Genome-wide analysis reveals Sall4 to be a major
regulator of pluripotency in murine-embryonic
Jianchang Yanga, Li Chaib, Taylor C. Fowlesa, Zaida Alipioa, Dan Xua, Louis M. Finka, David C. Warda,1, and Yupo Maa,1
aDivision of Laboratory Medicine, Nevada Cancer Institute, One Breakthrough Way, Las Vegas, NV 89135; andbDepartment of Pathology, Joint Program
in Transfusion Medicine, Brigham and Women’s Hospital/Children’s Hospital Boston, Harvard Medical School, 75 Francis Street, Boston, MA 02115
Contributed by David C. Ward, September 18, 2008 (sent for review June 2, 2008)
Embryonic stem cells have potential utility in regenerative medi-
cine because of their pluripotent characteristics. Sall4, a zinc-finger
transcription factor, is expressed very early in embryonic develop-
ment with Oct4 and Nanog, two well-characterized pluripotency
regulators. Sall4 plays an important role in governing the fate of
stem cells through transcriptional regulation of both Oct4 and
Nanog. By using chromatin immunoprecipitation coupled to mi-
croarray hybridization (ChIP-on-chip), we have mapped global
gene targets of Sall4 to further investigate regulatory processes in
bound by the Sall4 protein on duplicate assays with high confi-
dence, and many of these have major functions in developmental
and regulatory pathways. Sall4 bound approximately twice as
many annotated genes within promoter regions as Nanog and
approximately four times as many as Oct4. Immunoprecipitation
revealed a heteromeric protein complex(es) between Sall4, Oct4,
and Nanog, consistent with binding site co-occupancies. Decreas-
ing Sall4 expression in W4 ES cells decreases the expression levels
of Oct4, Sox2, c-Myc, and Klf4, four proteins capable of reprogram-
ming somatic cells to an induced pluripotent state. Further, Sall4
bound many genes that are regulated in part by chromatin-based
epigenetic events mediated by polycomb-repressive complexes
and bivalent domains. This suggests that Sall4 plays a diverse role
in regulating stem cell pluripotency during early embryonic devel-
opment through integration of transcriptional and epigenetic
induced pluripotent stem cells ? epigenetic regulation ? Oct4 ?
Nanog ? Sox2
(sal) (1–3). In Drosophila, sal is a homeotic gene essential in the
development of posterior-head and anterior-tail segments (4).
Human SALL4 mutations are associated with the Duane-radial
ray syndrome (Okihiro syndrome), a human autosomal-
dominant disease involving multiple organ defects (3, 5, 6). Sall4
homozygous knockout mice die at an early embryonic stage (7,
8). Our group and others have recently shown that mouse Sall4
plays an essential role in maintaining the self-renewal and
pluripotent properties of ES cells and in governing the fate of the
inner-cell mass through transcriptional modulation of Oct4 (also
known as Pou5f1) and Nanog (8–10).
ES cells are derived from the inner cell mass of the developing
embryo, and ES-cell pluripotency is regulated in part by Oct4,
Sox2, and Nanog, as well as through 2 polycomb-repressive
complexes (PRCs) (11, 12). Sall4 is expressed by cells of the early
embryo, exhibiting an expression pattern similar to Oct4 (8, 9).
In recent studies, Sall4 has also been used as part of a gene
signature for pluripotency and an enhancer for somatic cell
reprogramming (13, 14). However, the complete mechanism
whereby Sall4 controls pluripotency and differentiation in ES
cells is unknown. The studies reported here demonstrate that
Sall4 interacts with core transcription factors, genes in multiple
all4 is a zinc-finger transcription factor that was originally
cloned based on sequence homology to Drosophila spalt
signal transduction pathways, and genes relating to epigenetic
processes associated with PRCs as well as bivalent histone
methylations. These observations suggest that Sall4 is an essen-
tial regulator of cell pluripotency and differentiation.
Sall4 Is a Major Transcriptional Regulator in ES Cells.Agrowingbody
of evidence has shown that Sall4 plays a vital role in maintaining
ES cell pluripotency and in governing ES cell-fate decisions (9,
10, 14, 15). This prompted us to investigate the global down-
stream targets of Sall4 in mouse ES cells. By using a duplicate
set of ChIP-on-chip assays, we performed a global analysis of
chosen because it was previously used to generate a conditional
Sall4 knockout ES-cell line (9). The majority of transcription
factor binding sites in humans are known to occur ?1–2 kb of the
transcription start site (15). Thus, promoter tiling arrays
(NimbleGen, build MM8) spanning 2.5 kb of promoter regions
(2 kb upstream and 500 bp downstream from the transcription
start site) were selected for hybridization to chromatin-
immunoprecipitated DNA obtained by using an affinity-purified
anti-Sall4 antibody (16).
Successful ChIP assays critically depend on the specificity of
the antibody used. Therefore, we rigorously characterized the
antibody used in these immunoprecipitation assays. First, west-
ern blot analysis was used to compare the Sall4 antibody
preparation with a commercially available anti-HA antibody to
demonstrate specificity for either WT Sall4 or a Sall4-HA fusion
protein. Initially, in mouse fibroblast cells transfected with
Sall4-HA, we were able to detect the fusion protein by using an
anti-HA antibody, whereas in untransfected fibroblast cells, no
Sall4 band was detected [supporting information (SI) Fig. S1A,
Lanes 0 and 1]. Although no expression was observed in fibro-
blasts, experiments in W4 ES cells were able to detect expression
of endogenous Sall4 [Lanes 2 and 3 (14)]. The endogenous band
observed in ES cells was also successfully absorbed (Lanes 4
Next we sought to determine whether our antibody was
applicable in ChIP experiments. ChIP-PCR of DNA fragments
obtained by using the anti-Sall4 antibody was able to detect
enrichment of the peaks identified by the ChIP-on-chip assay. By
using heterozygous Sall4 ES cells overexpressing Sall4-HA,
Author contributions: J.Y. and Y.M. designed research; J.Y., Z.A., and D.X. performed
research; J.Y., L.C., T.C.F., Z.A., D.X., L.M.F., D.C.W., and Y.M. analyzed data; and J.Y., L.C.,
T.C.F., L.M.F., D.C.W., and Y.M. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The data reported in this paper have been deposited in the Gene
1To whom correspondence may be addressed. E-mail: email@example.com or yma@
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
December 16, 2008 ?
vol. 105 ?
immunoprecipitation of the HA-tag identified 88% (23:26) of
the genes identified by the anti-Sall4 antibody (Fig. S1B). This
suggests that our anti-Sall4 antibody is both sensitive and specific
for the Sall4 protein when used in immunoprecipitation. We
have also used this antibody for immunohistochemistry to detect
Sall4 protein in different tissue samples (Fig. S1C) and for flow
cytometry to identify cell populations corresponding to leukemic
blasts in patient bone marrow samples that uniquely express
Sall4 [Fig. S1D (16)].
Following binding site determination by NimbleGen, the
ChIP-on-chip duplicate assays identified roughly 5,200 Sall4-
bound genes in array 1, and 4,400 Sall4-bound genes in array 2.
The overall false discovery rate was ?0.20. Comparison of the
data from arrays 1 and 2 showed that 3,223 gene promoters gave
positive hybridization signals on both arrays. Of the 1,000 genes
exhibiting the most intense hybridization signals on array 2, 947
were also positive on array 1. When only the top 200 genes were
considered, the concordance rate was 98.5%. In contrast, when
the 800 lowest intensity signals on each array were analyzed, only
37.2% (array 1) and 52.6% (array 2) of the signals were
concordant in both assays. Therefore, we selected only the 3,223
genes that were positive on both arrays for further analysis.
We next validated a subset of the putative Sall4 binding sites
by using a ChIP PCR strategy. A total of 55 genes were
interrogated. Primer pairs were prepared for a randomly se-
lected set of hybridization positive genes with varying degrees of
signal intensity. If a selected gene did not initially produce an
amplicon level above background, a new primer set was designed
200–300 bases distal to the first primer site, and the quantitative
real-time PCR (Q-RT-PCR) assay was repeated. In some cases,
a third primer set was used before designating that gene to be a
false positive. In addition, ChIP-PCR using primers located
adjacent to true positive loci were shown to give negative
amplification results, further demonstrating the specificity of
Sall4 binding-site identification. Based on the Q-RT-PCR data
(52:55 positive), we concluded that ?94.5% of the 3,223 genes
common to both arrays are true positive SALL4 binding sites in
the mouse ES cell line (Fig. S2).
The full list of the 3,223 Sall4-bound genes and their respective
array hybridization data can be found within the supplemental
data (Dataset S1) and on the gene expression omnibus (GEO)
as accession number GSE11305. The number of promoter
sequences binding Sall4 is quite high, but other transcription
factor proteins, such as E2f1, have been reported to bind over
5,000 gene promoters (17). Myc has recently been reported to
bind a similar number of genes in mouse ES cells (18).
We then sought to determine the distribution of Sall4 binding
sites within the mapped regions of the genome by using DAVID
(19). Analysis of over-represented annotations for promoter
regions bound by Sall4 revealed significant representation of a
broad variety of genes that may be important for stem cell
functions (Fig. 1A). These include developmental genes and
genes necessary for signal transduction and other regulatory
processes. Further classification of the developmental genes
revealed over-representation of genes associated with organ
development, pattern specification, and brain development (Fig.
1B). Sall4 bound to promoter regions of 11 members of the Hox
gene family, and 42 other homeobox or homeobox-like genes
(Table S1). The binding of Sall4 to promoter regions of vital
developmental genes and others that govern ES cell fate support
the phenotypic consequence of Sall4 reduction in ES cells. This
also suggests that Sall4 plays a vital role in ES cells that may be
similar to Oct4 and Nanog. This hypothesis is supported by 3
lines of evidence: (a) Sall4 is expressed very early in the
developing embryo and is subsequently down-regulated in most
differentiated tissues, (b) both over- and under-expression of
Sall4 cause ES-cell differentiation, demonstrating the necessity
for tight regulation of Sall4 expression, and (c) the finding that
Sall4 modulates expression of both Oct4 and Nanog (9, 10).
However, the magnitude of the Sall4 transcriptional network is
quite striking and suggests that Sall4 may play a central role in
Sall4 Targets Important Signals That Control ES-Cell Differentiation
and Lineage Specification. Numerous signaling pathways play
important roles in maintaining pluripotency during embryogen-
esis. For example, the Wnt signaling pathway has important roles
in embryogenesis and cancer (20–23). TGF-? signaling is nec-
essary to maintain ES cell pluripotency, and PTEN signaling
plays important roles in the maintenance of hematopoetic stem
cell self-renewal. Fig. 1C shows the number of genes bound by
Sall4 within several developmentally important pathways and
examples of the genes bound within each pathway. This suggests
that Sall4 may play a broad role in regulation of ES-cell
pluripotency through interactions with key signaling pathways.
Magnitude of the Sall4 Transcriptional Network in ES Cells. Recently,
ChIP-on-chip studies have been performed on the gatekeeper
0 100 200300 400500600700
Map4k4, Tlr4, Tlr7, Traf2
Birc2, Birc4, Casp6, Tnf
Dkk1, Frat1, Tcf4, Wif1
Akt3, Casp3, FoxG1, FoxO1
Fos, Pitx2, Smad3, Tgfb1
Brca1, Casp6, Ccnd1, Ccnd2
Abl1, Abl2, Pdgfra, Elk1
Dyrk1a, Hhip, Prkaca, Stk36
included various potential regulatory and developmental annotations. Anal-
ysis was done with DAVID, and the x axis represents the gene number. (B)
Further classification of developmentally important genes over-represented
in the Sall4 binding pool. For the organ development annotation, the over-
the gene number. P values are inset following each bar and were calculated
genome. (C) Sall4 binds promoter regions belonging to a variety of pathways
that have definitive roles in development, suggesting that Sall4 may control
a wide variety of developmental processes. Listed genes are only representa-
presented because of the low number of genes within each pathway. Classi-
Sall4 is a major regulator in mouse ES cells. (A) Sall4 bound to
Yang et al.PNAS ?
December 16, 2008 ?
vol. 105 ?
no. 50 ?
genes, Oct4 and Nanog. This enabled us to compare genes bound
by Oct4 and Nanog with those bound by Sall4. Interestingly,
ChIP-on-chip assays showed that Oct4 had 783 promoter binding
sites, whereas Nanog had 1,284 binding sites within the mouse
genome (18). The ChIP-on-chip data presented here revealed
that Sall4 bound ?3,200 gene promoters. Given the similar
expression patterns of the transcription factors Sall4, Oct4, and
Nanog, this is remarkable. These observations suggest that Sall4
may play a similar, but broader role in regulating ES-cell
properties. However, the roles of each in vivo are not completely
Interaction and Co-Occupation of Sall4 with Oct4 and Nanog in ES
a regulatory complex in ES cells (10). Liang et al. (24) recently
showed that Sall4 forms a complex (or complexes) with both
Oct4 and Nanog by using mass spectrometry and immunopre-
cipitation of endogenous proteins. We have confirmed these
observations by immunoprecipitation experiments using ES cells
transiently transfected with Sall4-HA. Western blotting detected
an overexpression of Sall4-HA protein by both anti-HA (Fig.
2A) and anti-Sall4 antibodies (data not shown). Immunoprecipi-
tation with an anti-HA antibody produced a unique endogenous
45-kDa protein, Oct4, in the precipitate. By contrast, an IgG-
negative control failed to generate the Oct4 band in the same
extract, indicating a specific Sall4–Oct4 interaction. By using the
same method, the Sall4–Nanog interaction was confirmed in the
same anti-HA-pulldown precipitate (Fig. 2A).
Because these transcription factors physically interact, one
would expect them to colocalize to some of the same gene
promoters (25). A gene bound by any two of these proteins we
will refer to as ‘‘co-occupied’’. However, Oct4 and Sall4 co-
occupied only 92 common genes representing 12% of genes
bound by Oct4. Similarly, Sall4 binding was identified at 198
Nanog target genes, representing only 15% of Nanog’s bound
genes (Fig. 2B). This suggests that Sall4–Oct4 and Sall4–Nanog
interactions may form functional complexes only at select pro-
moter regions (9, 10). There are only 45 genes that are co-
occupied by Oct4, Sall4, and Nanog (Table S2). However, this
group includes developmentally important genes, such as Dkk1,
Msx2, Fbxl10, and Epc1.
Sall4?/?ES Cells Exhibit Decreased Expression of iPS Genes. Recent
studies have shown that ectopic expression of Oct4, Sox2, Klf4,
and c-Myc is capable of reprogramming somatic cells to confer
a pluripotent state, termed induced pluripotent stem (iPS) cells
(26). It has previously been demonstrated that Sall4 binds to
protein binds to the promoter regions of Oct4, c-Myc, Sox2, and
Klf4 through ChIP-PCR (Fig. 3A).
To determine the relationship between binding and Sall4
function, we used Q-RT-PCR to measure mRNA levels from
Sall4?/?ES cells. Expression levels of all 4 transcription factors
decreased in Sall4?/?ES cells indicating that Sall4 plays an
activating role on these genes (Fig. 3B). Because Sall4 is not
expressed in the majority of differentiated tissues including
fibroblasts, this suggests that exogenous expression of Sall4
may play a role in reprogramming somatic cells to confer a
pluripotent state. This hypothesis has recently been supported
by others (27).
Sall4 Binds to Genes Associated with H3K27 Methylation Domains as
well as to Target Genes of PRC1 and PRC2. Numerous studies have
implicated epigenetic modifications as a means for regulating
stem cell pluripotency (28–30). We have previously shown that
Bmi-1, a polycomb group member, is a downstream target of
SALL4 (31), thus, we focused on other polycomb-associated
genes for analysis. Although various covalent modifications can
(A) Transient transfection of W4 ES cells with a Sall4-HA construct exhibited
protein expression detected by both anti-HA and anti-Sall4 antibodies (the
latter data not shown) in the cell extract (left). Oct4 and Nanog are detected
by using respective antibodies in the whole ES cell extract (input). Immuno-
precipitation of Sall4-HA with an anti-HA antibody revealed both Oct4 and
Nanog bands, whereas immunoprecipitation with an IgG antibody detected
neither protein. (B) Venn diagram showing the overlapping target genes of
These complexes may function in the regulation of stem cell pluripotency.
Sall4c-Myc Klf4Sox2 Oct4
WT ES cells
Fold enrichment relative to
c-Myc Klf4 Sox2
sion of 4 key transcription factors, Oct4, Sox2, c-Myc, and Klf4, produces iPS
cells. (A) Sall4 binds to promoter regions of Oct4, Sox2, c-Myc, and Klf4 as
The Sall4/Gapdh ratio in control cells was set at 1. The values are the mean of
triplicate reactions, and the bars indicate SD.
Decreased expression of iPS genes in Sall4?/?ES cells. Ectopic expres-
www.pnas.org?cgi?doi?10.1073?pnas.0809321105Yang et al.
influence chromatin remodeling, here we investigate the com-
bined roles of Sall4, methylation of histone 3 on lysine 27
(H3K27), and PRCs. PRCs are key modulators of stem-cell
pluripotency and consist of 2 distinct groups (11, 12). PRC1
consists of ?10 proteins, including Bmi1, Rnf2, PhcI, and the
HPC proteins, whereas PRC2 contains Ezh2, Eed, Suz12, and
RbAp46:48 (32). Representative ChIP-on-chip assays have been
performed for PRC1 genes Rnf2 and Phc1, and PRC2 group
members Suz12 and Eed (11). PRCs maintain ES-cell pluripo-
tency by facilitating H3K27 methylation, a modification that
represses gene expression (11). The majority of H3K27 trim-
ethylation and PRC binding sites are ?1 kb of the transcription
start site, and both are frequently present on gene promoters.
However, not all H3K27 methylated domains are associated with
PRCs. Thus, Sall4 may bind and potentially regulate expression
of a subset of genes associated with H3K27 methylated domains.
Binding of Sall4 to Genes Associated with Histone Methylations
(GAHMs) occurred at 17% (422:2557) of previously identified
H3K27 methylation domains within 1 kb of annotated-
transcription start sites of mouse ES cells. We expect PRCs to be
associated with some of the Sall4-bound GAHMs. Sall4, PRC1,
and PRC2 co-occupied 160 GAHMs (Fig. 4A). There were also
GAHMs co-occupied by Sall4 and one of the polycomb proteins,
with 29 and 69 bound by PRC1 and PRC2, respectively. Inter-
estingly, Sall4 bound 164 GAHMs that were not occupied by
either PRC1 or PRC2. To determine the function of this subset
of genes, we categorized them based on overrepresentation by
using DAVID. As expected, GAHM-H3K27 and PRCs had
extremely significant roles in development (P ? 0.001). Surpris-
ingly, GAHM-H3K27 and Sall4 also had notable roles in devel-
opment (P ? 0.07; Fig. 4B). This reveals a system in which
regulation of GAHM-H3K27 may be controlled by dynamic
involvement of both Sall4 and PRCs.
Many Sall4 Targets Harbor Bivalent Domains. It has been reported
that dual epigenetic markers, coined ‘‘bivalent domains’’, con-
sisting of methylations at H3K27 and at histone 3 on lysine 4
(H3K4), exist for a large set of developmental genes within
Highly Conserved Noncoding Elements (HCNEs) (33). ES-cell
pluripotency is hypothesized to be maintained, in part, through
a balance of H3K4 gene activation and H3K27 gene repression
at these bivalent domains (33). To explore the role that Sall4 may
play in this epigenetic mechanism, Sall4 binding sites were
compared with bivalent domains identified within HCNEs. We
found that Sall4 co-occupied 27% (37:135) of Genes Associated
with Bivalent Histone Methylations (GABHMs), including 11
Hox gene family members (Fig. 5, Table S3). In contrast, Oct4
and Nanog each bind only 12% of GABHMs (Fig. S3). Surpris-
ingly, there are no genes that are bound by Sall4, Oct4, and
Nanog. Only 11 of the GABHMs are co-occupied by any 2
proteins, suggesting that Sall4, Oct4, and Nanog may play
independent roles in methylation regulation. Further, these 3
transcription factors account for binding to only 39% of iden-
tified bivalent domains. It remains to be determined what other
genes emerge as further epigenetic regulators.
We have shown that Sall4 binds ?3,200 genes within their
promoter regions in mouse ES cells. An analogous ChIP-chip
assay preformed by using chromatin-precipitated DNA obtained
by using Oct4 and Nanog antibodies revealed 783 and 1,284
bound genes, respectively. Given the similar gene expression
patterns of Sall4 and Oct4, the magnitude of Sall4 binding is
remarkable. Although extensive functional studies need to be
done on Sall4, Oct4, and Nanog, it appears that the role of Sall4
may be significant in determining stem cell fate.
Sall4, Oct4, and Nanog have been shown to form heteromeric
protein complexes that may regulate ES-cell gene expression in
complex ways. Transient combinatorial binding of Sall4, Oct4,
and Nanog may determine cell fate with different combinations
of these proteins controlling different aspects of pluripotency.
Although trimeric protein complexes may exist, there are rela-
0 1020 30 40 50 6070 80 90
Sall4 BoundSall4 Bound
by PRC1 (Rnf2, Phc1) and PRC2 (Suz12, Eed). One hundred sixty-four of these
genes are associated with Sall4, PRC1, and PRC2 (inner orange circle) with 69
PRC2-bound genes and 29 PRC1-bound genes also bound by Sall4 (outer
polycomb group proteins (outer gray circle). (B) Two hundred fifty-eight
genes are bound by one or more polycomb group protein(s) and Sall4. Of
these, 81 have developmental functions that display significantly over-
represented (P ? 0.001) binding to genes associated with various develop-
that two or more mechanisms may interact to regulate cell fate through
annotations are not mutually exclusive, and P values were determined by
using Fisher’s Exact Test.
The role of Sall4 in H3K27 methylation regulation (A) Sall4 binds to
diagram displaying the GAHMs bound by Sall4 within HCNEs. Notably, the
majority of Sall4-bound genes within characterized HCNEs are marked by
bivalent histone methylation domains including a cluster of homeobox genes
(see Table S3).
Sall4 target genes are associated with bivalent domains. Venn
Yang et al. PNAS ?
December 16, 2008 ?
vol. 105 ?
no. 50 ?
tively few genes bound by all 3 transcription factors. The
Sall4–Oct4 complex binds genes that have statistically signif-
icant roles in some developmental processes associated with
stem cell activities (P ? 0.05). In contrast, although Sall4–
Nanog complexes bind genes that have similar developmental
and transcriptional functions, this dimeric protein combina-
tion also binds genes important for organ development and
pattern specification at statistically significant frequencies
(P ? 0.05; Fig. S4 and Table S4). This data suggests that the
binding of these 3 transcription factors at select promoter
regions may dynamically control transcription required for the
stem cell state, although it is likely that many other proteins
also play important roles.
Another interesting observation is that down-regulation of
Sall4 also causes down-regulation of Oct4, Sox2, Klf4, and
c-Myc, 4 genes that induce reprogramming of somatic cells to
induced pluripotent stem cells. This suggests a mechanism by
which Sall4 could be a key regulator for the reprogramming
process. This interpretation was recently supported by Wong
et al., who used cell fusion to demonstrate that Sall4 can
enhance somatic cell reprogramming (27). Nevertheless, the
importance of Sall4 in somatic cell reprogramming and the
role it plays in regulating this gene quartet remain to be
Interestingly, recent work by Dr. Austin Smith’s group has
suggested that inhibiting a cell’s intrinsic signaling pathways is
those that proceed through mitogen-activated protein kinases
(ERK1/2) and glycogen synthase kinases (GSK3). Work from
our lab and from others suggests that Sall4 may interact with
Stat3, and preliminary data indicate that Stat3 is an upstream
regulator of Sall4. This would implicate Sall4 in this complex
regulatory loop. How Sall4 interacts with other microenviron-
mental signals is unknown at this time.
Important questions remain to be answered regarding evi-
dence for an Oct4–Sall4–Nanog complex and the regulatory
role that it may play in ES cell pluripotency maintenance.
Further, evidence presented here indicates that Sall4 may play
an important role in regulating chromatin remodeling. This
connects the independent processes of transcriptional regula-
tion and epigenetic regulation and may provide insights into an
integrated control process involved in determining stem cell
Materials and Methods
Core Facility, University of Iowa) were cultured with irradiated mouse embry-
(9). For W4 clone EA231, Sall4?/?ES cells were cultured with the antibiotic
G418 at a concentration of 125 ?g/ml.
ChIP-on-chip Assays. A complete ChIP-on-chip assay protocol was provided by
NimbleGen Systems, Inc. In brief, W4 ES cells were cross-linked with formal-
dehyde and lysed, and then the DNA was sheared by sonication. A sonication
regime consisting of 8 pulses lasting 20 seconds each were used with 90
seconds in-between spent on ice. The Misonix Sonicator 3000 was used at
with an affinity-purified anti-Sall4 antibody (16), ChIP-purified DNA was
blunt-ended, ligated to linkers, and subjected to low-cycle PCR amplification.
Resultant ChIP-DNA was then hybridized to duplicate promoter tiling arrays
(RefSeq arrays, build MM8) each containing 19,457 promoter annotations
containing 2.5 kb of each RefSeq promoter region. The promoter region is
covered by 50–75 mer probes at roughly 100-bp spacing dependent on the
extracted according to NimbleGen standard procedures. Data extraction was
done by using NimbleScan, which searches for 4 or more probes above a
specified cutoff value ranging from 90–15% using a 500-bp sliding window.
The cutoff value is a percentage of the hypothetical maximum determined by
using the mean plus 6 standard deviations and is decreased in 1% increments
from 90–15%. The data are then randomized 20 times to evaluate the
possibility of false positives, and each peak is assigned a false discovery rate
performed by using ChIP-PCR analysis of the amplicons applied to the arrays
(Fig. S1A). Negative control primers were designed adjacent to Sall4-bound
peaks (Fig. S2B).
Coimmunoprecipitation and Western Blotting. For Oct4–Sall4 and Nanog–Sall4
interactions, plasmid pcDNA3/Sall4-HA was transfected into W4 ES cells to
express the Sall4-HA protein by using Lipofectamine 2000 reagent (Invitro-
gen). Coimmunoprecipitations were performed following the Catch and Re-
lease v2.0 High Throughput Immunoprecipitation Assay Kit (Upstate) as rec-
ommended. For western blots, the membrane was incubated with Oct-3:4
(H-134), Nanog (M-149) (both from Santa Cruz Biotechnology, Inc), or Sall4
antibodies at a 1:300 dilution at 4 °C overnight. Detection was done by using
SuperSignal West Pico solutions (Pierce).
of Health Grants R01HL087948, NIH R21CA131522, and P20 RR016464 (to
Y.M.), The Leukemia and Lymphoma Society Special Fellow Award (to J.Y.),
and Harvard Stem Cell Institute (L.C.).
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