THE MYC-INTERACTING ZINC FINGER PROTEIN-1:
DNA AND PROTEIN INTERACTIONS IN HUMAN EMBRYONIC STEM CELLS
A Project
Presented to the faculty of the Department of Biological Sciences
California State University, Sacramento
Submitted in partial satisfaction of
the requirements for the degree of
MASTER OF ARTS
in
Biological Sciences
(Stem Cell)
by
Dana Anne Burow
SPRING
2012
THE MYC-INTERACTING ZINC FINGER PROTEIN-1:
DNA AND PROTEIN INTERACTIONS IN HUMAN EMBRYONIC STEM CELLS
A Project
by
Dana Burow
Approved by:
__________________________________, Committee Chair
Thomas Landerholm
__________________________________, Second Reader
Christine Kirvan
__________________________________, Third Reader
Jan Nolta
________________________
Date
ii
Student: Dana Burow
I certify that this student has met the requirements for format contained in the University
format manual, and that this thesis is suitable for shelving in the Library and credit is to
be awarded for the thesis.
_________________________, Graduate Coordinator
Ronald Coleman
Department of Biological Sciences
iii
_________________
Date
Abstract
of
THE MYC-INTERACTING ZINC FINGER PROTEIN-1:
DNA AND PROTEIN INTERACTIONS IN HUMAN EMBRYONIC STEM CELLS
by
Dana Burow
Stem cells can divide indefinitely and maintain their capability to differentiate
into many cell types, the key features of self-renewal and pluripotency, which explains
their importance to regenerative medicine. Embryonic stem cells (ESCs), the most highly
pluripotent of all stem cells, have the added potential of tumorigenesis. This is thought to
be driven in part through a shared gene expression program regulated by the transcription
factor, Myc.
Myc, first characterized as a potent oncogene, is shown to maintain pluripotency
and self-renewal in mouse ESCs. Myc regulation of pluripotency and self-renewal is
evident by its role in the generation of induced pluripotent stem (iPS) cells. Myc is
thought to regulate target gene expression both locally through classical mechanisms, and
globally through euchromatin remodeling. In this way, Myc can affect gene expression
on a large enough scale to reprogram differentiated cells into iPS cells. Miz-1, a
transcription factor named for its interaction with Myc, is thought to form a co-repressor
complex with Myc, silencing Miz-1 target genes including those associated with
differentiation and proliferation. Miz-1 contains BTB/POZ and 13 C2H2 zinc fingers
iv
and is thought to bind initiator sequences (INR) in the core promoters of target genes
thereby modulating their expression. Still, relatively little is known about the function of
Miz-1 as a transcriptional regulator and recent epigenetics analysis in hESCs suggest
Miz-1 binds alternative sequences, not associated with the INR of target gene promoters.
Using a Miz-1 maltose binding protein (MBP) fusion protein tag system, this
study implemented an in vitro, high-throughput DNA binding assay and Multiple em for
Motif Elicitation (MEME) analysis to identify putative Miz-1 DNA biding motifs de
novo. The consensus motifs, ATCGAT and GATTACCGA were then confirmed by
electrophoretic mobility shift analysis (EMSA) and further bioinformatics analysis
revealed motif occurrences in functionally relevant gene ontology clusters including:
transcription regulation, growth, chromatin, and developmental genes. MBP pull-down
mass spectrometry analysis also identified interesting Miz-1 protein cofactors from hESC
nuclear extracts that are associated with reported Miz-1 functions.
Miz-1 DNA and protein interactions highlighted in this study confirm its role as a
master transcriptional regulator, cofactor and antagonist of Myc in hESCs. Though, the
findings also underline the importance of further characterization of pluripotency and
self-renewal in hESCs so that potential therapies may be safe and effective.
___________________________, Committee Chair
Thomas Landerholm
___________________________
Date
v
ACKNOWLEDGEMENTS
I would like to acknowledge the support and dedication of the faculty mentors in the
Department of Biological Sciences at Sacramento State University. I would also like to
recognize the Knoepfler lab at UC Davis for their guidance and the opportunity to be a
part of the lab for the past year. Thanks to the Segal lab at the UC Davis Genome Center
for their advice and collaboration on Bind-n-Seq. Finally, thanks goes out to my
wonderful family and friends who’s love and support has helped me accomplish my
goals.
vi
TABLE OF CONTENTS
Page
Acknowledgements ............................................................................................................ vi
List of Tables ................................................................................................................... viii
List of Figures .................................................................................................................... ix
INTRODUCTION ...............................................................................................................1
METHODS ..........................................................................................................................7
Cloning, Recombinant Protein Expression and Purification7
Bind-n-Seq: in vitro DNA Binding Assay and de novo Motif Finding8
Electrophoretic Mobility Shift Assay9
Bioinformatics Analysis of Motifs Identified by Bind-n-Seq.9
MBP-Miz-1 Pull-down Mass Spectrometry Analysis9
RESULTS ..........................................................................................................................11
Miz-1 Expression and Purification by MBP11
De novo Motif Finding by Bind-n-Seq11
Bioinformatics Analysis of Motifs Identified by Bind-n-Seq13
EMSA Supports Miz-1 Binding ATCGAT and GATTACCGA16
MBP-Miz-1 Pull-down Mass Spectrometry Analysis16
DISCUSSION ....................................................................................................................25
Literature Cited ..................................................................................................................30
vii
LIST OF TABLES
Table
Page
1. Full-length Miz-1 Motif Consensus Sequences Identified by Bind-n-Seq14
2. Zinc finger Miz-1 Motif Consensus Sequences Identified by Bind-n-Seq15
3. Putative Miz-1 DNA Binding Motifs17
4. Gene Ontology Clusters Identified by DAVID Analysis18
5. MBP-Miz-1 Mass Spectrometry Analysis by Scaffold 323
viii
LIST OF FIGURES
Figure
Page
1. SDS-PAGE Detection of Protein from Purification by MBP. ......................................12
2. DAVID Analysis of Motif-Containing Miz-1 Bound Genes. .......................................19
3. EMSA Indicates Miz-1 Binding Motif-containing Oligonucleotide Probes20
4. MBP-Miz-1 Pull-down of c-Myc22
5. Current Model Proposed for Miz-1 Target Gene Regulation24
ix
1
INTRODUCTION
Stem cells are the focus of regenerative medicine and may hold the key to
unlocking cures for diseases ranging from neurological disorders to cancer and HIV
infection. In the early 1980s, embryonic stem cells (ESCs) were first isolated from
mouse embryos and shown to grow indefinitely while maintaining pluripotency, the
ability to differentiate into many different cell types (Evans, 1981). Since then there have
been significant gains in our understanding of these key features of stem cells: selfrenewal and pluripotency. A complex network of transcription factors mediate stem cell
differentiation. In 2006, scientists were able to reverse this process and generate induced
pluripotent stem cells, termed iPS cells, from differentiated fibroblast epithelial cells in
mice (Takahashi & Yamanaka, 2006) and later in humans (Takahashi et al., 2007). The
induction of pluripotent stem cells (iPS cells) demonstrated by Takahashi and Yamanaka
requires the forced expression of a set of transcription factors, including the protooncogene, c-Myc (Takahashi & Yamanaka, 2006).
Stem and cancer cells have many of the same properties, postulated to be driven
in part through a shared gene expression program regulated by the transcription factor, cMyc. Myc was first characterized through its association as a potent oncogene, though
the mechanisms of oncogenic activation were initially vague (Eisenman, 2001).
Members of the Myc family of transcription factors are well documented as being
deregulated in many cancers. This deregulation of Myc through translocation and
duplication events results in increased Myc expression and allows for oncogenic
activation (Meyer, 2008), whereas the normal, high levels of endogenous expression in
2
ESCs plays a key role in mediating pluripotency and self-renewal (N. V. Varlakhanova,
Cotterman, R.F., deVries, W.N., Morgan, J., Donahue, L.R., Murray, S., Knowles, B.B.,
Knoepfler, P.S., 2010). Pluripotency and self-renewal properties, the foundation of stem
cells, are implicated in their cancerous counterpart, teratoma, as early as 1960 (Pierce,
1960). Teratoma tumors contain all three germ layers and continue to be challenging to
overcome with current cancer therapies. Despite the promise of stem cell therapies, there
still remains the problem of ESC-related tumorigenesis.
Myc is a key player in the generation of iPS cells and though alternate routes to
iPS cell formation have been presented in recent years, the endogenous role of c-Myc is
implicated in the shift to pluripotency and self-renewal in each of these cases. A 2008
study by Nakagawa was able to generate iPS cells without the forced expression of cMyc (Nakagawa M., 2008). Though they generally have significantly reduced
reprogramming efficiency, it is suggested that endogenous c-Myc expression in these iPS
cells is able to mediate the shift to pluripotency and self-renewal through its activation by
the other exogenous factors used in reprogramming, including Oct4, Sox2, and Klf4
(Stadtfeld, 2010). This notion is supported by a study demonstrating that neural stem
cells can be converted to iPS cells through exogenous Oct4 expression alone since the
other defined factors are endogenously expressed in neural stem cells (Kim, 2009). In
2010, Stadtfeld have also linked the original set of iPS cell forming factors to another
proven set (Oct4, Sox2, Nanog, and Lin28) through the deregulation of c-Myc by Lin28
(Stadtfeld, 2010). The importance of Myc in the mediation of pluripotency and selfrenewal in stem cells cannot be overlooked.
3
The Myc protein family is a group of transcription factors containing the basic
helix loop helix leucine zipper (bHLHZ) domain. Myc, like other members of the
bHLHZ superfamily of transcription factors, can heterodimerize with another bHLHZ
protein, Max, and bind the enhancer box (E-box) sequence, CACGTG, activating target
genes (Chaudhary, 1999). Myc can also have repressive action on gene expression
through association with and inhibition of the activating function of the Miz-1
transcription factor (Peukert, 1997). In this way, Myc modulates gene expression to in
turn regulate diverse cellular processes including: metabolism, cell cycle, differentiation,
apoptosis, senescence, and DNA replication (Grandori, 2000).
In more recent studies, Myc is shown to regulate global euchromatin structure and
is tightly associated with certain histone modifications (Knoepfler et al., 2006). Myc and
the cofactor transformation/transcription domain-associated protein (TRRAP) are known
to recruit histone acetyl transferases (HAT) (McMahon, 2000). HATs are wellcharacterized proteins that function in the acetylation of amino-terminal lysine residues
of histone proteins, especially those located near transcriptional start sites (Knoepfler,
2007). Myc is shown to regulate euchromatin on a global scale and to some extent
independent of its role as a classical transcription factor (Cotterman et al., 2008). In a
recent review, a model is proposed that defines Myc’s role in the self-renewal of stem
cells and tumorigenesis through both local transcriptional activation, by classical
mechanisms, and global euchromatin structure, whereby overexpression results in
tumorigenesis (Knoepfler, 2007). In iPS cell formation, c-Myc is hypothesized to allow
Oct4 and Sox2, two other defined factors in iPS cell formation, to bind to genomic targets
4
through its mediation of global histone acetylation (Takahashi & Yamanaka, 2006).
While Myc is most well known for its transcriptional activating function, interest in its
repressive functions is growing and is implicated in its regulation of stem cell
pluripotency and self-renewal.
Miz-1, named for its interaction with Myc (Myc-interacting zinc finger protein-1),
was first characterized in 1997 and found to function strongly in growth arrest (Peukert,
1997). Miz-1 is a BTB/POZ (BR-C, ttk and bab/pox virus and zinc-finger) domaincontaining transcription factor that is thought to directly bind core promoter initiator
(INR) sequences and recruit the co-activator protein, p300, in order to activate target
genes, such as negative regulators of cell cycle control and growth and positive regulators
of differentiation (Kime & Wright, 2003; Seoane et al., 2001; Staller, 2001; M. Wanzel,
Herold, S., Eilers, M., 2003; Wu, 2003). Myc can bind Miz-1 through its HLH domain
and is thought to repress Miz-1 gene activation through competition with p300 (Staller,
2001). Recent epigenetic studies also support the hypothesis that the mechanism by
which Myc represses expression of differentiation genes, thereby maintaining
pluripotency and self-renewal, is related to Miz-1. Co-immunoprecipitation using anti-cMyc antibody suggests recruitment of Histone Deacetylase 2 (HDAC2) and DNA
(cytosine-5)-methyltransferase 3a (DNMT3a) to form a repressor complex (Varlakhanova
unpublished data). The DNA CpG methyltransferase, DNMT3a, is known to interact
with Myc by means of Miz-1, forming a corepressor complex that functions at the
promoter of target genes like p21Cip1, a cyclin-dependent kinase inhibitor (Brenner et
al., 2005) and Mad4, another transcriptional regulator of proliferation and differentiation
5
(Kime & Wright, 2003). This confirms that the mechanism by which Myc represses
differentiation gene expression in hESCs is through Miz-1.
Biological and epigenetic characterization of Miz-1 in hESCs demonstrates that
Myc and Miz-1 function coordinately in the regulation of pluripotency and self-renewal
through their repression of differentiation associated genes. Myc and Miz-1 also display
antagonistic roles in the regulation of genes important to stem cell pluripotency, selfrenewal and differentiation. Genome-wide chromatin immunoprecipitation-microarray
(ChIP-chip) analysis demonstrates that Myc also occupies nearly 30% of Miz-1 targets,
and that these are predominantly differentiation associated genes, including many
members of the Hox gene family (N. V. Varlakhanova, et al, 2011). Additionally, parallel
ChIP-chip analysis of activating euchromatin marks, including acetylation of lysine 9 on
histone 3 (AcH3K9) and trimethylation of lysine 4 on histone 3 (H3K4me3), and Miz-1
DNA binding show a significant overlap between genes involved in cellular metabolism
and growth. Conversely, genes not associated with active euchromatin marks and those
associated with the inactivation mark trimethylation of lysine 27 on histone 3
(H3K27me3) were predominately differentiation associated genes, including many Hox
genes (N. V. Varlakhanova, et al, 2011). Myc knockdown in hESCs results in an
upregulation of differentiation associated genes and a downregualtion of pluripotency and
growth associated genes, while conversely Miz-1 knockdown in hESCs results in a
downregulation of differentiation associated genes and an upregualtion pluripotency and
growth associated genes (N. V. Varlakhanova, et al, 2011). Interestingly and contrary to
the current literature (Kime & Wright, 2003; Seoane, et al., 2001; Staller, 2001; M.
6
Wanzel, Herold, S., Eilers, M., 2003; Wu, 2003), which describes Miz-1 binding
localized to core promoter INR sequences, the recent work of Varlakhanova demonstrates
that the global distribution of Miz-1 binding is predominantly localized to regions more
than 1000 bases upstream of the transcriptional start sites of target genes (N. V.
Varlakhanova, et al, 2011). It is important to note that unlike the Varlakhanova study,
previous studies only analyzed Miz-1 regulation of few candidate genes and did not
assess global genomic binding. Cis-Regulatory Element Annotation System (CEAS)
analysis (Ji X, 2006) of Miz-1 ChIP-chip data from the Varlakhanova study failed to
identify potential DNA binding motifs for Miz-1, however, Miz-1 INR sequenceindependent DNA binding is of clear significant to the global function of Miz-1. INRindependent Miz-1 binding represents more than half of total Miz-1 genomic binding
sites, and identification of novel Miz-1 DNA binding motifs is central to furthering our
understanding of this important Myc antagonist.
Understanding the interworking of the complex Myc network of transcription
factors, including Miz-1, that mediates stem cell self-renewal, pluripotency and
differentiation will help to further our knowledge of both stem and cancer cell biology.
Teasing out the subtle differences between Myc-mediated pluripotency and self-renewal
in stem cells and that in cancer cells is of vital importance in furthering stem cell based
therapies so that they are both safe and effective, and may also lead to novel cancer
treatments. The present work identifies putative Miz-1 DNA binding motifs and potential
protein cofactors and serves as a platform for further investigation into specific Miz-1
DNA and protein interactions in hESCs.
7
METHODS
Cloning, Recombinant Protein Expression and Purification. A plasmid vector coding for
a N-termal fusion of E. coli maltose binding protein (MBP) to full-length human Miz-1
was cloned by restriction ligation using pMAL-c5G (New England Biolabs Ipswich, MA)
and Miz-1 cDNA generated from H9 hESC mRNA. The sequence encoding the 13 C2H2
zinc fingers (nucleotides 805-2379) of Miz-1 cDNA were cloned by Gateway (Invitrogen
Life Technologies, Carlsbad, CA) into a plasmid vector coding for an N-terminal GSTMBP tag (Segal Lab, UC Davis Genome Center, Davis, CA). Transformed E. coli
BL21STAR (Invitrogen Life Technologies) were grown at 37°C and 225rpm. Expression
of the MBP-hMiz-1 fusion constructs was induced at 2.5 hours by Isopropyl β-D-1thiogalactopyranoside (IPTG). Cells were harvested 5 hours post-induction by
centrifugation (3500rpm, 20 min, 4°C) and lysed in Zinc Buffer A [ZBA; 10mM Tris (pH
7.5), 90mM KCl, 1mM MgCl2, 90μM ZnCl2, 5mM DTT] by sonication (6 rounds: 30 sec
(high), 30 sec rest). Protein lysate was isolated by centrifugation (20,000rpm, 30 min,
4°C) then incubated at 4°C with amylose linked agarose beads (New England Biolabs)
for 20 min. Protein lysate was cleared by gravity flow and beads subsequently washed
with 10 column volumes of ZBA. MBP-hMiz was eluted in 3 mL ZBA and 10 mM
maltose then dialyzed in 2 L ZBA overnight to deplete free maltose. MBP-hMiz-1 protein
was concentrated using Amicon Ultra Filter units (Millipore, Billerica, MA). Purity and
quantity of the MBP-hMiz fusion protein was assessed by SDS-PAGE and Bradford
Assay (Thermo Fisher Scientific, Waltham, MA).
8
Bind-n-Seq in vitro DNA Binding Assay and de novo Motif Finding. MBP-hMiz-1
proteins at various concentrations (Tables 1-2) were bound to random oligonucleotides
with barcodes in Bind-n-Seq (BnS) binding buffer [BnSBB; 0.12μg/μL Herring Sperm
DNA, 100μM ZnCl2, 5mM DTT, 5% BSA] for 30 minutes with agitation at room temp.
Binding reactions were then washed 6X for 10 min each with BnS wash buffer [BnSWB;
10mM Tris (pH 8.5), 100μM ZnCl2, 1mM MgCl2, 5mM DTT] under various KCl salt
concentrations. Bound oligonucleotides were eluted for 10 min in EB buffer (Qiagen,
Hilden, Germany) and 10mM maltose. Quantitative PCR was performed by the Opticon
Monitor system and SYBR green detection (Program: 94°C for 4 min initial denaturation,
26 cycles of 94°C for 30 sec, 63°C for 30 sec and 72°C for 1 min) to determine optimal
amplification cycles for each set of oligonucleotides. Oligonucleotides were amplified
using iProof DNA Polymerase (Bio Rad, Hercules, CA), cleaned by PCR Purification Kit
(Qiagen), quantified by NanoDrop (Thermo Fisher Scientific), and 100ng of each
sampled pooled for sequencing. Amplified, pooled oligonucleotides with barcodes were
sequenced on the MiSeq (Illumina, San Diego, CA) at the UCD Genome Center Core
facility and reads sorted and filtered for quality by the MiSeq platform software.
Bioinformatics were performed by the Segal Lab at the UC Davis Genome center. Sorted,
filtered reads were analyzed in randomly sampled clusters of 10,000 reads by MEME.
Intermediate motifs were matched back to the original dataset and subsequent rounds of
MEME performed to generate the most enriched motifs for each BnS condition (table 1).
De novo motif finding was performed at the UC Davis Genome Center by the Segal lab.
9
Electrophoretic Mobility Shift Assay. EMSA was performed by binding MBP-hMiz to
synthesized and hybridized probes (5’ CAAAAGTGCGATCGATGCTGCGTGGT 3’
and 5’ CAAAAGTGCGGATTACCGAGCTGCGTGGT 3’) and poly(deoxyinosinicdeoxycytidylic) acid nonspecific competitor in ZBA for 20 min at room temperature then
visualized on Novex 6% DNA Retardation polyacrylamide gels (Invitrogen Life
Technologies) by SYBR green DNA stain and SYPRO ruby protein stain (Invitrogen Life
Technologies) at 300nm UV transillumination.
Bioinformatics Analysis of Motifs Identified by Bind-n-Seq. Gapped Local Alignment of
Motifs (GLAM2) (Frith, 2008) analysis was performed on motifs identified by BnS for
each full-length and zinc-finger Miz-1 constructs. Then, GLAM2 results were searched
against Miz-1 ChIP-chip sequence data (N. V. Varlakhanova, et al, 2011) for motif
occurrences by Find Individual Motif Occurrences (FIMO) (Grant, 2011) analysis with a
p-value cut-off of 0.0001. The subsequent motif-containing gene list was analyzed using
the Database for Annotation, Visualization and Integrated Discovery (DAVID) (Huang,
2009a, 2009b) with a p-value cut-off of 0.001 for identification of enriched gene
ontology clusters.
MBP-Pull down Mass Spectrometry Analysis. MBP-hMiz-1 was bound to amylose linked
agarose beads (New England Biolabs) and in vitro transcribed and translated c-Myc (TNT
Translation/Transcription Kit, Promega) in ZBA for 20 minutes then washed with 10
volumes ZBA, eluted with LDS sample loading buffer (Invitrogen Life Technologies)
and detected by Western Blot against human c-Myc. MBP-hMiz-1 was bound to amylose
linked agarose beads (New England Biolabs) and H9 human embryonic stem cell nuclear
10
extracts in ZBA for 20 minutes then washed with 10 volumes ZBA and eluted in ½X
ZBA and 10mM maltose. Samples were submitted to the UC Davis Proteomics Core for
Mass Spectrometry analysis. Data was analyzed by Scaffold 3 (Proteome Software, Inc.,
Portland, Oregon).
11
RESULTS
Miz-1 Expression and Purification by MBP. Induction of recombinant MBP-hMiz-1
protein expression by IPTG under the Tac promoter in E. coli is an efficient and effective
means of robust protein production in vitro. The MBP tag allows for efficient purification
by amylose-linked agarose beads and elution with maltose. SDS-PAGE and Bradford
Assay confirm MBP-Miz-1 purity and concentrations of greater than 2 μM, important for
subsequent implementation in the in vitro DNA binding assay.
De Novo Motif Finding by Bind-n-Seq. Bind-n-Seq is a high-throughput, in vitro DNA
biding assay that allows for the systematic and rapid detection of DNA binding motifs in
parallel. While other protein-DNA binding approaches have been identified and widely
implemented, including ChIP-chip, ChIP-Seq, protein-binding microarrays (PBM),
cyclical amplification and selection of targets (CAST), systematic evolution of ligands by
exponential enrichment (SELEX) and even one and two-hybrid systems, Bind-n-Seq has
distinct advantages over these other analyses. In vivo approaches including ChIP
technologies, and one and two-hybrid systems are powerful but experimentally complex
and limited in their application, by for example, the availability of ChIP quality
antibodies. In vitro analyses including, PBM, CAST and SELEX are limited in scope by
the size of the DNA library available for analysis and/or the labor required to successfully
execute the technique. Bind-n-Seq, through its simple design and implementation of
next-generation sequencing technology, overcomes challenges of experimental
complexity and scope. Short, randomly generated
12
Figure 1. SDS-PAGE Detection of Protein from Purification by MBP. Significant
amounts of recombinant protein was obtained by purification by MBP, while little
carryover of bacterial proteins is evident.
13
oligonucleotides (21bp binding region) with barcodes are bound to MBP-protein
constructs and amylose-linked agarose beads, washed and eluted with maltose and
identified by massively parallel sequencing to generate approximately 100,000 reads per
sample, while maintaining the ability to run up to 64 samples in parallel (Zykovich,
2009). In this study, MBP fused to Full-length Miz-1 and MBP fused to Miz-1 zinc
fingers (residues 269-793) constructs were each analyzed by Bind-n-Seq across 5
different binding buffer and wash buffer conditions. The 5 most highly enriched
consensus sequence motifs identified for each the full-length and zinc-finger construct
and condition are presented in Tables 1 and 2 respectively. All motifs had significant
enrichment of greater than 5-fold and up to 25-fold over background. Interestingly,
conditions of higher stringency (higher salt concentration) did not see the lowest
enrichment, rather, conditions of low protein concentration produces the lowest
enrichment values. Further Gapped Local Alignment of Motifs (GLAM2) analysis (Frith,
2008) was performed on each set of consensus sequences identified for the respective
protein constructs and the results are presented in Table 3. The motifs GATTACCGA and
ATCGAT were identified as the most significant matches for the full-length and zinc
finger constructs respectively.
Bioinformatics Analysis of Motifs Identified by Bind-n-Seq. Motifs retrieved from BnS
were analyzed by GLAM2 to generate a list of principal motifs for each full-length and
zinc finger constructs. The two top-scoring motifs are shown in Table 3. The MEMEformatted motifs from GLAM2 were subsequently used to search Miz-1 ChIP-chip data
by Find Individual Motif Occurrences (FIMO). FIMO analysis revealed 3052 and 2411
14
Table 1. Full-length Miz-1 Motif Consensus Sequences Identified by Bind-n-Seq. The five
most highly enriched consensus sequences for each binding condition of the BnS assay
for the full-length Miz-1 construct is shown along with the enrichment score.
Binding Condition
50nM [protein]
1mM [salt]
50nM [protein]
50mM [salt]
50nM [protein]
100mM [salt]
5nM [protein]
100mM [salt]
350nM [protein]
100mM [salt]
Consensus Sequence
ATAATCGAT
GATTACCGA
CGATTAATCG
ATTACCGATC
AATCGATCTC
ATCGGTAATC
GATTACCGA
ATCGGCAATC
ATCGGTATTC
GGCTTACCGA
ATCGGTAATC
GATTACCGA
GGATTACCGA
AGATTACCGA
GATTGCCGAA
GATTACCGA
AGATTGCCGA
ATCGGTAATC
GATTGCCGA
ATCGATTAA
GATTACCGA
ATCGATTAC
GATTGCCGA
AATCGATTA
TAATCGATTA
Fold-Enrichment
17.417
16.667
14.5
14.312
13.6
20.867
19.31
17.5
16.952
15.529
14.125
13.478
12.8
11.8
11.667
9.808
9.6
9.375
6.333
5.871
11.162
11.029
11
10.514
9.474
15
Table 2. Zinc finger Miz-1 Motif Consensus Sequences Identified by Bind-n-Seq. The five
most highly enriched consensus sequences for each binding condition of the Bind-n-Seq
assay for the zinc finger Miz-1 construct is shown along with the enrichment score.
Binding Condition
50nM [protein]
1mM [salt]
50nM [protein]
50mM [salt]
50nM [protein]
100mM [salt]
5nM [protein]
100mM [salt]
120nM [protein]
100mM [salt]
Consensus Sequence
ATCGATTAAT
TAATCGATTA
ATAATCGATC
ATCGGTAATC
ATCGATTAA
AAAAATCGAT
ATCGGTAATC
ATCGGCAATC
ATCGATTAAA
ATCGATTAC
AACATCGAT
GATTGCCGA
AGTAATCGAT
CATCGATCG
ATCGATCGAT
ATCGATCGAT
ATCGATCGA
GATTGCCGA
ATCGGTACTC
ATCGGTACTC
ATCGGTATC
ATCGATTG
AATCATCGAT
GATTGCCGA
GATTACCGA
Fold-Enrichment
25.053
23.71
19.611
16.909
16.81
26.2
17.267
16.053
16
14.2
16.421
16.31
15.053
14.864
13.333
19.6
16
14.227
12.2
11
10.75
9.619
8.833
7.781
6.892
16
motif occurrences with a p-value less than 0.0001 respectively for the full-length and zinc
finger constructs. Database for Annotation, Visualization and Integrated Discovery
(DAVID) analysis of Miz-1 bound genes containing motif occurrences identified by
FIMO shows enriched clusters of functionally related genes. Significant gene ontology
clusters are outlined for both full-length and zinc finger constructs in Table 4 and
includes genes involved in cell growth, differentiation, including Hox genes, and other
developmental associated genes, regulation of transcription and DNA binding and
chromatin structure including acetylation.
EMSA Supports Miz-1 Binding ATCGAT and GATTACCGA. Electrophoretic mobility
shift analysis is a long-standing, robust method of confirming protein-DNA interaction
and serves as a means of validating the motifs ATCGAT and GATTACCGA affinity for
the human Miz-1 protein. Poly-dI/dC non-specific inhibitor oligonucleotide was added to
the binding reactions and results were analyzed by polyacrylamide DNA retardation gel
electrophoresis. SYBR green DNA stain revels that ATCGAT and GATTACCGA
containing probes were bound and shifted in the gel in the presence of full-length Miz-1
protein, while reactions not containing Miz-1 protein ran unaltered.
MBP-Miz-1 Pull-down Mass Spectrometry Analysis. MBP-Miz-1 pull-down experiments
were performed in order to screen for novel Miz-1 cofactors in hESCs. To assess the
possibility of performing a pull-down experiment with MBP-Miz-1 constructs, MBPMiz-1 proteins were incubated with a known interactor, c-Myc, and analyzed by western
blot (Figure 3). c-Myc is shown to bind MBP-Miz-1 full-length construct, but does not
17
Table 3. Putative Miz-1 DNA Binding Motifs. Consensus seed motifs from Bind-n-Seq
were analyzed by GLAM2 and the highest scoring motifs for the full-length and zincfinger constructs are shown.
Construct
Fulllength
Zinc
fingers
Consensus
GATTACCGA
Motif
G(A/C)(T/A)(T/A)(A/G)(C/T)CGA
Score
198.362
ATCGAT
ATCG(A/G)(T/C)
188.623
18
Table 4. Gene Ontology Clusters Identified by DAVID Analysis. Gene lists generated by
FIMO for full-length and zinc finger constructs respectively were submitted for DAVID
analysis, the most significant gene ontology clusters are shown (p ≤ 0.001) along with the
percentage of genes that comprise the cluster and the enrichment score.
Gene Ontology Cluster
Positive regulation of cell
proliferation
Miz-1
Construct
% Total
Genes in
GO
category
P-Value
FoldEnrich
ment
FL
8
2.50x10-05
3.5
-04
1.6
Regulation of transcription
FL
23
4.10x10
Regulation of cell proliferation
FL
9.9
7.80x10-04
2.3
4.2
8.90x10
-04
4.5
1.30x10
-04
3.4
2.30x10
-04
1.9
3.20x10
-04
1.7
3.80x10
-04
5.1
4.80x10
-04
2.7
-07
1.7
Embryonic organ development
Transcription cofactor activity
Transcription regulator activity
DNA binding
Transcription corepressor activity
Transcription factor binding
FL
FL
FL
FL
FL
FL
7
16.4
22.1
4.2
8
Nucleus
FL
36.2
7.80x10
DNA-binding
FL
18.8
2.90x10-05
Homeobox
Phosphoprotein
Acetylation
FL
FL
FL
5.6
48.8
22.5
2
5.70x10
-05
4.7
1.30x10
-04
1.3
2.00x10
-04
1.7
-04
1.8
Transcription regulation
FL
17.8
7.00x10
Chromatin
ZF
0.5
4.20x10-04
-04
2.3
5
Transcription factor activity
ZF
1.2
6.00x10
Transcription regulator activity
ZF
1.6
7.70x10-04
1.9
4.8
3.60x10
-05
1.4
1.80x10
-04
1.6
9.30x10
-04
1.7
Phosphoprotein
Nucleus
Acetylation
ZF
ZF
ZF
3.2
2.1
19
DAVID Analysis of Motif-Containing Miz-1 Bound Genes
chromatin
transcription regulation
acetylation
phosphoprotein
Homeobox
DNA-binding
nucleus
transcription factor binding
Miz-1 ZF
transcription corepressor activity
Miz-1 FL
DNA binding
transcription regulator activity
transcription cofactor activity
embryonic organ development
regulation of cell proliferation
regulation of transcription
positive regulation of cell
proliferation
0
1
2
3
4
Fold-Enrichment
5
6
Figure 2: DAVID Analysis of Motif-containing Miz-1 Bound Genes. DAVID analysis
gene ontology clusters of genes both bound by Miz-1 and containing motifs.
GATTACCGA
Miz FL +
GATTACCGA
ATCGAT
Miz FL +ATCGAT
STD
20
100bp
Figure 3. EMSA Indicates Miz-1 Binding Motif-containing Oligonucleotide Probes.
Oligonucleotide probes and polydI/dC nonspecific competitor nucleotide DNA was
incubated with or without full-length Miz-1 protein and subsequently separated by nondenaturing polyacrylamide get electrophoresis and DNA visualized by SYBR green,
300nm UV transillumination. Protein-containing lanes show a retardation of the short
oligonucleotides containing the motifs ATCGAT and GATTACCGA.
21
bind the MBP-Miz-1 zinc-finger construct. Although the zinc-finger construct lacks the
BTB/POZ domain, it does contain the reported c-Myc interaction domain (Sakamuro &
Prendergast, 1999). Stringent washing conditions with high ionic strength buffer (2 M
NaCl) did not disrupt the interaction between MBP-Miz-1 and c-Myc, however,
treatment with detergent abolished any interaction. Less stringent washing with ZBA
yielded background bands in the bead-only control. The MPB-Miz-1 pull down mass
spectrometry analysis was performed with an MBP only control and the results are
summarized in Table 5. Significant matches were identified in the full-length Miz-1
construct, while the MBP only and Miz-1 zinc finger constructs yielded far fewer
peptides, all present in the MBP only control. Miz-1 recombinant protein was readily
detected and comprised the most abundant protein in the samples, as expected. Other
ribosomal and collagen associated proteins identified as top hits in the scaffold 3 analysis
can be disregarded as background from the hESC nuclear extracts and contamination
respectively. However, nucleophosmin (NPM) and developmental pluripotencyassociated protein 4 (DPPA4) are of interest as putative Miz-1 cofactors, but further
validation by western blot is needed.
22
Figure 4. MBP-Miz-1 Pull-down of c-Myc. Western blot of MBP-Miz-1 Pull-down with
c-Myc (72 kDa) input under increasingly stringent washing conditions reveals specific
binding under high salt. Under 2M NaCl salt washing condition c-Myc appears to
maintain interaction with Miz-1. NP-40 detergent abolishes any interaction and ZBA
wash buffer yields higher background.
23
Table 5. MBP-Miz-1 Mass Spectrometry Analysis by Scaffold 3. Percent probability of
proteins identified by MS is given for targets of interest. Two putative cofactors were
identified by MS. DPPA4 is of interest for its role in mediating pluripotency in mouse
embryonic stem cells and NPM is another target of interest with important known
transcriptional regulatory functions. Further validation by direct pull-down and western
blot is needed to confirm these interactions.
Protein
Coverage
Miz-1
NPM
DPPA4
9%
8%
8%
Protein
Identification
Probability
100%
100%
50%
24
Dppa4
Myc
Max
p300
Dnmt3a
HDAC
Miz-1
NPM
Miz-1
INR
ATCGAT
GATTACCGA
Growth
Cell cycle
Chromatin structure
Acetylation
Transcriptional control
Development
Hox
Figure 5. Current Model Proposed for Miz-1 Target Gene Regulation. Miz-1 gene
repression involves the recruitment of protein cofactors including Myc, HDAC and
Dnmt3A, while Miz-1 gene activation involves co-activators p300 and NPM. Adapted
from Varlakhanova et al, 2011.
25
DISCUSSION
Essential to the success of regenerative medicine therapies is our basic scientific
understanding of hESCs. The complex regulatory network that governs pluripotency and
self-renewal, distinguishing characteristics of hESCs, is an important topic of basic stem
cell research. c-Myc, the well-studied oncogene, is also a key player in the maintenance
of pluripotency and self-renewal in stem cells (N. V. Varlakhanova, Cotterman, R.F.,
deVries, W.N., Morgan, J., Donahue, L.R., Murray, S., Knowles, B.B., Knoepfler, P.S.,
2010). Myc regulation of pluripotency and self-renewal is evidenced by its function in the
generation of iPS cells (Takahashi, et al., 2007; Takahashi & Yamanaka, 2006). Myc
regulates target gene expression both locally by classical mechanisms, and globally
through euchromatin remodeling (Knoepfler, et al., 2006). In this way, Myc can affect
gene expression on a large enough scale to reprogram differentiated cells into iPS cells.
Miz-1, named for its interaction with Myc, is known to bind initiator sequences in the
promoters of target gene core promoters thereby modulating their expression (Kime &
Wright, 2003; Peukert, 1997; Seoane, Le, & Massague, 2002). In the current model
(Figure 5), Miz-1 is thought to form a co-repressor complex with Myc, silencing Miz-1
target genes (Peukert, 1997), and alternately, Miz-1 forms a co-activating complex with
p300 and NPM, activating target genes (M. Wanzel, Herold, S., Eilers, M., 2003; M.
Wanzel, Russ, A.C., Kleine-Kohlbrecher, D., Colombo, E., Pelicci, P.G., Eilers, M.,
2008). Still, relatively little is known about the function of Miz-1 as a transcriptional
regulator and recent gene expression and epigenetic analysis in human ESCs suggest
26
Miz-1 binds alternative sequences, not associated with the initiator sequences of target
gene promoters (N. V. Varlakhanova, et al, 2011).
CEAS analysis of Miz-1 ChIP-chip data failed to identify putative motifs for Miz1 DNA binding. While not surprising given the size and complexity of the data, finding
motifs is still vital to the study of Miz-1 in hESCs. An in vitro approach to finding DNA
binding motifs is an efficient and comprehensive alternative way to examine Miz-1-DNA
binding. The production of MBP-Miz-1 fusion protein allows for flexible analysis using
both protein-DNA and protein-protein biochemical assays. Bind-n-Seq overcomes
problems associated with other motif-finding approaches including limitations on in vivo
detection and sensitivity, and time and labor-intensive in vitro approaches. Instead, Bindn-Seq employs massively parallel sequencing and a MBP purification scheme to renovate
de novo motif finding into a high-throughput in vitro assay. Identification of novel human
Miz-1 DNA binding motifs by Bind-n-Seq assay (Zykovich, 2009) has revealed highly
enriched DNA binding motifs for Miz-1 relating to both full-length and zinc-finger
containing protein constructs. Currently, the Bind-n-Seq assay is optimized for the study
of zinc-finger containing proteins and even then, results highly depend on the specific
properties of each protein fusion construct. Additionally, Bind-n-Seq analysis is likely to
only identify the most highly enriched DNA motifs, while there may be several motifs for
a given protein based on its structural conformation. Because of these limitations, Bind-nSeq serves best as a stepping off point for further analysis of important protein-DNA
interactions, and it is important to incorporate other analyses to corroborate Bind-n-Seq
findings. The consensus motifs identified for Miz-1, ATCGAT and GATTACCGA, were
27
both highly enriched over background and were further analyzed for their ability to bind
Miz-1 protein in vitro and their presence near Miz-1 bound genes, determined from
Varlakhanova (N. V. Varlakhanova, et al, 2011) hESC ChIP-chip data. EMSA analysis
confirmed Miz-1 binding ATCGAT and GATTACCGA containing DNA probes in the
presence of poly-dI/dC non-specific competitor oligonucleotide. Probing Miz-1 ChIPchip data by FIMO reveled an abundance of motif-containing sequences related to the
Miz-1 ChIP-chip peaks. In total, over 2000 and 3000 sequences contained significant
matches to the motifs identified by GLAM2 respectively. Of these thousands of hits,
many are sequences corresponding to genes that were present in replicate in the ChIPchip data or belong to genes that have yet to be fully annotated. Respectively, 166 and
212 well-annotated genes identified by FIMO were submitted for DAVID analysis for the
zinc finger and full length constructs. Significant, functionally related, gene ontology
clusters had overlap between both constructs including: acetylation, phosphoprotein, and
transcriptional regulators. The full-length construct revealed more significant matches in
both FIMO and DAVID analysis, yet it is important to note that there is significant
overlap between both constructs. Across both constructs, the most highly enriched gene
ontology clusters from DAVID analysis include: chromatin, homeobox, transcription
corepressor, embryonic organ development, and cell proliferation associated genes. These
results are in agreement with known functional roles of Miz-1 in hESC and support the
hypothesis that Myc maintains hESC pluripotency and self-renewal in part through a corepression program with Miz-1.
28
Additionally, the motif, ATCGAT, is of particular interest because of its
similarity to known transcription factor binding motifs, including c-Myc. ATCGAT is a
palindromic sequence like that of the Myc E-box, CACGTG. However, ATCGAT has
not yet been previously associated with other human proteins in the literature.
Palindromic motifs in DNA are common and not unique to just transcription factor
binding motifs. They can be highly conserved across many species and important for
mobile, repetitive DNA elements like transposons. These shared features of palindromic
DNA motifs may imply an important function and significance for the DNA-binding
motifs of master transcriptional regulators like Myc and Miz-1.
The MBP-Miz-1 fusion protein construct also allowed for detection of novel Miz1 protein interactors by MBP pull-down mass spectrometry analysis. Pull-down of hESC
nuclear extracts followed by mass spectrometry identification of proteins revealed a
couple candidate cofactors for Miz-1 including DPPA4 and NPM. DPPA4 is not well
studied, however, it has been shown to be important to mESC pluripotency and selfrenewal, whereby overexpression resulted in cell proliferation and inhibition of
differentiation (Masaki, 2007). Alternately, NPM is a better-characterized protein that
functions in diverse cellular processes from histone assembly and cell proliferation to
regulation of important tumor suppressors like p53 and ARF (Okuwaki, 2008;
Swaminathan V., 2005; Wang H.F., 2011). The coordinate role of Miz-1 and NPM is
only characterized by their association in a co-activating complex (Figure 4), and further
investigation is of particular interest. Additional biochemical analysis, like direct
immunoprecipitation or pull-down and western blot detection, will need to be conducted
29
in order to validate putative protein cofactors. Like the Bind-n-Seq assay, MBP pulldown mass spectrometry analysis serves as a great starting point for more in-depth
biochemical and in vivo studies.
Bind-n-Seq has revealed important INR-independent DNA binding functions of
Miz-1 and MBP pull-down mass spectrometry has identified interesting putative
cofactors of Miz-1 in hESCs, while supporting the antagonistic functions of Myc and
Miz-1 in hESCs. Though these important analyses require further validation and study in
vivo, the work represents an essential advance in the understanding of an important
master transcriptional regulator and Myc antagonist in hESCs. Continued study of the
basic regulation of pluripotency and self-renewal in hESCs is vital to our understanding
of their purpose and potential in regenerative medicine so that therapies may be safe and
effective.
30
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