Cell, Vol. 122, 947–956, September 23, 2005, Copyright 2005 by Elsevier Inc. DOI 10.1016/j.cell.2005.08.020
Core Transcriptional RegulatEmbryonic stem (ES) cells are derived from the inner
cell mass (ICM) of the developing blastocyst and can
be propagated in culture in an undifferentiated state
while maintaining the capacity to generate any cell type
in the body. The recent derivation of human ES cells
*Correspondence: young@wi.mit.edu
6
These authors contributed equally to this work.
1998; Schöler et al., 1990). Studies in a broad range of
eukaryotes have shown that transcriptional regulators
that have key roles in cellular processes frequently reg-
ulate other regulators associated with that process
(Guenther et al., 2005; Lee et al., 2002; Odom et al.,
2004). It is likely that the key stem cell regulators bind
and regulate genes encoding other transcriptional reg-
ulators, which in turn determine the developmental po-
tential of these cells, but we currently lack substantialin Human Embryonic Stem C
Laurie A. Boyer,
1,6
Tong Ihn Lee,
1,6
Megan F. Cole,
1,2
Sarah E. Johnstone,
1,2
Stuart S. Levine,
1
Jacob P. Zucker,
3
Matthew G. Guenther,
1
Roshan M. Kumar,
1
Heather L. Murray,
1
Richard G. Jenner,
1
David K. Gifford,
1,4,5
Douglas A. Melton,
3,5
Rudolf Jaenisch,
1,2
and Richard A. Young
1,2,5,
*
1
Whitehead Institute for Biomedical Research
9 Cambridge Center
Cambridge, Massachusetts 02142
2
Department of Biology
Massachusetts Institute of Technology
Cambridge, Massachusetts 02139
3
Howard Hughes Medical Institute
Department of Molecular and Cellular Biology
Harvard University
Cambridge, Massachusetts 02138
4
MIT Computer Science and Artificial Intelligence
Laboratory (CSAIL)
32 Vassar Street
Cambridge, Massachusetts 02139
5
Broad Institute of MIT and Harvard
One Kendall Square, Building 300
Cambridge, Massachusetts 02139
Summary
The transcription factors OCT4, SOX2, and NANOG
have essential roles in early development and are re-
quired for the propagation of undifferentiated embry-
onic stem (ES) cells in culture. To gain insights into
transcriptional regulation of human ES cells, we have
identified OCT4, SOX2, and NANOG target genes
using genome-scale location analysis. We found, sur-
prisingly, that OCT4, SOX2, and NANOG co-occupy a
substantial portion of their target genes. These target
genes frequently encode transcription factors, many
of which are developmentally important homeodo-
main proteins. Our data also indicate that OCT4,
SOX2, and NANOG collaborate to form regulatory cir-
cuitry consisting of autoregulatory and feedforward
loops. These results provide new insights into the
transcriptional regulation of stem cells and reveal
how OCT4, SOX2, and NANOG contribute to pluripo-
tency and self-renewal.
Introduction
Mammalian development requires the specification of
over 200 unique cell types from a single totipotent cell.ory Circuitry
ells
provides a unique opportunity to study early develop-
ment and is thought to hold great promise for regenera-
tive medicine (Pera and Trounson, 2004; Reubinoff et
al., 2000; Thomson et al., 1998). An understanding of
the transcriptional regulatory circuitry that is responsi-
ble for pluripotency and self-renewal in human ES cells
is fundamental to understanding human development
and realizing the therapeutic potential of these cells.
Homeodomain transcription factors are evolutionarily
conserved and play key roles in cell-fate specification
in many organisms (Hombria and Lovegrove, 2003).
Two such factors, OCT4/POU5F1 and NANOG, are essen-
tial regulators of early development and ES cell identity
(Chambers et al., 2003; Hay et al., 2004; Matin et al.,
2004; Mitsui et al., 2003; Nichols et al., 1998; Zaehres
et al., 2005). Several genetic studies in mouse suggest
that these regulators have distinct roles but may func-
tion in related pathways to maintain the developmental
potential of these cells (Chambers, 2004). For example,
disruption of OCT4 or NANOG results in the inappropri-
ate differentiation of ICM and ES cells to trophectoderm
and extra-embryonic endoderm, respectively (Cham-
bers et al., 2003; Mitsui et al., 2003; Nichols et al., 1998).
However, overexpression of OCT4 in ES cells leads to
a phenotype that is similar to loss of NANOG function
(Chambers et al., 2003; Mitsui et al., 2003; Nichols et
al., 1998; Niwa et al., 2000). Knowledge of the set of
genes regulated by these two transcription factors
might reveal why manipulation of OCT4 and NANOG
results in these phenotypic consequences.
OCT4 is known to interact with other transcription
factors to activate and repress gene expression in
mouse ES cells (Pesce and Schöler, 2001). For exam-
ple, OCT4, a member of the POU (PIT/OCT/UNC) class
of homeodomain proteins, can heterodimerize with the
HMG-box transcription factor, SOX2, to affect the ex-
pression of several genes in mouse ES cells (Botquin
et al., 1998; Nishimoto et al., 1999; Yuan et al., 1995).
The cooperative interaction of POU homeodomain and
HMG factors is thought to be a fundamental mecha-
nism for the developmental control of gene expression
(Dailey and Basilico, 2001). The extent to which ES cell
gene regulation is accomplished by OCT4 through an
OCT4/SOX2 complex and whether NANOG has a role
in this process are unknown.
OCT4, SOX2, and NANOG are thought to be central
to the transcriptional regulatory hierarchy that specifies
ES cell identity because of their unique expression pat-
terns and their essential roles during early development
(Avilion et al., 2003; Chambers et al., 2003; Hart et al.,
2004; Lee et al., 2004; Mitsui et al., 2003; Nichols et al.,

Cell
948Figure 1. Genome-Wide Location Analysis in Human Embryonic Stem Cells
(A) DNA segments bound by transcriptional regulators were identified using chromatin immunoprecipitation (ChIP) and identified with DNA
microarrays containing 60-mer oligonucleotide probes covering the region from −8 kb to +2 kb for 17,917 annotated transcription start sites
for human genes. ES cell growth and quality control, ChIP protocol, DNA microarray probe design, and data analysis methods are described
in detail in Experimental Procedures and Supplemental Data.
(B) Examples of OCT4 bound regions. Plots display unprocessed ChIP-enrichment ratios for all probes within a genomic region. Genes are
shown to scale below plots (exons and introns are represented by thick vertical and horizontal lines, respectively), and the genomic region
represented is indicated beneath the plot. The transcription start site and transcript direction are denoted by arrows.
knowledge of the regulatory circuitry of ES cells and
other vertebrate cells.
To further our understanding of the means by which
OCT4, SOX2, and NANOG control the pluripotency and
self-renewal of human ES cells, we have used genome-
scale location analysis (chromatin immunoprecipitation
coupled with DNA microarrays) to identify the target
genes of all three regulators in vivo. The results reveal
that OCT4, SOX2, and NANOG co-occupy the promot-
ers of a large population of genes, that many of these
Results and Discussion
OCT4 Promoter Occupancy in Human ES Cells
DNA sequences occupied by OCT4 in human H9 ES
cells (NIH code WA09; Supplemental Data) were iden-
tified in a replicate set of experiments using chromatin
immunoprecipitation (ChIP) combined with DNA micro-
arrays (Figure 1A and Supplemental Data). For this pur-
pose, DNA microarrays were designed that contain 60-
mer oligonucleotide probes covering the region from −8target genes encode developmentally important ho-
meodomain transcription factors, and that these regu-
lators contribute to specialized regulatory circuits in
ES cells.kb to +2 kb relative to the transcript start sites for
17,917 annotated human genes. Although some tran-
scription factors are known to regulate genes from dis-
tances greater than 8 kb, 98% of known binding sites