNF711 mutations have been found in X-linked intellectual disability and most of
those mutations are frameshift mutations, which suggests ZNF711 loss of function may play
a role in neurodevelopmental disorders. Other than its function as a transcriptional activator,
not a lot is known about the mechanism by which ZNF711 influences transcription. In this
study, I used LN-229 cells, a glioblastoma cell line which does not express ZNF711, to
perform an overexpression study. RNA-seq analysis revealed that thousands of genes showed
upregulated expression after transfection of a ZNF711 expression construct, with some genes
involved in bone, neuron, and muscle development. Furthermore, ChIP-seq data revealed that
although ZNF711 binds identically as ZFX, it can directly regulate a different set of genes.
Further studies investigating the mechanisms by which ZNF711, but not ZFX, can activate
specific target genes are underway.
1
CHAPTER I. Introduction
Transcription factors (TF) are one of the critical factors in transcription regulation.
Transcription factors bind to the gene promoter region near the transcription start site (TSS) or
to enhancer elements (distal regulatory sequences from the TSS that activate gene expression)
and help in the recruitment of co-regulators [1, 2]. Promoter regions consist of sequence
elements such as a CpG island or a TATA box; the majority of promoters in the human genome
are CpG islands [1]. One of the largest site-specific transcription factor families is the C2H2
zinc finger family. A C2H2 zinc finger protein (ZFP) is characterized by having a DNA-binding
domain containing zinc fingers, a protein structure formed by two cysteines and two histidines
bound together by a zinc ion [3]. The majority of the human C2H2 ZFPs contain a KRAB
repressor domain that recruits histone methylase complexes to create silenced chromatin [4, 5];
thus, most C2H2 ZFPs are repressors of transcription [6].
The ZFX family of transcription factors are members of the large C2H2 zinc finger
family and have been shown to bind to CpG island promoters [4]. There are three members of
the ZFX family: ZFX, ZFY, and ZNF711 (Figure 1.1).
Figure 2.1 ZFX family members. Shown is a Treefam (http://www.treefam.org/) alignment of the ZFX family. (Ni et al. 2020).
Unlike the majority of ZFPs, the ZFX family members do not have a KRAB repressor
domain. The ZFX family members have an acidic N-terminal domain, a nuclear localization
sequence, and a zinc finger DNA-binding domain at the C-terminal of the protein (Hsu &
Farnham, unpublished data) [1]. Based on their amino acid sequence, ZFX and ZFY are highly
2
similar, having 99% similarity in the zinc finger domain (consisting of 13 zinc fingers) and
96% overall similarity. However, ZNF711 has only an 87% similarity to ZFX in the zinc finger
domain due to the fact that it has only 11 zinc fingers (fingers 3 and 7 have amino acid changes
that eliminate the correct structure of these two fingers). The N-terminal transactivation domain
of ZNF711 has also diverged from ZFX, resulting in an overall similarity of 67% of ZNF711
to ZFX (Figure 1.2). ZFX and ZNF711 are both encoded on the X chromosome and ZFY is
encoded on the Y chromosome [1].
Figure 1.2 ZFX family gene structures. Dashed lines indicate conserved zinc finger between family members [1]. This
schematic is taken from Ni and Farnham (2020).
Alterations in zinc finger transcription factors can lead to human diseases, including
cancers and developmental disorders. Of the 3 ZFX family members, only ZFX has been
associated with tumorigenesis. In fact, the high expression of ZFX found in many types of
cancers (including prostate cancer, breast cancer, colorectal cancer, glioma, renal carcinoma,
gastric cancer, gallbladder adenocarcinoma, non-small cell lung carcinoma, and laryngeal
squamous cell carcinoma) has been associated with poor patient survival [8-14]. However, we
have previously shown that the removal of either ZFX or ZNF711 in HEK293T cells causes
reduced cell growth and major defects in proliferation were observed with the simultaneous
knockdown of both ZFX and ZNF711. Effects of loss of ZFY on cell proliferation have not
been performed.
It is known that C2H2 zinc finger proteins can be involved in brain development and
function [18]. Interestingly, somatic mutations in both ZFX and ZNF711 (but not ZFY) have
previously been linked to intellectual disability. For ZFX, exome and genome sequencing have
revealed frameshift and missense mutations with clinical findings that include developmental
delay/intellectual disability, behavioral abnormalities, hypotonia, and congenital anomalies
[16]. Interestingly missense variants in the ZFX DNA-binding domain demonstrated
differential expression of a small set of target genes relative to wild-type ZFX in cultured cells,
3
suggesting both a gain and loss of transcriptional activity [15]. Frameshift and missense
mutations in ZNF711 have also been identified in families with X-linked intellectual
disabilities. Because the ZNF711 mutations mostly are frameshift mutations that lead to
premature stop codons, this suggests that X-linked intellectual disability may be caused by
ZNF711 loss of function [15-17].
Our previous lab studies have shown that ZFX and ZNF711 binding patterns are almost
identical in 22Rv1 prostate cancer cells and HEK293T kidney cells. Both proteins bind
downstream of transcription start sites (at ~+250) and function as transcriptional activators [1].
However, an analysis of the function of ZNF711 in neuronal cells has not been performed. To
better understand the role of ZNF711 in neuronal functions, we need to have a deeper
understanding of the mechanism of action of ZNF711. Therefore, the aim of my dissertation is
to identify target genes and protein partners of ZNF711. I have chosen to use the LN-229
glioblastoma cell line for my studies, as these cells are derived from brain tissue. Notably, the
LN-229 cell line has lost the expression of endogenous ZNF711 but still expresses ZFX.
Therefore, I can transfect these cells with wildtype or mutant ZNF711 expression constructs
and not have to worry about the competing effects of endogenous ZNF711 in my experiments.
4
CHAPTER II. Methods
Overall design study
I used LN-229 (female cells) which do not express endogenous ZNF711 and transfected
them with a ZNF711-miniTurbo plasmid (Figure 2.1). This plasmid consists of full length
cDNA of ZNF711 (having a FLAG tag) fused with the miniTurbo biotinylating protein. I
sequenced the plasmid before transfection to make sure the plasmid did not have any mutations.
I used this expression vector for transactivation experiments (to examine the effects of ZFN711
on the transcriptome), ChIP-seq (to identify the direct target genes of ZNF711), and TurboID
experiments (to identify protein interactors).
Figure 2.1 ZNF711-miniTurbo fusion protein. The fusion protein contains the ZNF711 coding
sequence fused to a FLAG tag for use in ChIP assays and the miniTurbo sequence for use in
Turbo-ID experiments. This construct is cloned into the pCMV6 expression vector.
I also created another plasmid that has the ZNF711 N-terminus sequence fused onto the ZFX
zinc finger domain, followed by the FLAG and miniTurbo sequences (Figure 2.2) and ZFX Nterminus sequence fused onto the ZNF711 zinc finger domain, followed by FLAG and mini
Turbo sequence. These two plasmids allow me to investigate the different functions of the
ZNF711 and the ZFX N-termini (which only have 37% similarity) in transcriptional regulation.
Figure 2.2 ZNF711-Nterminus_ZFX_miniTurbo fusion protein. The fusion protein consists of
the ZNF711 N-terminus sequence followed by ZFX zinc finger domains and the FLAG
sequence for ChIP and miniTurbo sequence for Turbo-ID. This construct is cloned into pCMV6
expression vector.
5
Figure 2.3 ZFX-Nterminus_ZNF711_miniTurbo fusion protein. The fusion protein consists of
the ZFX N-terminus sequence followed by ZNF711 zinc finger domains and the FLAG
sequence for ChIP and miniTurbo sequence for Turbo-ID. This construct is cloned into the
pCMV6 expression vector.
Plasmid cloning
The ZNF711-miniTurbo plasmid was cloned by Shannon Schreiner using Gibson
Assembly. I cloned the ZNF711-Nterminus_ZFX_miniTurbo plasmid and the ZFXNterminus_ZNF711_miniTurbo plasmid. I designed primers for Gibson Assembly to make
four fragments; the ZNF711 N-terminus, the ZFX N-terminus, and the ZFX or ZNF711 zinc
finger domain plus FLAG and miniTurbo sequences and the rest of the vector (Table 2.1).
Fragments were produced using PCR with 2X Platinum SuperFi Master Mix (Invitrogen). To
make sure the fragments were the right length and confirm only a single band is formed for
each pair of primers, gel electrophoresis was performed using a 1% agarose gel, 100 volts for
60 minutes (Figure 2.3). After confirming the size of the fragments, the PCR products were
cleaned up using AmpureXP (Beckman Coulter) following the manufacturer’s protocol.
Gibson Assembly was achieved using two fragments with 2X Gibson Assembly Master Mix
(NEB #E2611), following the manufacturer’s protocol. The ligated product was then
transformed into competent cells (CopyCutter EP1400 Chemically Competent E. coli; Lucigen
#C400CH10), following the manufacturer’s protocol. Cells were plated and incubated
overnight at 37oC on kanamycin agar plates (Teknova) to select for colonies containing the
plasmid.
Table 2.1. Primers used for cloning of ZNF711 N-terminus ZFX miniTurbo plasmid
Name Sequence
ZFX_ZF_Vec.fwd TATCCTTGCATGATTTGTGGGAAGAAGTTTAAGTCGAGAGG
ZFX_ZF_Vec.rev GGCGATCGCGGCGGCAGATCTCC
ZNF711_Nterm.fwd CTGCCGCCGCGATCGCCATGGACAGCGGTG
ZNF711_Nterm.rev
ACTTCTTCCCACAAATCATGCAAGGATACACCGTCAATGGC
TGCCC
ZFX_N-term.fwd GATCTGCCGCCGCGATCGCCATGGATGAAGATGGGCTTG
6
ZFX_N-term.rev
GTGCAAATGTGGCAGGGGTAGACAGTCAAAGGATGTCCAT
C
ZNF711_ZF_vec.f
wd
TACCCCTGCCACATTTGCACAAAAAAGTTTAAATCCCGCGG
C
ZNF711_ZF_vec.r
ev GGCGATCGCGGCGGCAGATCTCCTCG
Figure 2.4 Gel visualization of PCR product. The ladder used is Tri-Dye 1kb plus ladder (NEB
#N3270S). Lane 1 is the ZFX zinc finger vector fragment (6.8 kb), lane 2 is the ZNF711 Nterminus fragment (1.19 kb), lane 5 is the ZNF711 zinc finger vector fragment (6.8 kb), and
lane 6 is the ZFX N-terminus fragment (1.3 kb).
Cell culture
Human glioblastoma LN-229 (ATCC #CRL-2611) cells were obtained from ATCC
(https://www.atcc.org/). Cells were cultured in DMEM media supplemented with 10% fetal
bovine serum (Gibco by Thermo Fisher) plus 1% penicillin and 1% streptomycin at 37◦C with
5% CO2.
7
Transient transfections
LN-229 cells were seeded into a six-well plate for RNA-seq experiments (1 well per
replicate), 15 cm plates for ChIP-seq and TurboID experiments (2 plates per replicate). During
log phase growth, cells were transfected with the wt or mutant ZNF711-miniTurbo plasmid or
empty vector as a control using Lipofectamine 3000 (ThermoFisher), according to the
manufacturer’s protocol. The cells were incubated for 24 hours and then harvested for RNAseq and ChIP-seq experiments. For Turbo ID, the cells were incubated for 48 hours .
Pre-experimental controls
To confirm that addition of the miniTurbo domain did not affect the transcriptional
activity of ZNF711, I transfected LN-229 cells with the wt ZNF711 miniTurbo plasmid and
isolated total RNA using TRIzol Reagent (Thermo Fisher) 24 hours after transfection. Total
RNA was converted to cDNA using iScript (Bio-Rad) and RT-qPCR was carried out using
pairs of primers to monitor expression of endogenous genes (Table 2.1). Because ZNF711
target genes in LN-229 were not known, I used a known LN-229 ZFX target gene for a positive
control (because previous experiments using HEK293T cells showed that ZNF711 and ZFX
share many target genes). Therefore, LONRF2 was used as a test target gene and GAPDH was
used as a control gene (Figure 3.1 A). In addition, I also tested the DNA binding activity of
the ZNF711 miniTurbo clone. For this experiment, I transfected LN-229 cells, waited 48 hrs,
and harvested the cells and performed a ChIP assay. ZNF711 ChIP-qPCR was performed using
a known ZFX target gene (LRRC41) to check the binding of ZNF711 to the genome and the
DEAD region (which does not contain any known regulatory regions) was used as the negative
control (Figure 3.3 A) (Table 2.2).
Table 2.2. Primers used for RT-qPCR and ChIP-qPCR
Name Sequence Assay Usage
LRRC41 F CGG TCG CTT
AGT CAG TTT GG ChIP
PCR
Check ChIP enrichment
LRRC41 R
CAG ATT GGA
GAG CGA GGG
AA
Check ChIP enrichment
DEAD F
CTC CAC ACC
ACA ACA AAG
GTG C
ChIP
PCR Control for ChIP PCR
8
DEAD R
GCT GTC ATT
ACT TGC ACT
TTG
Control for ChIP PCR
LONRF2 F
GGA GCT GGC
TCC TGA TGA
TAA RTqPCR
Check transfection & transactivation
of ZNF711 in LN-229 cells
LONRF2 R CTG CAC TGG
CAT CTT GGA GA
Check transfection & transactivation
of ZNF711 in LN-229 cells
GAPDH F AAT CCC ATC
ACC ATC TTC CA RTqPCR
Control for RT-qPCR
GAPDH R CTC CAT GGT
GGT GAA GAC G Control for RT-qPCR
RNA sequencing (RNA-seq)
Cells were transfected with the ZNF711Turbo plasmid and total RNA was extracted 24
hours later using TRIzol Reagent (Thermo Fisher), following the manufacturer’s protocol.
RNA integrity was checked using a 2100 Bioanalyzer (Agilent) with RNA integrity number >
8.0 considered as good. RNA-seq libraries for cells transfected with empty vector and
ZNF711_miniTurbo were prepared and sequenced by Novogene using the Illumina NovaSeq
platform. Sequencing data was aligned to the human genome GRCh38 using STAR and
quantified using FeatureCount. RNA-seq experiments were performed in four replicates, and
to determine if they were good replicates, PCA analysis was performed using R (DESeq2 &
ggplot) (Figure 3.1 B). Differential expression analysis was done using R (DESeq2) with fold
change +/- 1.5 and q-value cut-off of 0.05. Gene ontology analysis was done using ShinyGO
(http://bioinformatics.sdstate.edu/go/).
Chromatin Immunoprecipitation Sequencing (ChIP-seq)
Cells were transfected with the ZNF711-miniTurbo plasmid. Two days later,
crosslinking was done by adding formaldehyde to the cell culture media, followed by adding
cold glycine after 15 minutes to stop crosslinking. Cells then were harvested by scraping the
cells from the plate by adding cold Dulbecco’s Phosphate-Buffered Saline (DPBS). After that,
cells were lysed using cell lysis buffer then nuclei lysis buffer. Chromatin fragmentation was
achieved by sonicating the chromatin solution for 10 cycles using Diagenode Bioruptor Pico.
Then, ChIP assays were performed using 100 ug chromatin and 15 ul FLAG antibody
(SIGMA). Following the manufacturer's protocol, ChIP-seq libraries were prepared using the
KAPA HyperPrep kit (Roche #KK8502). ChIP-seq libraries were sequenced by Novogene
using the Illumina Novaseq platform. Sequencing data was aligned to human genome GRCh38
9
using Bowtie2, followed by peak calling using MACS2. ChIP-seq experiment was done using
two replicates; to determine if the replicates are good IDR analysis was performed [19].
Downstream analyses was performed using R and HOMER for annotation and CEAS for
generating the heatmap. Gene ontology analysis was done using ShinyGO
(http://bioinformatics.sdstate.edu/go/).
Turbo-ID
Cells were transfected with the ZNF711-miniTurbo plasmid (3 replicates) and
incubated for 48 hours at 37oC. Cells then treated with biotin followed by 15 minutes of
incubation. Cells were scraped from the plate using cold PBS and lysed using cell lysis buffer
with protease inhibitor followed by RIPA with protease inhibitor. Protein concentration was
quantified from the nuclear lysates. Tagged proteins were pulled down using MyOne
Streptavidin Magnetic Beads (Dynabeads #65601) and digested with trypsin (Promega). The
digested protein extract was then analyzed using mass spectrophotometry (samples were
analyzed by the UC Davis Proteomics Core Facility). Mass spectrophotometry data was
selected using a Padj < 0.05 and a log2 fold change > 0.50, followed by filtering out
contaminants and cytoplasmic proteins using the CRAPome (https://reprintapms.org/?q=wk_1_1_search) and uniport (https://www.uniprot.org/). An interaction map was
made using STRING (https://string-db.org/) for physical interactions.
10
CHAPTER III. Functional Characterization of ZNF711
In order to investigate the function of ZNF711 in glioblastoma cells (LN-229), I
performed RNA-seq experiments using the ZNF711-miniTurbo plasmid in LN-229 cells and I
analyzed the data myself. I also re-analyzed the ZFX and ZFY RNA-seq data from LN-229
that was previously obtained by another lab member (Yao Liu), so that all RNA-seq data
analyses were performed using the same pipeline. I performed the ChIP-seq experiments using
the ZNF711-miniTurbo plasmid in LN-229 cells and I analyzed the data myself. I also reanalyzed ZFX ChIP-seq data from LN-229 cells that was previously obtained by another lab
member (Emily Hsu) so that all ChIP-seq data analyses were performed using the same
pipeline. I performed the Turbo-ID experiments using the ZNF711-miniTurbo plasmid in LN229 cells and analysed the data.
Effects on the transcriptome after ZNF711 transfection.
In order to investigate the effects on the transcriptome mediated by ZNF711, I
performed RNA-seq experiments using LN-229 cells transfected with ZNF711-miniTurbo and
an empty vector as a control. I performed RT-qPCR to check the transactivation activity of
ZNF711 using LONRF2 as the target gene (LONRF2 is known to be up-regulated by ZFX and
ZFY in LN-229 cells, suggesting it may also be up-regulated by ZNF711). Indeed, I saw an
upregulation of LONRF2 when ZNF711 was expressed, demonstrating that the fusion protein
was active and that my transactivation was successful (Figure 3.1A). I performed the RNA-seq
in 4 replicates; to determine if the replicates were reproducible, I did a PCA analysis which
showed that the experimental and control groups form 2 separate clusters. Indeed, the control
group and experimental group separate from each other with 92% variance between groups and
3% variance within each group (Figure 3.1 B).
11
Figure 3.1 Experimental controls for ZNF711 RNA-seq in LN-229. (A) RT-qPCR of LN-229
cells transfected with the ZNF711-T plasmid or empty vector as a control; 4 replicates were
performed. LONRF2 expression was normalized using GAPDH expression. (B) PCA plot of
the 4 replicates from the control group and the experimental group (transfected with the
ZNF711-miniTurbo plasmid).
I then used the RNA samples for RNA-seq analyses. After sequencing and mapping the
reads to the genome, I first examined the genomic regions encompassing the ZNF711 gene. I
expected to see ZNF711 as one of the highest expressed genes in the RNA-seq datasets, as the
amount expressed by the introduced plasmid should be very high, as compared to expression
of endogenous genes. However, there were no reads associated with ZNF711 in the RNA-seq
datasets. I knew that my transfection had worked because of the RT-qPCR assays; therefore,
there must have been another reason why I did not detect ZNF711. I realized that the ZNF711
cDNA used in this experiment had been codon-optimized by the company. Although the same
amino acids were encoded as in endogenous ZNF711 , the codon optimization resulted in major
changes in the RNA sequence and, as a result, the sequenced tags could not be aligned to the
12
genome. Therefore, I chose a region of the codon optimized RNA and searched the RNA-seq
reads (before mapping them to the genome). Using this method, I could now identify a huge
number of ZNF711 codon-optimized sequence reads, thus reassuring me that I had achieved
high expression of ZNF711 in the transfected cells (Figure 3.2 A).
After determining that my RNA-seq experiments were of high quality, I created volcano
plots showing genes that were up or down-regulated after introduction of ZNF711-miniTurbo
into LN-229 cells (Figure 3.2 B). I identified 2484 up-regulated genes and 1433 downregulated genes. Because ZFX has been shown to be a transcriptional activator, it is likely that
ZNF711 is also an activator. Thus, the up-regulated genes should include direct targets and
indirect targets whereas the down-regulated genes are indirect targets downregulated due to
changes in the cellular phenotype caused by activation of ZNF711 target genes. Some of the
most up-regulated genes, such as FGFR3, RBM38, EPAS1, EPHB2, and LAMA5, are involved
in bone development, myogenic differentiation, angiogenesis, and neuronal growth [20-24].
13
Figure 3.2 ZNF711 effects on the transcriptome. (A) Number of codon-optimized ZNF711
sequence reads from sequencing data. (B) Volcano plot of expression data from LN-229 cells
transfected with ZNF711-T plasmid compared with control cells transfected with empty vector;
up-regulated (red) and down-regulated (blue) genes are shown (using a Padj < 0.05 and fold
change of +/- 1.5).
14
To understand the roles of all the up- and down-regulated genes, I performed Gene
Ontology (GO) for biological process (Figure 3.3). Based on the GO analysis, ZNF711 upregulates a set of genes involved in neurogenesis, neuron differentiation, generation of neurons,
and central nervous system development and the down-regulated genes are involved in mRNA
metabolism.
Figure 3.3 Gene Ontology (GO) biological process analysis of ZNF711 up-regulated and downregulated genes ranked by FDR values.
Up-regulated genes
Down-regulated genes
15
ZNF711 has different effects on the transcriptome than ZFX and ZFY
Because ZNF711 has less overall similarity with the other two family members, ZFX
and ZFY, it is possible that ZNF711 regulates a different set of genes compared to ZFX and
ZFY. To investigate this, I analyzed ZFX and ZFY RNA-seq data in LN-229 that was obtained
by a previous lab member (Yao Liu). I compare the up-regulated genes and the down-regulated
genes from the three family members. I created volcano plots identifying 1725 and 1845 upregulated genes along with 1084 and 1177 down-regulated genes upon introduction of ZFXminiTurbo and ZFY-miniTurbo into LN-229 cells, respectively (Figure 3.4 A). I then compared
the up-regulated and down-regulated genes for all 3 family members and found that ZNF711
up-regulates a large set of unique genes (1157 genes) whereas ZFX and ZFY generally
upregulate a similar set of genes (1351 genes) and the same goes for the down-regulated genes
(Figure 3.4 B).
Figure 3.4 Comparison of ZNF711, ZFX, and ZFY effects on transcriptome. (A) Volcano plots
of expression data from LN-229 cells transfected with ZFX-miniTurbo and ZFY-miniTurbo
plasmid compared with control cells transfected with empty vector. Showing up-regulated and
16
down-regulated genes with Padj < 0.05 and fold change of +/- 1.5. (B) Overlaps of ZNF711,
ZFX, and ZFY up-regulated and down-regulated genes in LN229.
To invesitagte the roles of genes that are “unique” or “common” between ZNF711 and
the other two family members, I performed GO analyses using 1157 genes only up-regulated
in ZNF711, 970 genes upregulated by all three family members, and 381 genes only upregulated by both ZFX and ZFY. Based on the GO analyses, genes that are up-regulated only
by ZNF711 are involved in axonogenesis and neural migration and projection and the genes
that are up-regulated by all three family members also have similar involvement in
neurogenesis (Figure 3.5).
Figure 3.5 Gene Ontology (GO) biological process analysis. Genes only up-regulated by
ZNF711 (1157 genes), genes up-regulated by ZNF711, ZFX, and ZFY (970 genes), all genes
up-regulated by both ZFX and ZFY (1351), and genes up-regulated by ZFX & ZFY but not by
ZNF711 (381 genes), ranked by FDR values.
17
ZNF711 binds downstream of the TSS and binds to the same promoters as does ZFX
The ZNF711 up-regulated genes from the RNA-seq experiment include both direct and
indirect target genes. In order to identify ZNF711 direct target genes, I performed ChIP-seq to
determine genes directly bound by ZNF711. To profile ZNF711 binding sites, I performed two
replicates of ChIP-seq experiments using LN-229 cells transfected with ZNF711-miniTurbo
plasmid. Very few studies of ZNF711 target genes have been performed and none of these were
using LN-229 cells. However, in 22Rv1 prostate cancer cells, previous lab members have
shown that ZNF711 and ZFX bind to the same promoters [4]. Therefore, I performed q-PCR
to check the ZNF711 ChIP enrichment using LRRC41 as the target promoter, because previous
experiments in the lab have shown that ZFX binds to LRRC41 in LN-229 cells. The ZNF711
ChIP-qPCR shows enrichment of LRCC41 after immunoprecipitation using a FLAG antibody,
demonstrating a successful ChIP (Figure 3.6 A). I then prepared libraries and sent them out for
sequencing. I aligned the sequence files and using merged replicates files I identified ZNF711
genomic binding sites. As expected from the previous studies of ZFX and ZFY, ZNF711 is
predominantly localized to promoter regions (Figure 3.6 B). To more precisely determine
where in the promoter region ZNF711 binds to, I made a tag density plot showing ZNF711
binds to downstream of the TSS (Figure 3.6 C). I then compared ZNF711 and ZFX binding to
see if they bind to the same promoters. I used LN-229 ZFX ChIP-seq data from our lab
(courtesy of Emily Hsu) and re-analyzed it using the same method as used for the ZNF711
ChIP-seq data. Although ZNF711 lacks zinc finger 3 and 7, it binds to the same promoters at
the same location downstream of the start site as ZFX (Figure 3.6 D & E). The binding of ZFX
looks stronger in the heatmap compared to ZNF711 binding; this could be caused by ZFX ChIP
is from endogenous protein whereas ZNF711 ChIP is from transfected cells (and not all cells
may have received the plasmid).
18
Figure 3.6 ZNF711 genomic binding. (A) qPCR of ZNF711 ChIP using FLAG antibody. (B)
ZNF711 binding distribution. (C) Tag density plot of ZNF711 binding at promoter regions (+/-
2kb from TSS). (D) Heatmap of ZNF711 and ZFX ChIP-seq data at promoters, ranked from
high to low ZFX binding. (E) Browser tracks of ChIP analysis of ZNF711 transfected cells and
endogenous ZFX.
ZNF711 directly regulates genes involved in neurogenesis
By combining the ZNF711 RNA-seq and ChIP-seq data, I can identify genes directly
regulated by ZNF711 (i.e. direct target genes). To do this, I overlapped the set of genes upregulated by ZNF711 and the set of promoters bound by ZNF711. Using this method, I
identified 1295 direct target genes of ZNF711 (Figure 3.7 A). I also identified ZFX direct target
1.0 1.5 2.0 2.5 3.0
−2kb 0bp 2kb
LN229_ZNF711_rep2
@hg19.bed.gz
LRCC41
0
100
200
300
400
500
Fold Enrichment
ZNF711 Rep 1
ZNF711 Rep 2
A B
C D
E
High
Low
-3kb 3kb
TSS TSS
ZFX ZNF711
ZNF711
ChIP-seq Tag Density
Other Promoter peaks +/- 2kb from TSS
1.0 1.5 2.0 2.5 3.0
-2kb TSS 2kb
19
genes using the same method, resulting in 633 direct target genes of ZFX (Figure 3.7 B).
Because ZNF711 and ZFX genomic binding is similar overall, I want to see if their direct target
genes are also similar. I had previously shown (Figure 3.4 B) that 1159 genes were up-regulated
by both ZNF711 and ZFX. Having the ChIP-seq data, I can now determine how many of these
commonly up-regulated genes are direct targets for ZNF711, ZFX, or both factors. Based on
the overlap between ZNF711 and ZFX direct target genes, I have shown that these two family
members have 399 direct target genes in common, 896 direct target genes specific to ZNF711,
and 234 direct target genes specific to ZFX (Figure 3.7 C). This unique set of direct target
genes of ZNF711 are involved in neurogenesis, neuron differentiation, generation of neurons,
axon development, axonogenesis, and neuron projection, meanwhile ZFX unique direct targets
are involved in development process (Figure 3.7 D).
20
Figure 3.4 Comparing ZNF711 and ZFX direct target genes. (A) Identification of ZNF711
direct target genes. (B) Identification of ZFX direct target genes. (C) Overlap between ZNF711
A B
Up-regulated genes
Up-regulated genes
Genes with
promoter
bound by
ZNF711
Genes with
promoter
bound by
ZFX
Direct target genes
Direct target genes
ZFX direct target genes
ZNF711 direct target genes
A
B
C
D ZNF711 only direct target
ZFX only direct target
21
and ZFX direct target genes. (D) Gene Ontology (GO) biological process analysis of ZNF711
(896) and ZFX (234) unique direct target genes ranked by FDR values.
22
CHAPTER IV. Identification of ZNF711 Interaction
Complexes
Based on the RNA-seq result, ZNF711 and ZFX regulate a different set of direct target
genes. To understand the cause of this specific regulation, I performed proteomics analysis
using TurboID followed by mass spectrophotometry to investigate ZNF711 interacting proteins
based on proximity biotinylation. ZNF711 interacts with 5 complexes; the integrator complex,
the survival of motor neurons (SMN) complex, the COMPASS complex, the DNA replication
factor C complex, and the ISWI-type complex (Figure 4.1). The integrator complex is known
to be a critical player in small nuclear RNAs biogenesis and help RNA polymerase II in
promoter-proximal pause-release on protein coding genes. Its mis-regulation may lead to
developmental defects and disease [25,26]. The SMN complex (SMN protein along with
Ddx20 protein) contributes to splicing regulation as the assemblysome of the spliceosomal
small riboucleoproteins (snRNPs), some of which may be important to motor neuron [27,28].
The DNA replication factor C complex consist of RFC proteins that act as clamp loader during
DNA replication process [29]. The last complex, ISWI-type complex is involved in chromatin
assembly and in nucleosomes spacing and sliding to regulate transcription along with DNA
replication, recombination, and repair [30].
23
Figure 4.1 ZNF711 interaction map. ZNF711 interacts with 5 complexes; Integrator complex
(yellow), SMN complex (blue), COMPASS complex (red), DNA replication factor C
complex (pink), ISWI-type complex (green) and histone deacetylase complex (dark green).
Since ZNF711 and ZFX regulate different set of direct target genes, I also want to
investigate if their interacting partners differ as well. I compared ZNF711 interacting partners
from the analysis I did with ZFX interacting partners from previous study done by our lab.
Based on the comparison, they interact with different proteins with only 12 proteins in
common. Previous study done by our lab also show that ZFX interacts with COMPASS
complex but not with the rest of ZNF711 interacting complexes (Hsu et al, Nucleic Acids
Research, in press, 2024).
24
Figure 4.2 Overlap between ZFX and ZNF711 interacting proteins. ZFX TurboID data from
Hsu et al, Nucleic Acids Research, in press, 2024.
25
CHAPTER V. ZFX Family Member Domain Swapping
In Chapter III, I showed that ZNF711 regulates genes in common with ZFX but that it
also has a set of unique direct target genes. ZNF711 and ZFX have a high similarity in their
DNA binding domains (67% overall and 87% in zinc finger domain) with the last 3 fingers of
ZFX (which are identical in ZNF711) being sufficient for recruitment to the genome [1]. This
high degree of similarity results in a very similar genomic binding pattern. Thus, the
regulation of a unique set of target genes by ZNF711 is unlikely to be due to differences in
the DNA binding patterns. However, the N-terminal domain of ZNF711 has only a 37%
similarity with ZFX (Figure 5.1). Therefore, I hypothesized that the differential regulation of
genes by ZNF711 as compared to ZFX and ZFY may be conferred by protein-protein
interactions mediated by the N-terminus of ZNF711.
Figure 5.1 ZFX family N-terminus amino acids alignment.
In order to investigate this hypothesis I have swapped the N-terminus of ZFX with the
N-terminus of ZNF711 (Figure 5.2) to determine if the N-terminus of ZNF711 confers the
ability to regulate a set of unique genes compared to ZFX (and vice versa). Using these
mutants, I have transfected cells and performed RNA-seq to compare the target genes of the
mutants to wt ZNF711 and wt ZFX. I am currently awaiting the sequencing data for these
final experiments.
Figure 5.2 Experiment design for N-terminus swapping.
1 2
2 3 4
6 7
5
8 9
9
13
10 11 12
26
CHAPTER VI. Discussion
In this study, I aimed to characterize ZNF711 function in glioblastoma cells to begin
to understand its involvement in causing X-linked intellectual disability. Previous studies in
the lab suggest that ZNF711 can act as transcriptional activator [1,4], but ZNF711 function
in neuronal cells had not yet been performed. Therefore, I performed RNA-seq and ChIP-seq
to provide a list of genes regulated by ZNF711 in LN-229 glioblastoma cells. Based on my
RNA-seq analyses, I showed that ZNF711 upregulates a set of genes that are involved in
neurogenesis, neuron differentiation, generation of neurons, and central nervous system
development, supporting the previous suggestion that it may be involved in
neurodevelopmental processes [16-18]. Interestingly, more than 50% of the genes upregulated by ZNF711 are not regulated by ZFX or ZFY. This set of ZNF711-specific genes
is involved in axonogenesis and neural migration, further supporting ZNF711 as a regulator
of neurogenesis.
To begin to understand how the genes uniquely up-regulated by ZNF711 are selected,
I performed ChIP-seq. Because ZNF711 has 87% similarity to ZFX in their DNA binding
domains they are likely to have many binding sites in common. Based on the ChIP-seq
analysis, I found that ZFX and ZNF711 bind to essentially the same set of promoters in LN229 cells. Using this information, I then determined ZNF711 direct target genes by combining
genes that are up-regulated and genes that are bound by ZNF711 at the promoter region; this
eliminates indirect target genes that are up-regulated simply due to the effects of ZNF711 on
the cell physiology. I performed the same experiment for ZFX to compare the direct target
genes of these two family members. From the comparison of ZNF711 and ZFX direct target
genes, I can conclude that the majority of ZNF711 direct target genes are not ZFX direct target
genes in LN-229 cells. This unique set of ZNF711 direct target genes is involved in
neurogenesis, axonogenesis, and neuron projection, again supporting previous results that
ZNF711 regulates genes involved in neurogenesis which ZFX unique direct target genes are
not involved in.
To further investigate how ZNF711 can regulate different target genes than ZFX (even
though they bind to the same genomic sites) I performed TurboID and found that most of the
proteins identified using the ZNF711-Turbo protein are different than the proteins identified
using the ZFX protein. As the N-terminus is the part of the proteins that is the least conserved,
I created mutant expression constructs having swapped N-termini. I hypothesize that the unique
27
set of ZNF711 target genes will be activated by the construct having the ZNF711 N-terminus
fused to the ZF DNA binding domain. I am currently awaiting the sequencing results that will
test this hypothesis.
28
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021-02151-x
Abstract (if available)
Abstract
The ZNF711 gene is a member of the ZFX family of C2H2 zinc finger proteins. It is known that ZFX family members act as transcriptional activators and bind to CpG islands promoter. ZNF711 mutations have been found in X-linked intellectual disability and most of those mutations are frameshift mutations, which suggests ZNF711 loss of function may play a role in neurodevelopmental disorders. Other than its function as a transcriptional activator, not a lot is known about the mechanism by which ZNF711 influences transcription. In this study, I used LN-229 cells, a glioblastoma cell line which does not express ZNF711, to perform an overexpression study. RNA-seq analysis revealed that thousands of genes showed upregulated expression after transfection of a ZNF711 expression construct, with some genes involved in bone, neuron, and muscle development. Furthermore, ChIP-seq data revealed that although ZNF711 binds identically as ZFX, it can directly regulate a different set of genes. Further studies investigating the mechanisms by which ZNF711, but not ZFX, can activate specific target genes are underway.
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Pangemanan, Jemima Andrea
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Identification of target genes and protein partners of ZNF711 in glioblastoma cells
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