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  Dynamic transcription programs during ES cell differentiation towards
  E& x% D! r# v% X) z: u  x    mesoderm in serum versus serum-freeBMP4 culture
   Stephen J Bruce1,2 , Brooke B Gardiner1,3,4 , Les J Burke1,4 , M Milena
   Gongora1,3 , Sean M Grimmond1,3,4  and Andrew C Perkins1,2,3,4
   1Institute for Molecular Bioscience, University of Queensland, Brisbane, 6 |! j5 U8 f! M8 f% M2 d, p# p# q
   Queensland, Australia
   2Wesley Research Institute, Queensland, Australia/ j- K1 I& Y; @2 T
   3ARC Special Research Centre in Functional and Applied Genomics, Australia, D* `+ J0 \* B1 q" X, k1 p
   4Australian Stem Cell Centre, Melbourne, Australia
   author email corresponding author email3 @6 n4 [- W3 p6 Z0 _
   BMC Genomics 2007, 8:365doi:10.1186/1471-2164-8-365
   The electronic version of this article is the complete one and can be 3 B. W7 L$ ]3 c! j* \
   found online at: http://www.biomedcentral.com/1471-2164/8/365
         Received:28 May 2007
         Accepted:10 October 20070 v( j. g& f( n5 O3 k0 D7 [- ]
         Published:10 October 2007
! @! M# h9 u6 g4 n" N3 B# s. b! e" ~
   © 2007 Bruce et al; licensee BioMed Central Ltd.
   This is an Open Access article distributed under the terms of the Creative
   Commons Attribution License (http://creativecommons.org/licenses/by/2.0), 0 U" }5 J5 m! r# v' N8 {4 Z# c
   which permits unrestricted use, distribution, and reproduction in any
   medium, provided the original work is properly cited.
   Abstract
   Background
   Expression profiling of embryonic stem (ES) cell differentiation in the
   presence of serum has been performed previously. It remains unclear if 4 ^9 r; i6 x6 M, I+ _) d
   transcriptional activation is dependent on complex growth factor mixtures 9 z) ?* s3 \4 b0 A$ e8 _4 Z
   in serum or whether this process is intrinsic to ES cells once the stem
   cell program has been inactivated. The aims of this study were to - b5 g. O- Y* R& h2 C) m
   determine the transcriptional programs associated with the stem cell state
   and to characterize mesoderm differentiation between serum and serum-free , T6 @$ a" y  L0 B
   culture./ y: C" i4 c% N2 h& y
   Results
   ES cells were differentiated as embryoid bodies in 10% FBS or serum-free
   media containing BMP4 (2 ng/ml), and expression profiled using 47 K " e) R" O/ L  n. d- w4 z
   Illumina(R) Sentrix arrays. Statistical methods were employed to define 2 k5 t; W# k$ M- W+ L9 u4 q$ O
   gene sets characteristic of stem cell, epiblast and primitive streak
   programs. Although the initial differentiation profile was similar between - C, {& S  C" q" |
   the two culture conditions, cardiac gene expression was inhibited in serum
   whereas blood gene expression was enhanced. Also, expression of many ( x# s, x) v" M- G
   members of the Kruppel-like factor (KLF) family of transcription factors
   changed dramatically during the first few days of differentiation. KLF2 ) I' H7 p8 n  h3 J
   and KLF4 co-localized with OCT4 in a sub-nuclear compartment of ES cells,
   dynamic changes in KLF-DNA binding activities occurred upon
   differentiation, and strong bio-informatic evidence for direct regulation
   of many stem cell genes by KLFs was found.
   Conclusion
   Down regulation of stem cell genes and activation of epiblast/primitive 5 R8 f+ v5 w( ~) w6 c
   streak genes is similar in serum and defined media, but subsequent
   mesoderm differentiation is strongly influenced by the composition of the
   media. In addition, KLF family members are likely to be important # Q/ B% g7 b3 o8 r% T
   regulators of many stem cell genes.% ~! O6 y+ \" `
   Background( \/ M2 d, {. o- [  i8 O
   Embryonic stem (ES) cells isolated from the inner cell mass of the early ( x6 S! e. u5 ?- `- u" v
   mammalian blastocyst-stage embryo retain pluripotency when cultured on
   mouse embryonic fibroblasts (MEFs) in the presence of leukemia inhibitory 9 b( \5 |; g: C4 t
   factor (LIF) [1]. In the absence of LIF and MEF attachment, ES cells 3 O! j$ m7 d3 w1 T3 s% J3 Q% m
   spontaneously differentiate into multi-cellular aggregates termed embryoid 5 Q! C% X+ S( H
   bodies (EBs). Although the spatial complexity of organogenesis is not
   established during EB maturation, the dynamics of gene expression closely ( [4 @% t9 M# i+ M% Q6 ]
   mimic those which characterize early stages of mouse development [2-5].
   Thus, ES cell differentiation is an excellent model system for the
   discovery of genes involved in developmental processes.  ~/ {; z% I) K3 R
   Many studies have attempted to identify genes that define the stem cell 5 H" s& D# q' ^4 D7 f. _' ^
   state by mining for genes co-expressed in ES cells and various other stem
   cell populations such as hematopoietic and neural stem cells [6,7], or ES ) z1 n" D6 [' P" n+ g. `
   cells and trophoblast stem cells [8]. Some of these studies have been
   criticized because they were unable to define a similar cohort of stem
   cell genes [9]. Also, this approach is likely to find co-expression of : h3 t6 L9 w8 w3 L5 w
   'housekeeping' genes that are irrelevant to the stem cell state. More
   recently, dynamic expression profiling during ES cell differentiation has $ g5 P/ ?& z0 y% ?3 X
   been undertaken using various EB culture techniques [10,11] and the
   addition of exogenous growth factors [7]. Most of these studies followed
   differentiation for a short time period (up to 6–8 days) due to inherent - G- `  p: s6 C" O  F0 b
   limitations of liquid or adherent cultures cell differentiation systems.9 o9 g( e8 R1 S0 r& d( O
   Various platforms have been used to determine gene expression profiles,
   including differential display, SAGE [12] and slide micro-arrays, such as
   those provided by Affymetrix, Agilent and Compugen. In this study we 7 L4 U! T, g" j+ _. u
   employed Illumina® Sentrix Mouse-6 oligo bead arrays [13], which have ~47 , y/ t; r' S( O0 d& q- h- V- N; @
   K probes that are largely based on the MEEBO set of transcripts, plus ESTs
   discovered as part of the FANTOM2 transcriptome project [14], some
   alternatively employed exons, and a small subset of non-coding
   transcripts. This is a new, rich and sensitive platform for expression
   profiling [15].
   ES cells were differentiated in methylcellulose cultures, which allowed
   consistent EB development over 16 days in either serum [2,16] or defined
   serum-free media containing BMP4 (2 ng/ml), termed serum-freeB4L hereafter ' R" k$ I& B+ D6 C5 H# M, l/ o
   [17]. The dynamics of loss of stem cell gene expression and activation of $ C; O3 J: A. `4 Z
   epiblast and primitive streak gene expression programs were similar in
   serum and serum-freeB4L but QT clustering revealed a significant
   difference in mesoderm outcomes from day 6 onwards. In particular, cardiac
   gene expression was greater in serum-freeB4L whereas activation of the 0 w2 a# b2 u4 O+ ^. w/ b) g
   blood program was enhanced in serum.
   High expression levels of Kruppel-like factor (KLF) family members: Klf2, , [5 G" z& m  W( @8 A
   Klf4, Klf5 and Klf9 was detected in undifferentiated ES cells, and ; r% y- A6 ^# z# _+ l' D, @
   confirmed by quantitative real time RT-PCR, indirect immuno-fluorescence 7 V1 o5 q& ~7 U+ E2 M9 P* d) g* R
   and DNA-binding assays. Expression was down regulated rapidly upon
   differentiation and a different set of Klfs: Klf3, Klf16 and Klf1/Eklf,
   were activated upon ES cell differentiation. Also, KLF-binding sites were & v# h: {5 l  ]! r
   markedly over represented in the proximal promoters of most stem cell
   specific genes, suggesting possible direct regulation by KLFs. We suggest : B$ S) a- I: Z8 g
   a model in which the balance between self-renewal and differentiation is
   regulated by competitive occupancy of the proximal promoters of key stem & U* T. E3 N( Q; P5 C1 q
   cell genes such as Zfp42/Rex-1, Nanog, Pou5f1(Oct4), Lefty1 and Lefty2, 8 O4 b5 v9 r7 c1 {0 S& z
   and others such as the Klf genes themselves.4 e# y# e5 _8 Z
   Results( v  V, r4 r8 b5 e9 i
   Differentiation of ES cells in serum and serum-freeB4L culture
   The directed differentiation of embryonic stem cells into desirable cell 2 U; w' o) e/ P5 f
   types will depend upon the addition of specific growth factors to defined
   media and reproducible physical conditions of culture. To establish a
   baseline for such studies, we undertook a comparison of murine ES cell 8 [5 p( Y$ `+ ~! `! M6 v" ^8 B
   differentiation using an embryoid body (EB) methylcellulose culture system 8 M( j; ?7 U" @$ R0 V7 T/ ]
   containing either 10% FBS [2] or a chemically defined BSA-based media 6 i) S8 O% y" E( \' i1 V5 |
   containing 2 ng/ml of BMP4, termed serum-freeB4L [17]. Triplicate . E) ^/ v0 T/ H" P* R! h" C
   experiments were performed in which ES cells were harvested after feeder
   depletion, and set up in parallel EB cultures in serum versus 7 X9 Z$ \; p- v3 v. o+ m% Q. ~
   serum-freeB4L. RNA was collected at regular intervals up to 16 days,
   quantitative RT-PCR was performed on a panel of genes representing defined
   stages of development to establish the robustness of the culture system
   (Figure 1), and expression profiling was performed using Illumina® Sentrix / Y, I9 ~4 K0 ]4 U/ N$ l& D0 p
   Mouse-6 bead arrays (see Methods and [13]).
   Figure 1. Dynamic gene expression during EB differentiation in serum and ! X* g( I6 m/ b$ [9 Y& }
   serum-free media containing BMP4 [2 ng/ml]. Expression of stem cell, 2 G8 s; W( J6 w! i+ ~# X$ A
   primitive streak and late mesoderm genes were analysed by quantitative ; N/ z, A+ j1 w! |4 E; V" L2 O9 D; W
   RT-PCR in undifferentiated ES cells (0, grey bar) and in EBs collected for
   up to 16 days in serum (white bars) or defined media (black bars) from the 9 O& T' h, X3 o- [* d
   same starting ES cell populations. Bars represent the means of three 7 n; [# V) e, K
   (serum) or two (serum-freeB4L) biological replicates. The Y-axis
   represents a log scale normalized relative to the housekeeping gene HPRT. / \; p6 j/ ~$ C* j" A
   Error bars indicate standard deviation.
   Changes in gene expression were extremely reproducible across ES
   differentiation experiments with overall correlation of global gene : \, D# ]  c, W+ [
   expression for biological replicates in the order or 0.95–0.96. " h* S8 ~5 v# Y8 k  }
   Approximately 30% of the probes were differentially expressed at one or 9 z% m: H( ]  w6 d( Z
   more stages of EB differentiation as determined by one-way Welch ANOVA % C: O1 D! s, Z6 `9 T
   with multiple testing corrections using the Benjamini and Hochberg False
   Discovery Rate algorithm (7,967 probes with a p-value of < 0.01 by Welch
   t-test) (Figure 2A).0 ]9 t& H( f: Q) y# p5 K* w
   Figure 2. Gene expression profiles during 16 days of EB differentiation in
   serum and serum-free media containing BMP4 [2 ng/ml]. (A) GeneSpring
   representation of the subset of genes from the 48 K Illumina array that " f' ^3 \; ^2 `. T
   showed significant differences in gene expression at 2 or more time
   points. Each line represents the mean normalised expression of an
   individual Illumina probe. Colours represent relative expression (red 4× 4 N/ y; |( ?0 T6 T8 Y3 P9 s0 T1 [
   increased; green 0.25× reduced) compared to a gene mean of 1 (black) with
   expression in undifferentiated ES cells (day 0) as the decision point for # U2 i  e% b/ x
   colour coding. The expression profiles of T (brachyury), Hba (α-globin),
   and Igf2 are indicated for reference. (B) Clustering (tree) of the genes
   that showed significant (p < 0.01) changes in gene expression upon
   differentiation by One-way Welch ANOVA. Expression levels are heat map / w+ U; H$ l9 t; H
   colour coded (inset) with high expression at Day 0 coloured red and low
   expression coloured green.
   To mine for genes with similar patterns of expression across the time 4 H' I/ x7 @+ D6 f2 D# t2 {# m" U
   course, hierarchical clustering was performed on the 7,967 probes and . j4 L: p& ?! C2 {. ]
   represented graphically. A large percentage of these (~30%) displayed   X+ _; K' j7 s% W" O5 N
   highest expression in undifferentiated ES cells and subsequent reduced
   expression over time (labeled stem in Figure 2B). A second major cluster 7 Q6 p* l7 T/ N3 f) j2 e. D
   showed marked induction after ~day 4–6 of differentiation (labeled 1 {' Q$ s' [. l6 j+ K3 ?& x+ v9 v$ O
   mesoderm in Figure 2B).! F& K5 p7 R- s
   Putative stem cell genes sets& X0 G* e6 X! W9 ?. Z8 h9 b
   Previous studies have identified genes important to the stem cell state by
   comparing expression profiling data from different stem cell populations
   such as ES cells, haematopoietic stem cells (HSCs) and neural stem cells
   [6,18]. We used two alternative approaches to identify specific stem cell
   specific gene expression. Firstly, genes with dynamic expression patterns # ]- v  @- V- L
   which correlated closely with Oct4, Sox2 or Nanog expression profiles / L+ X+ v4 J$ [# i" U% ^* O
   (Pearson correlation > 0.9) were determined as previously described [6,7].
   Second, genes rapidly down regulated ≥3 fold by day 3 of differentiation, " b4 U8 ~2 S5 I& B. C6 e
   a time point where ES cell clonogenicity decreases dramatically, were 1 _9 k% G! F9 s' m- z7 a9 `
   determined. Not surprisingly there was very little overlap between these $ @) \! l- T, i. T$ Q8 C
   gene lists, primarily because Oct4 and Nanog expression persists during EB ; D7 I1 O8 i- v, ?0 g6 k4 m
   differentiation longer than many other stem cell genes (Figure 1). Full
   lists of genes ranked according to similarity of expression (Pearson   K. ]/ i6 _& X0 m  X* v7 r
   correlation >0.9) with expression patterns for Oct4, Nanog or Sox2 are
   provided in Additional files 1, 2, 3, respectively.  _! S0 V+ x" a) q1 n! u; f
   Additional file 1. Oct4 gene list. The data provided lists all genes
   expressed during 16 days of embryoid body differentiation with similarity
   to Oct4 (Pearson correlation >0.9).
   Format: DOC Size: 630KB Download file
   This file can be viewed with: Microsoft Word ViewerAdditional file 2.
   Nanog gene list. The data provided lists all genes expressed during 16 7 _0 G3 ^3 t: D* V3 B, n) J
   days of embryoid body differentiation with similarity to Nanog (Pearson * ^! ^: e$ l+ D+ {1 x
   correlation >0.9).
   Format: DOC Size: 111KB Download file# w6 T4 u; f" T% Z& b$ W! F
   This file can be viewed with: Microsoft Word ViewerAdditional file 3. Sox2
   gene list. The data provided lists all genes expressed during 16 days of
   embryoid body differentiation with similarity to Sox2 (Pearson correlation $ Y) n3 i+ m2 p3 s: K( v& x
   >0.9).0 j* b/ ^7 I8 ~) t' B
   Format: DOC Size: 237KB Download file- {2 @7 R7 U4 t  a& I
   This file can be viewed with: Microsoft Word Viewer
   Only fifty-nine genes were down regulated ≥3 fold by day 3 of EB
   differentiation (Table 1). Many of these have been identified as stem cell # _# ]: ?& `0 E1 n/ w& b
   markers by other groups using alternative strategies [6,7,19]. Information 6 O6 K( R+ n% J9 }' h+ W( i* s* _
   was compiled on their known expression patterns and/or gene knockout ' o* F; s' Q8 a/ f8 @" ~
   phenotypes via literature searches as referenced in Table 1. Insufficient 3 G* e, v6 h8 l  ~
   space exists to discuss every gene and most have been previously 5 t) O8 g( v; `- w5 T2 C
   identified as ES cell enriched transcripts. Osteopontin is secreted by
   osteoblasts in the bone marrow niche where it is thought to play an
   important role in the maintenance of HSC quiescence and maintenance of
   'stemness' [20]. It has also been discovered by other groups as a strong : ^; y8 L! b" H2 A0 s: G; X6 X
   marker of ES cells and is regulated directly by the key stem cell . \- n/ Z8 E7 b# o. p: T
   transcription factors, Oct4 and Sox2 [21]. Thus, osteopontin is likely to 2 N0 S! V6 C( L+ x3 D
   be a useful marker of ES cell pluripotency and may play autocrine or 7 j# R7 B% e2 a3 n; X
   paracrine roles in maintenance of stemness. The second most rapidly down : A) q; i3 F' S
   regulated gene was estrogen related receptor β, Esrrb, which had
   previously been identified as a stem cell specific gene and shown to be ( M/ w3 C$ f8 Y9 s+ D$ I
   critical for maintenance of the stem cell state [19]. F-box 15 (Fbxo15), a
   member of the large F-box family of genes [22] was also rapidly down ; x$ b% D" x9 Y
   regulated. Fbxo15 is known to be highly expressed in ES cells, is
   regulated by Oct4 and Sox2, but is dispensable for ES cell self-renewal 2 Q! U. V* d0 f& W. y0 F, |
   [23]. Recently, the stem cell specificity of this locus was used to screen
   for cDNAs capable of transforming mouse fibroblasts into ES cell
   populations [24]. Other notable rapidly down regulated genes included the   }" R" l; k# @
   nodal inhibitors Lefty-1 and Lefty-2, and Zfp42/Rex-1, which are also well & e3 G+ Q% B3 ~* g5 q
   established as stem cell markers [19].+ @3 ^5 T8 ?) S" \& R. t5 t# C7 w
   Table 1. Genes rapidly down regulated (>3 fold in 3 days) upon ES cell
   differentiation1 k# }5 j7 R# x; v& M, j; @% B" ?( _! f' G
   Oct4, Nanog and Sox2 are not included in Table 1, even though each is
   required for the maintenance of ES cell pluripotency [25-27] due to their
   persistent expression during the first 3 days of EB differentiation 6 F: ~1 T# G  l1 `
   (Figure 1 and 5A, and Additional file 1). Indeed, Oct4 expression is known
   to persist beyond the inner cell mass stage of development in vivo, with - j4 U! D2 ?- t
   transcripts detected in the epiblast and later in the primitive streak, ! h; ^' F  m) y
   before it becomes further restricted to primordial germ cells [10,28]. 779 ' N5 T1 B% g& d: c& b( H( x( r
   genes with a similar pattern of expression to Oct4 (Pearson correlation of
   > 0.9) were identified (Additional file 1). As expected this list also
   includes many well known stem cell genes such as Sal-like 1, nodal, + Y" M, }( [6 T& x, q3 {; u
   chromobox homolog 7 (Cbx7), Dnmt3b and Sprouty homolog 4 [19]. We also / L3 L9 C" x5 ^& V3 z
   mined for stem cell genes by looking for similarity (Pearson correlation
   of > 0.9) with Nanog and Sox2 gene expression profiles. These provided 8 i0 y) H0 m: P7 M; a
   overlapping but not identical sets of putative stem cell specific genes + ?0 o1 J+ R( ]& k; R
   (Additional files 2, 3). Full data for the 16 days of differentiation can 0 ?) n8 n) Q' y
   be interrogated and mined for specific differentiation outcomes via Signet 3 O8 l, Y$ B' d% q& o
   (Login_Bruce) (see Methods).# n7 R: y# I) g  h* n
   Tightly co-ordinated waves of gene expression suggest sequential " \# U; t9 x  b
   activation of epiblast, primitive streak and mesoderm differentiation
   programs- S/ D" l* r& F' H  d" T( }: b2 K
   Many alternative differentiation programs occur simultaneously during ES % H! B, ^9 N2 M! {. C
   cell differentiation. Therefore, it is difficult to find gene
   syn-expression patterns that identify specific programs using standard / l8 Q5 J" c( X( X3 q% `, P$ |
   clustering methods. An alternative approach is to search for expression
   patterns that closely resemble genes of known function. For example, the T ( q5 M7 ?; ^6 b. [- U
   box gene, brachyury, is a well characterized specific marker of the
   primitive streak [29]. Brachyury expression is transient in vivo, a
   feature recapitulated during ES cell differentiation [2]. Using qRT-PCR,
   brachyury was induced ~1000 fold from D0 to day 4 and then rapidly : `2 u+ V+ C8 ?; U3 e# L- ^9 ^
   silenced to baseline levels by D6 (Figure 1). The dynamics of brachyury
   expression were similar between serum and serum-freeB4L media (Figure 1 / Y) n- C8 Z9 N, D3 e$ Q4 z
   and 3A), but serum-free media was unable to support brachyury induction in
   the absence of BMP-4 (data not shown). Thus, BMP4 (or related factors in : x! e+ H6 Y# p- t
   serum such as activin) is essential for mesoderm generation. Thirty genes
   were identified with a similar expression pattern to brachyury (Pearson
   correlation >0.9) (Table 2) including many genes with established / b. M  e% d& r" Q
   expression or critical functions during primitive streak formation, such & ]& R. R9 m9 J4 {
   as Mix1 [30], Lim1 [31], Sp5 [32] and eomesodermin [33]. Transient % I8 A8 W% V% e3 w+ M
   expression of many of these 'streak-specific' genes was validated by 8 \: _6 r/ d: @6 D% s' w
   qRT-PCR (Figure 3B and data not shown). In all cases there was a close 9 N& g/ s( R5 t/ q8 S
   correlation between the qRT-PCR results and the profiling data (Figure ; Q1 B6 |0 T' v2 d* Z
3A).0 l- v; D4 P& s
   Figure 3. Mining for genes expressed in patterns consistent with primitive & r5 H9 l% N$ D* ?
   streak, and epiblast differentiation programs. (A) GeneSpring
   representation of epiblast and streak gene expression profiles during EB 1 s' g# ?* L- ^/ }6 G2 Y2 G
   differentiation in 10% serum or serum-freeB4L culture. The up-regulation * x2 {0 n0 z& N
   of genes between days 2–4 were similar under both conditions. (B) qRT-PCR
   analysis of Mixl, Lim1, Cdx4 and Riken clone 8430415E04Rik. The Y-axis 8 H, q  Q( D3 k$ i& ~
   represents expression relative to the housekeeping gene HPRT. Error bars 8 D' y1 l2 H" O" k/ k
   indicate ± SD from three biological replicates. (C) 102 genes transiently ( Q5 s: a% |& C& G* i3 G
   up regulated during the first 4 days of ES cell differentiation were
   clustered using a tree algorithm and Pearson correlation of > 0.9. The - l3 \- ?- E$ `# C
   'Brief' group represents gene with very transient expression at day 3. (D) : m& _7 ]! F( ?. r/ ~' Q
   Coding region for 8430415E04Rik. The shaded areas represent the three Heat 2 E% U4 j# l+ h, j
   domains.Table 2. Brachyury-like (Primitive Streak) gene list (Pearson 0 N. k7 F& b& X  P
   correlation >0.9 with brachyury); W+ ?  \; p+ W2 ?5 B* K* [. V7 A1 B
   Interestingly, four novel transcripts were identified in this list (Table : }6 I0 b4 q9 U( A
   2). One of these, 8430415E04Rik, was detected by two independent probes, ( s0 r* l, e5 v3 B' [; b* k
   strongly supporting its 'streak-specific' expression; by qRT-PCR there was
   ~50 fold induction from day 2 to day 3 of differentiation and a decrease ! k: }; V1 K- G& Y" ]1 D( ~3 I
   to near baseline levels by day 6 (Figure 3B). The gene contains 24 exons, ' l! a$ _; N$ Z6 P: z3 X. k; z
   and the full length cDNA encodes for a protein of 868 amino acids (Figure
   3D) which is highly conserved in vertebrates (Additional file 4). It has 9 @& v( k) U8 F2 d. j
   little homology to other proteins apart from three predicted HEAT domains
   or armadillo domains (grey shading in Figure 3D). Drosophila armadillo ; B$ f, ]0 n8 e; q
   interacts with β-catenin via its HEAT/armadillo repeat [34], which / j% o, K, C9 V6 F, o2 ~
   suggests the protein encoded by 8430415E04Rik may also be involved in ; \2 \/ @, t" s( \% d6 g/ A6 `
   protein-protein interactions.
   Additional file 4. Sequence alignment of RIKEN clone 8430415E04RIK. The ( _- I1 b( L  Y" G0 s% }
   data provides an alignment of RIKEN clone 8430415E04RIK using human,
   mouse, chick and zebrafish sequences.
   Format: DOC Size: 31KB Download file6 F* G5 V: M& @3 \: l5 ]
   This file can be viewed with: Microsoft Word Viewer
   The list of primitive streak genes in table 2 is smaller than that % r/ P: I. o7 M9 T' J9 z3 l8 W
   previously published [10] due to the very stringent criteria used to 7 q: N8 @4 `; K" B( Z
   determine brachyury-like patterns of expression. Thus, to find further " a" G  O4 P6 N" ~! v
   genes characteristic of epiblast, early and late primitive streak / [5 _: f. E3 e, `- W. s' o" ~" X& w) c
   programs, probes were identified which were transiently up and down
   regulated at least two fold with peak expression at day 2, 3 or 4 of EB + [) y. G+ @+ d! k- f, Z9 f
   differentiation. Gene lists were combined (102 Illumina probes) and 6 y9 M) [6 a. o* }, I' Q8 N
   clustered into epiblast, early, mid and late streak groups plus a unique 9 k8 d' d7 v3 x6 D3 Y5 J: [
   group in which expression was very transient at day 3 of differentiation ( Q5 _. U: F. R% r
   (designated 'brief' at the bottom of Figure 3C). Also, lists were
   generated by searching for similarity of dynamic expression profiles to 4 O/ @" w3 s6 B2 D# e7 R/ d$ K2 r
   known genes which are accepted specific markers of specific stages of ' Y3 d3 f  s5 t
   mouse development. For example, Fgf5 is specifically expressed in the 6 j7 H! o$ z1 r' ?% G' _# n
   epiblast [35], and Wnt5a is expressed in the late primitive streak [36]. ; _& P' k0 S! Y/ P
   The Fgf5-like (epiblast) list includes the methyltransferase, Dnmt3b, ; s: n! D# l2 e+ ^4 c
   which was massively up-regulated in the first 2 days of EB differentiation ; k' o$ a2 s; _; s5 T
   (Additional file 5) and is known to be essential for mesoderm " w: v* E( _3 q. v
   differentiation [37]. This list also includes Pim2, Zic2, Wnt8a, the SP1 : [3 c7 x9 a" a2 S3 c: |& `
   (and possibly SP5) cofactor Crsp2, and Irf1. The Wnt5a-like (late
   primitive streak) list contains 76 genes including many homeobox genes ! i% |! |$ h  Z7 o/ g% A
   (Hoxb2, Hoxd1, Hoxb6 and Hox8/Msx2), Cdx2, Hand1, Tbx3, Bmp4 and the VEGF
   receptor Flt1 (Additional file 6). Many are expressed at E10 of mouse ! n4 B9 n9 w- I! A
   development in the remnants of the primitive streak [36], suggesting the 0 _+ M- E4 G2 ^
   Wnt5a-like list includes many genes known to pattern the embryo following
   formation of the germ layers. There is a significant overlap between the ( P9 f; @$ x. C. w& z; q
   genes we identified as being epiblast-, early and late streak-specific and
   those recently identified using a similar EB differentiation system and
   Affymetrix arrays [10], and by other groups [2,38]. In addition, there are
   addition novel RIKEN cDNAs in these lists, which are likely to encode for / x8 c2 r9 K; e4 U9 @0 M
   interesting proteins worthy of further study (Additional files 5, 6).
   Additional file 5. FGF5 gene list. The data provided lists all genes 9 f+ [( b; E3 c
   expressed during 16 days of embryoid body differentiation with similarity ) ~* l( e2 e3 n7 a
   to FGF5 (Pearson correlation >0.9).: i3 c# v+ {/ ~- ?8 T8 P8 [
   Format: DOC Size: 69KB Download file
   This file can be viewed with: Microsoft Word ViewerAdditional file 6.
   Wnt5a gene list. The data provided lists all genes expressed during 16
   days of embryoid body differentiation with similarity to Wnt5 (Pearson
   correlation >0.9).) Q  j: X; }# n! t! a" V
   Format: DOC Size: 76KB Download file  o2 q, q0 H8 J. h
   This file can be viewed with: Microsoft Word Viewer
   Following the primitive streak wave of gene expression there was a
   dramatic activation of many genes (Figure 2A). Many of the most highly
   induced genes, such as globin genes, heme synthesis enzymes and ; K. i' O+ L2 A- @. a7 P4 S
   haematopoietic transcription factors, are known to be expressed in the
   blood. qRT-PCR for globin genes confirmed > 10,000 fold induction between " R) ~0 y6 J' V, {% I# b
   days 3 and 8 with a steady decline thereafter (Figure 1). Other mesoderm
   and endoderm programs are also activated at this stage, so it is difficult 7 j8 D1 y) d' S% d7 j2 ]
   to mine for these different outcomes.
   Enhanced cardiac gene expression and reduced erythropoiesis in
   serum-freeB4L EB culture
   Overall there were very strong similarities between ES cell
   differentiation in serum and serum-free cultures supplemented with 2 ng/ml   ?& d0 l" ^8 L+ w
   BMP4. For example, all 29 of the primitive streak gene set, as defined
   from serum-containing cultures, were also expressed transiently and to a 0 w/ m) H! o( ]# V
   similar level in serum-freeB4L media (Figure 3A). Genes differentially # b9 T  C2 o1 d( V
   expressed at each EB time point (Day 1–16) were pooled and subjected to QT
   clustering to find sets differentially expressed between serum and
   serum-freeB4L EB culture. Using a minimal cut-off of five genes per group,
   nine gene sets were identified; seven showed higher expression in serum
   and two showed higher expression in serum-freeB4L (Table 3 and Figure 4)./ v* e( T& [) z5 u2 V2 |; N; {( X! l
   Table 3. Genes differentially expressed during ES cell differentiation in 1 }3 l+ w/ P6 b
   serum versus serum-free defined media (BMP4 2 ng/ml)Figure 4. QT
   clustering revealed sets of genes differentially expressed between serum
   and serum-freeB4L EB culture. QT clustering revealed nine sets of genes * _/ X) S( h) l5 a7 p- M# e
   with similar dynamic expression profiles (Pearson Correlation coefficient
   of > 0.9). In sets 1–7, expression is higher in serum (black sets). In
   sets 8–9, expression is greater in serum-freeB4L EB culture (red sets). % L% C+ g0 A& h7 u" S
   Some sets have been named based on the similarity of overall gene function
   during differentiation pathways (see Table 3)., i6 u" S/ G* c  G( F4 r
   The genes in set 9 were expressed more highly in serum-freeB4L media from 8 _$ ^( ^" o! x$ j5 V8 g
   day 6, and genes in set 8 showed up-regulation at day 12 and 16 in the
   absence of serum (Figure 4). Both sets are comprised almost exclusively of
   cardiac-specific genes such as myosin heavy chain 7 (Myh7), myosin light
   chain 7 (Myl7), the cardiac isoform of α-actin (Actc1), troponin (Tnnc2), ( W6 F4 c# h& \$ N% I7 [
   tropomysin (Tpm2), the nicotinic cholinergic receptor (Chrng), and cardiac + c! m) f& @( [- D
   muscle fast twitch 1 specific ATPase (Atp2a1) (Table 3). Further
   interrogation of the array data revealed a cohort of cardiac transcription - L3 X3 @* y1 h% g0 B9 H% m
   factors, including ISL1, MEF2C, Bop/Smyd1 and Hand2 [39], which were
   transiently up regulated 1.4 to 3.5 fold in serum-freeB4L media compared : T( H, p5 Q  y! ?" E' E; g
   to serum at day 6 (Signet Login_Bruce). Importantly, this elevated cardiac $ D/ |$ ]# l4 q+ i# }
   gene expression program correlated with increased numbers of spontaneously
   beating EBs in serum-freeB4L media (data not shown). Enhanced expression
   (~100 fold) of Actc1, Myl4 and Myl7 in serum-freeB4L media compared with % B% I/ r9 q, q; h+ G7 |2 a
   serum-containing media was confirmed by qRT-PCR (Figure 1). Taken 6 |1 Z1 @' @6 }9 T
   together, this data strongly suggests cardiac muscle cell differentiation + ]: D& R- B- q8 P8 i* E
   is either inhibited in serum or enhanced by the addition of BMP4 to   A8 {$ R+ ?& _- M
   serum-free culture.3 `8 c3 _) {: U3 [0 O6 s7 C9 n
   The genes in set 2 were expressed in both serum and serum-freeB4L , h* R& ?) _. ]+ Y6 I
   conditions immediately following primitive streak formation, but expressed
   to higher levels and slightly earlier in serum (Table 3 and Figure 4).
   Many of these genes are globins (both embryonic and definitive) suggesting ! x, c- S- o' X# |
   erythropoiesis is activated more robustly in the presence of serum (See
   Discussion). Alas2, a gene encoding the erythroid specific isoform of the
   first and rate limiting enzyme in the heme biosynthesis pathway, and the
   erythroid specific transcription factor, p45-NF-E2 are also in set 2 [40].
   qRT-PCR for α- and βh1-globin confirmed more rapid expression in serum
   versus SFB4L media (Figure 1), suggesting factors in addition to BMP4 3 _6 V1 j4 p6 z) O! @6 ~; K
   enhance activation of the blood program from a similar primitive streak " h' M& B) l$ ^" ]4 H
   platform.
   The genes in set 3 were expressed at a similar time in serum but expressed 2 w4 ~& b9 m; Q/ p
   at very low levels or not at all in serum-freeB4L media. Again, this set
   is highly enriched for erythroid specific genes such as alpha hemoglobin " |3 y! W2 n/ j) g# C
   stabilising protein (Ahsp) [41], Glycophorin A, erythroid Kruppel-like
   factor (EKLF/Klf1) [42] and mitochondrial solute carrier protein   c+ x& ^4 V2 y  `" }
   (mitoferrin) [43] (Figure 4 and Table 3). Genes in sets 1 and 5 are
   activated more robustly at late time points in serum cultures. Most of
   these are specifically expressed in macrophages. Together, these results
   suggest delayed and less robust primitive haematopoiesis occurs in SFB4L * ]: r1 i: {# w( P' r/ R
   culture compared with serum, an observation confirmed by less robust
   erythroid cell generation and 'redness' of the EBs from day 6–10 of 4 @3 G2 D8 j3 _5 e6 E
   differentiation (data not shown).
   Rapid changes in Kruppel-like factor gene expression upon ES cell , f( P$ K+ M$ \# m$ `9 I
   differentiation) n6 p- P, k9 ^, j! r8 s
   Three Kruppel-like factor genes, Klf2, Klf4 and Klf5, were rapidly down
   regulated during the first few days of EB development (bold type in Table
   1). All are members of the KLF family of transcription factors which are
   characterized by a similar C-terminal domain of three C2H2 zinc fingers 7 H; c8 @1 u5 E$ W
   which confers binding to CACC box elements in promoters and more distant
   regulatory elements [44,45]. Klf2 and Klf4 have N-terminal transcriptional 1 B5 g9 ]+ o) n7 s/ |( |. B
   action domains and act primarily as transcriptional activators, whereas 0 `$ [% g( z- B6 D2 K
   Klf5 is best known as a transcriptional repressor [46]. Until recently, : e5 ]4 o; u- E
   Klf2 and Klf4 were considered to have restricted expression patterns ; }$ {5 J  @0 V3 ~  F  w, Y
   [47,48] with gene knockout animals showing late developmental defects in & C) E/ q* j8 x( W. f1 ]2 _$ d
   vasculogenesis and skin differentiation, respectively [49,50]. Recently - j  a: y. a/ S- p
   however, Klf4 expression was noted in ES cells [19,51], and forced ! q$ u* V& B. q% ~
   expression was shown to enhance Oct4 expression and promote self-renewal
   [51].
   qRT-PCR confirmed the rapid down regulation of Klf2, Klf4 and Klf5 in both 6 f0 l0 U, I( b0 Y
   serum and serum-freeB4L differentiation conditions, although Klf5 4 P9 q+ m/ X6 d( D  r6 x
   expression was down regulated more slowly (Figure 5A and 5B, and Table 4).
   Klf2, Klf4 and Klf5 were all re-induced from day 3–5 of differentiation as 7 S: s! S2 ~$ t& Q: n$ g2 `8 u/ ^
   mesoderm and endoderm developmental pathways are activated (Figure 5A and
   5B). Sixteen of the 17 known KLFs were examined in detail throughout the : k" H+ Q7 c1 f3 f; D- Y7 v: B1 s4 B
   entire differentiation time course (Table 4). Klf9 was also steadily down " R5 i/ q1 V) S
   regulated (>10 fold) between ES cells and day 5 EBs in serum and
   serum-freeB4L media (Figure 5B). Klf3, a well characterized
   transcriptional repressor [52] via its ability to recruit the ! s8 M' w9 D3 @9 f& D0 I2 e2 t
   co-repressor, CtBP [53], displayed an interesting biphasic expression
   pattern with initial rapid down regulation then re-induction from days 5–6
   (Figure 5B). In contrast, Klf16 displayed little change in expression
   levels over the 16 days of EB maturation (Figure 5B).
   Table 4. Kruppel-like factor gene expression during ES cell
   differentiationFigure 5. Kruppel like factors (Klfs) are dynamically
   expressed during the first few days of ES cell differentiation. (A)
   GeneSpring plot of normalized gene expression for all KLFs detected during
   ES cell differentiation in serum or serum-freeB4L culture. Most of the ' S9 h6 f, q( Y
   genes are listed on the y-axis in order from their highest relative 4 {! O( Y- O+ t; F7 C8 o7 z
   expression in ES cells. There is dramatic up regulation of Klf1 (Eklf) 1 y" v) D. b1 I' |8 q9 U
   only in serum following day 6 of differentiation. Plots representing Nanog
   (red), Sox2 (red) and Pou5f1(Oct4) (green) are shown for comparison.
   Pou5f1 and Nanog gene expression persists at high levels for 2–3 days - n# }! b9 W: U7 I; N. V
   after Klf2, Klf4 and Klf5 are down regulated. (B) Validation of changes in / _6 l2 s) v% V- y
   gene expression of six members of the KLF family by quantitative real time " ]( M7 B; r' B& k- d( G8 D
   RT-PCR. Scheme as described for Figure 2B.4 X# [5 W8 ]" F8 p! W; X0 |
   Klf2 and Klf4 co-localize with OCT4 in the nuclei of ES cells (Figure 6A). . Y# o7 ]$ [/ l' R: q" [
   Interestingly, all three proteins were found to preferentially reside in a
   nuclear sub-compartment (possibly nucleoli for transcription factories),
   suggesting possible co-involvement in a protein complex or network. Also, . U1 ]9 B  m, M2 t2 D
   endogenous KLF2 was detected by specific CACC box DNA-binding activity in " d' Z* b5 u  h2 h
   undifferentiated ES cells, which was lost upon EB differentiation (Figure : b. Q2 p! h" H5 r5 Z' l
   6B). In contrast, endogenous ES cell KLF4 DNA binding activity was not
   detected using the p18INK4c promoter CACC box sequence, although
   recombinant KLF4 was shown to bind to this probe (data not shown). SP1 and
   SP3 are ubiquitously expressed CACC-box binding proteins, which are + h* O# g9 M( ?( B. Y, ~
   dominant in ES cell nuclear extracts (Figure 6B). We hypothesize KLF2,   D6 F  R8 [7 F, e3 f7 y
   KLF3, KLF4, KLF5 and KLF6 compete with SP1 family members for DNA binding ; ~, b, g' }4 e$ l) t* h
   to key cis elements in various stem cell genes to regulate expression in . e2 e: `+ C  P- s1 C
   positive and negative fashions (see Discussion).
   Figure 6. Kruppel like factor expression and DNA binding activity during & }. |9 x! |+ b$ U6 ^4 N
   ES cell differentiation. (A) Co-expression of Oct4 with KLF2 or KLF4 in ES 6 ^5 v+ Z' a; m" Y) \
   cells. Indirect immuno-fluorescence shows co-localization of KLF2 and KLF4
   with Oct4 in sub-nuclear compartments (possibly nucleoli). Individual ( j1 x* K& @9 m/ z6 M1 @. B2 e
   confocal images for OCT4, KLF2, KLF4, and DAPI are shown with the
   corresponding composite image. Scale bar 40 μm. (B) Electro-mobility gel
   shift assay showing changes in DNA binding activities at a conserved CACC
   box site in the p18-INK4c gene promoter. Nuclear extracts were generated 5 N, C; j! O# B' j  U: t0 T; g) c
   from ES cells or EBs differentiated for four days in serum. Super-shifts * F% @9 n5 z% G7 l5 y6 L
   were performed with specific antisera for SP1, SP3, KLF2, KLF3, and KLF4 0 M: s3 D( Y4 Z3 A) e
   (See Methods). There is strong binding of endogenous Sp1 to the CACC
   element in ES cells and EB cells. KLF2 DNA-binding activity is present in
   ES cells as determined by a specific inhibition of binding of the
   indicated DNA complex with a KLF2 antibody. This activity is lost upon ; h# @3 L9 M% y! W2 f
   differentiation into EBs. The identity of the CACC box binding activity in . A, @9 Q+ C- g2 p1 a( P( x
   EBs denoted CAC-X, and the binding activity in ES cells denoted CAC-Y, was
   not definitively identified using this panel of antibodies.
   Enrichment of KLF-binding cis elements in the proximal promoters of stem 9 B5 k# T9 o/ j, n
   cell specific genes' ^& m' ?: f2 k3 c* b+ T0 C
   To investigate whether Kruppel-like factors might directly regulate stem ( d# t, Y: u# C5 d2 u
   cell gene expression, and therefore play a role in the maintenance or loss ( i7 r5 J3 a* ~% j
   of 'stemness', the presence of KLF transcription factor binding sites * a; Y7 I/ G1 L8 a  |7 D$ L) r
   (TFBS) within the proximal promoters of all genes in Table 1 was
   determined. Although a position weighted matrix (PWM) for KLF4 binding
   site specificity has been published [54], this site does not resemble the ) T4 z) J0 |5 X7 w. i; d+ F% G* h1 m
   CACC box sequences known to bind other KLFs. Since KLF4 has identical # E' N. u6 P+ B5 u
   DNA-binding residues in each of its three C-terminal C2H2 zinc fingers to & t' r5 f/ s/ \4 r& _- r; K
   other members of the Kruppel-like factor family [45], it should bind to
   similar sequences. Based on the crystal structure of the zinc fingers of
   Zif268 and SP1 bound to DNA [55,56], and testing of specific binding of
   CACC box variants to recombinant KLF1, KLF3 and KLF17 [45,52,57,58], a # m2 x3 y6 z" w
   generic KLF TFBS has been proposed. Although a SELEX experiment for KLF1
   binding has not been published, experiments from our group suggest a C at . z3 c9 V6 j* m+ G# Y8 n
   position 1, an A or C at position 3, an A at position 5, and an A, T or C 7 X( d* i+ O6 a! {' ~% K
   at position 9 of the 9 bp consensus provides enhanced affinity for KLF1 4 I2 U+ W7 j0 b9 L' ~. L  o
   binding. Taken together, these studies have enabled the development of a
   PWM (called here KLF-A) that should predict KLF4, KLF2 and other KLF TFBSs
   (see Methods and Additional file 7).
   Additional file 7. Position Weighted Matrices. Provides published data on
   position weighted matrices for Oct, Sox, KLF-A, KLF4, Nanog, E-box, Gata-1 / n, Q1 Q+ O8 N$ {8 `+ }3 r! ]3 p
   and their variants.
   Format: DOC Size: 116KB Download file
   This file can be viewed with: Microsoft Word Viewer' {4 Z- [& `0 c; {/ r( J
   Clover [59] was used to determine if defined TFBSs were statistically
   over-represented in the stem cell lists. We searched for PWMs for KLF-A, , j. f( K, E& c: R& i, e0 `
   KLF4 [54], octamer, extended octamer, nanog, and Oct-Sox binary sites as
   well as negative control sites (gata-1 and E-box) in sequences 2 kb 9 P: {' k: R! G* l. X
   upstream of the TSSs of stem cell genes (Table 1), versus 2 kb of " u  r% P" I1 z6 c: Q
   sequences upstream of the entire murine transcriptome (See Additional file
   7 for details of PWMs). There was significant over-representation of KLF-A 2 j* X/ X! P/ [  u1 u4 D
   TFBS within the first 2 kb of promoter sequences from the stem list gene
   list (p value of < 0.01) (Figure 7A), as well as over-representation of ' U; n6 M) p  k
   octamer sites and extended Oct4-binding sites as defined from ChIP-PET
   data [60]. No statistically significant over-representation of KLF4 [54], " q: T7 O; c6 t* t) x2 N1 d$ D& P
   NANOG, GATA1 or E-box TFBSs were identified. All genes in the rapidly down & v3 o9 k* y. j0 ~1 x
   regulated gene list have at least one KLF-A type CACC site, or octamer or
   Oct4 [60] TFBS. A full list of the promoters with highlighted sites is 2 O# R$ [  S" }; `& b& z0 @
   available on request. Together, this data strongly suggests direct   C: \$ }# h' J  S
   transcriptional regulation of many stem cell genes by KLFs.  c$ n. G( X1 a/ S5 Z
   Figure 7. KLF and octamer binding sites are highly enriched in stem cell ) a# J7 @3 `- L5 k7 g1 k
   gene promoters. (A) A Clover analysis was used to identify
   over-represented transcription factor binding sites within the promoter . _/ p9 U2 i4 ?6 q  A* g% Q
   sequences of all stem cell genes identified in Table 1. Representative
   gene promoters are shown, indicating KLF-A binding sites (pink), octamer ! f/ _/ T5 |3 l' z. m( t% M0 G
   sites (ATGCWAAT) (green) and extended Oct4 binding sites (Oct4-Loh)(cyan) 7 ^) }2 v( d  m: j  P
   [60]. (B) Clover output for 2 kb of promoter sequence of murine
   Zfp42/Rex1. The positions and sequences corresponding to PWMs for KLF-A,
   octamer and Oct4 occupancy sites are indicated in the table and colour / x1 O+ @- F; Q
   coded in the sequence. The positions are relative to the transcriptional : f' {/ m* G  Y/ i% y
   start site. (C) ECR Browser output of conserved sequence identity between
   mouse and rat in the promoter and part of the first intron of the Zfp42
   gene. The blue arrows indicate the direction of transcription. Yellow - h5 l8 b1 d5 v# i9 I9 b( y
   indicates the extent of the first (non-coding) exon, pink indicates
   regions of sequence conservation in the first intron and red indicates
   regions of sequence conservation in the 5' upstream region. rVISTA was   U& Z3 r6 B8 _0 G3 {! Y
   used to find all of the KLF-A and octamer sites in the murine gene (Murine
   only) and conserved sites between mouse and rat (Conserved mouse, rat)./ e+ |5 L2 Q& z) ]! n. }
   The Zfp42/Rex1 gene promoter is presented as an example of output from the - M$ q- h& G. c# ^6 T
   Clover program (Figure 7B). It contains five KLF-A, one octamer and three ( P5 m/ Y. Y3 {. d
   OCT4 TFBS. The two CACC box elements within the first 250 base pairs of
   the TSS represent classical extended KLF-binding sites [42,61]. These ' W2 c4 q4 o! ]6 Y+ E- T) b6 }
   regions are the same as previously reported [62] although their functional * g2 P/ \; d  n4 _8 [. s
   importance has not been determined. Interestingly, two of the three KLF-A
   TFBS were evolutionarily conserved between the mouse and rat gene
   promoters (Figure 7C). Also, two further conserved CACC sites at -1.4 and
   -1.5 kb were identified within 500 bp of extended evolutionary & j9 V6 m  l- H
   conservation (>70% identity) (Figure 7C). This might act as a KLF ; r) p0 a, k9 D' m
   dependent enhancer. Most of the stem cell genes in list 1 have conserved + r7 [& e5 J% U: @1 y
   CACC box elements in their promoters [63-66]. In some cases these have
   been reported to bind the ubiquitous SP1, but our Clover analysis suggests ) M, |0 w' D8 N  W. {% O+ B
   they are also likely to bind KLFs (see Discussion).
   Discussion8 u/ i4 o+ t/ _- n
   Expression profiling of murine ES cell differentiation was undertaken over
   a 16-day time course. We compared gene expression in methylcellulose , g$ |8 x( t7 G3 R) A7 q0 X' w
   cultures containing serum versus chemically defined media containing LIF
   (1 U/ml) and low concentrations of BMP4 (2 ng/ml) [67]. The Illumina®
   Sentrix Mouse 6 bead array provided a sensitive and detailed platform for
   analysis of dynamic gene expression. Using various data mining approaches,
   lists of stem cell-enriched genes and genes that are induced during the in
   vitro equivalent of epiblast and primitive streak stages of 6 D" H: P4 v1 f! P* v
   differentiation were generated. In combination with other ES cell 3 u. k8 ?( B" E3 J  N  G
   profiling studies [7], our detailed expression data provides a useful " M% l  z) ~) w) W# \' \
   resource for future reverse genetic approaches (i.e. siRNA knockdown) to   y2 j* o8 s5 t0 X: F' t! q4 _
   study the function of these genes during ES cell differentiation and in
   vivo development. We also found a number of previously uncharacterized ; ~/ N& j4 D1 P$ k. Q- Q
   cDNAs (RIKEN clones) which could play important roles during development.- ]. V2 q3 |6 C1 h' m
   Importantly, the loss of pluripotency, measured by Oct4 gene expression, 9 R! j- {% B# j! b
   was comparable between serum and serum-freeB4L EB culture, following a
   predicted decrease over the first 6 days. Surprisingly, Oct4 gene 7 E! S, S' n9 d6 H4 T
   expression gradually increased following day 6, an observation independent 1 Q& j8 c, z1 b; j$ O
   of the cell lines used (data not shown). It remains undetermined if
   expansion of Oct4 positive ES cells persists as undifferentiated
   populations within our EB culture system. Previous studies have identified ; P5 O, i% |$ u1 l2 e# D% z
   the development of Oct4 positive primordial germ cells (PGC) following 12 8 z; C$ ]4 S2 O* Y7 }# |' l3 o
   days of ES cell differentiation [68-70], suggesting the Oct4 profile may . J* r" g+ i! s+ Y
   alternatively represent the expansion of non-ES cell populations. Although
   the expansion of some undifferentiated ES cells is possible, the array   N; S) q" o4 J* K* x5 c. H: h
   profile does not suggest global persistence or up-regulation of ES gene
   expression late in the EB program. Also, markers of mesoderm induction
   such as brachyury and Mixl, do not show persistent up-regulation following
   peak expression at day 4, suggesting EBs are unlikely to harbour cells % Y! }, U* Y" n. {# R5 M- U" }& p
   which are delayed or arrested from entering the differentiation program.* i3 U3 J, \! c
   We found significant differences in cardiac gene expression during EB . F9 d& N% A7 f4 O/ m) f5 F3 `- N
   differentiation in serum and serum-freeB4L culture. Our current
   understanding of cardiac development provides possible insights into this
   observation. Briefly, in vivo studies have revealed BMPs secreted from the
   anterior lateral plate, and ill-defined signals from anterior primitive
   endoderm, are key inducers of cardiac development [71-73]. The
   administration of recombinant BMP2 or BMP4 to chick explant cultures
   induces cardiac differentiation in non-cardiogenic mesoderm [71] and Bmp2 : C: c8 \/ N* g4 G; c
   knockout animals develop cardiac abnormalities [74]. Conversely, Wnts $ i  [) X& y5 Z
   secreted by the neural tube are strong suppressors of cardiogenesis.
   Together, these opposing signals act to establish the borders of the heart 7 s% }! V* U# ~( c# `+ I
   field [71,75]. By mimicking the environment that establishes cardiogenesis , T, V5 S2 l' v  \
   in vivo, assays capable of directing cardiomyocyte production from ES 9 u) z5 N% X. s0 o
   cells have been established. BMPs can efficiently enhance the cardiac
   program when added to EB culture [76,77], whereas BMP inhibition 7 P, s% [# n7 k* ?
   drastically suppresses this outcome. In our hands cultures supplemented
   with BMP4 (2 ng/ml) supported cardiomyocyte maturation (as determined by
   expression of cardiac specific genes) with greatest expression detected at # S. }$ `) @! j1 ~# x
   Day 8–10. This correlated with increased numbers of spontaneously beating
   EBs, and the timing of initial spontaneous contractions during murine
   embryogenesis [78]. The cardiac program was significantly reduced in
   culture containing 10% serum, an observation supported by a number of
   other studies [79,80]. This suggests the constituents of serum are 2 P, n5 C5 J, j/ P4 o9 L
   inhibitory to cardiomyocyte development. Although a detailed assessment of
   cardiomyocyte differentiation was not the focus of this analysis, the " D8 c3 J0 w* I8 f/ l3 c8 ]
   array output, qRT-PCR profiles and morphological observations described
   supports the usefulness of this assay in future investigations. In 7 @7 _$ I8 `! ^' K: M* d
   addition, the defined constituents of serum-freeB4L media provides an
   excellent opportunity to identify additional recombinant factors required
   to further expand cardiac progenitor cell production from EBs.) |" E/ S! P5 I+ N1 `' v0 |2 v! y7 j
   In contrast, the hematopoietic program was more pronounced within EBs " `% \7 M8 O& J" W% M
   grown in 10% serum than BMP4 alone (Figure 1 and 4). In particular, EKLF + W: j. ~6 |& d7 |; x1 i# j% ^
   (Klf1) expression was significantly reduced in serum-free media
   supplemented with BMP4 (2 ng/ml) compared with serum (Figure 5A and Table 9 B- }5 M! ?3 ^" |$ ~9 U
   4). EKLF is essential for regulation of a large cohort of erythroid   N' j$ {. J, {8 k1 H+ h9 ^
   specific genes [81,82], many of which were identified in sets 2 and 3 $ k5 D' N7 h( w/ u+ [9 K
   (Figure 4). Thus, it is likely that reduced EKLF expression in SFB4L media 1 Z8 Y% Q8 B! A& n9 |: [
   directly results in inhibition of a cascade of erythroid gene expression. % d1 {) k/ `# X8 e
   Previous work showed the addition of BMP (at 5 ng/ml) to serum-free ES
   cell culture induces EKLF expression and restores hematopoietic cell
   differentiation [83]. Although a weak hematopoietic program was observed + n% p8 l% r7 r4 S+ D
   in SFB4L culture, the concentration of BMP4 used was less than that used
   by Adelman et al. Thus, we suggest a robust cardiac (anterior-ventral 1 c5 q  W$ W; |. q
   mesoderm) gene expression program is induced by low conentrations of BMP4 9 y6 O) c; c  e+ H  F3 q2 R! }  h0 ?; Q6 s
   (or other BMPs), whereas a robust blood gene expression program
   (posterior-ventral mesoderm) requires >2 ng/ml of BMP4 or additional 7 N, r# r8 f( P; M
   growth factors. Recently, mesoderm derived progenitor cell populations for
   hematopoietic and cardiac lineages were studied during EB development
   using ES cells in which GFP is targeted to the brachyury locus [80]. / K0 t) j! ^0 q2 W6 s! i
   Within day 3.25 EBs, GFP+Flk1+ cells were shown to represent hematopoietic 7 C( A% j" M( e9 l* A
   precursors, whereas GFP+Flk1- cells were significantly enriched for : I7 Z* N* X6 H% K' t" R
   cardiac progenitor populations. It would be interesting to determine if
   the ratio of Flk1+/Flk1- within the brachyury positive population is
   altered between our two culture conditions.
   A number of the Kruppel-like factor family of transcription factors were
   dynamically expressed in the first few days of ES cell differentiation. It
   was initially surprising to find highly enriched Klf2 and Klf4 expression
   in ES cells, since both are considered markers of terminally
   differentiated cell types such as skin [50], gut [84], vascular smooth 3 o4 ^' j$ Q, M) V' }* q' h
   muscle [49,85] and lymphocytes [86]. However, recent evidence suggests ' G, A2 }( M$ S* s4 P
   KLFs may regulate stem cell function, since Klf4 is enriched in ES cells
   [19] and forced over-expression within these cells can maintain
   pluripotency in the absence of LIF [51]. Furthermore, Klf4 can bind the
   Lefty1 gene core promoter co-operatively with Oct4 and Sox2 [65]. Although
   Lefty 1 and 2 are best characterized as repressors of nodal, acting to 6 `: j9 h% J" Q$ Y% g$ y; s
   regulate left-right patterning [87], our results and those of others,
   suggest a possible redundant role for lefty proteins during the
   maintenance of ES cell pluripotency [65]. Similarly to the Oct4 expression
   profile, many of the KLF family members also increased in expression late
   in the differentiation program. As mentioned, the KLFs are expressed in
   diverse tissues during development. It is therefore expected that the 8 s$ E' L0 [; L; J/ p# O$ N. N! m( w
   profiles obtained reflect the generation and early specification of $ E/ T6 ?5 h+ P; ]* q5 V# j
   mesoderm and endoderm cells following primitive streak gene activation at / v+ }5 @# u4 H+ w- P
   day 4.7 f5 Z% o2 d* D: p/ q  O- N
   Based on the likely identical binding specificity of the KLF family,
   established transcriptional activation and repression roles of certain ; _% T! w) |4 V6 N* h( _
   family members [44], and bioinformatic evidence of a high prevalence of
   KLF TFBSs in many stem cell genes, we propose KLF competition for
   occupancy of these CACC box elements might determine self-renewal versus
   differentiation of ES cells. According to this model, high levels of KLF2
   and KLF4 expression in undifferentiated ES cells would lead to occupancy * f8 m; }1 N) a
   of CACC box elements in promoters of stem cell genes such as Pou5f1/Oct4,
   Nanog, Esrrb, Zfp42/Rex1, Lefty1 and Lefty2. Furthermore, the Klf2 and
   Klf4 genes themselves have CACC box elements in their proximal promoters ! _/ d+ V. H+ z; \# S3 d  g2 o. h+ `
   (Figure 7A), suggesting a positive feedback loop within ES cells is 1 r7 P; v. S" C# d4 i1 |6 b1 C7 l
   likely. Interestingly, gene knockout and lentiviral shRNA gene knockdown 0 }5 S2 O" r3 s% L7 \0 ]
   of Klf2 or Klf4, does not lead to an obvious stem cell defect
   [19,49,50,85], suggesting these two KLFs may have functionally redundant " L. s+ C* k3 y) A1 T0 H
   roles during the maintenance of pluripotency. Future analysis of KLF2/KLF4
   double knockout ES lines or the knockdown of both proteins using RNAi
   technologies will be necessary to validate this hypothesis. Upon ES cell 9 ^, t5 O, p/ g6 Y+ `2 d3 I9 m, e
   differentiation, down regulation of Klf2 and Klf4 was very rapid, whereas ; A# v" W$ V6 u1 c
   down regulation of Oct4 and Nanog was delayed for one to two days. We   \, I+ G& y: }0 U, |1 W1 h) F
   suggest loss of KLF2 and KLF4 binding to Pouf1/Oct4, Nanog, Esrrb and
   perhaps other stem cell promoters, could be directly responsible for their
   down regulation. Moreover, Klf5 was down regulated more slowly, and Klf3
   was up-regulated in the first two days of differentiation, suggesting - T/ _" h0 D# {8 L; ?  I
   these KLFs may function primarily as transcriptional repressors at the " Z; P8 s& K1 p. u
   Pouf1/Oct4, nanog, Zfp42 and Esrrb gene promoters. Once this 2 g, v9 C- s' f! a! A) s8 ?# c0 ?
   differentiation driving transcription network is activated we suggest
   KLF3, KLF5 and KLF9 can accelerate loss of the stem cell state by directly
   repressing expression of the Klf2 and Klf4 genes themselves. Therefore, a
   combination of cross regulation of transcriptional outputs, and 4 j/ U$ P9 Z' m# w- U. @$ q' C: K2 y- h
   competition for occupancy of key stem cell promoters by KLF proteins with $ ]0 b! U( X+ k0 o
   differing biochemical properties but identical DNA-binding capacities, may
   determine self-renewal versus differentiation outcomes. Again validation
   of this model requires functional analysis. It is important to note we
   have not proven occupancy of stem cell gene promoters by the KLFs in vivo. # o$ o8 L8 a$ ~4 ^
   Chromatin immuno-precipitation (ChIP) experiments in ES cells and
   differentiated populations are essential to validate this model, however
   ChiP grade antibodies are not currently available.
   This scenario is not necessarily limited to ES cell differentiation. A
   similar program may take place in many adult stem and progenitor cells
   populations. Certainly competition between Klf4 and Klf5 has been
   suggested to regulate cell growth [88], and Klf1 and Klf3 compete for
   binding and have opposing effects in blood cells [89]. Furthermore, an 1 k7 s5 [: [) U4 j. Y3 H8 i% ?( p
   imbalance between occupancy of key stem cell or proliferation gene
   promoters by KLFs, with differing activating and repressing functions, # b+ Y3 p' B- }5 `" ]" ^; ?$ T
   could be responsible for the development or progression of many common
   forms of cancer. For example, loss of hetero-zygosity for Klf4 and Klf5 ! d1 f! F, K& F3 u/ z
   has been found in colon, stomach, breast, prostate and liver cancers 4 R9 f' \1 Y5 n) |$ B0 g# _
   [90,91].
   In addition to the proposed complex transcriptional interplay between KLFs ! m8 m: o3 h9 M: X: B5 e
   and stem cell gene promoters, direct protein-protein interactions and 0 k& J8 f2 t( E; d1 T0 j
   protein networks involving KLFs are possible. We showed sub-nuclear
   localization of KLF2 and KLF4 with OCT4 in ES cells (Figure 6A), and KLF4
   can co-operate with OCT4 and SOX2 to drive expression of the Lefty1 core
   promoter [65]. Also, very recent biochemical purification of ! U' I& }  N/ ~! k
   NANOG-interacting proteins discovered KLF4 as part of a NANOG-OCT4 network
   in ES cells (Stuart Orkin, personal communication). This is also 9 K$ o5 i( U: q6 C0 s
   consistent with the ability of KLF4 to co-operate with OCT4, SOX2 and
   c-MYC to drive de-differentiation of fibroblasts into ES-like cells [24].
   Conclusion8 s/ {! h# L" L
   Defining the genetic regulation of ES cell self renewal and 9 B3 V. q! {) G0 u) ?
   differentiation will be instrumental for the development of future cell 6 J- h5 b( f( N. H  o, v& F
   based therapies. Using 47 K Illumina® Sentrix bead arrays, the 2 p0 n, t+ o9 |$ `4 R
   differential expression of genes during 16 days of ES cell differentiation
   was determined. Hierarchical gene clustering and correlation statistical
   analyses lead to the identification of a small cohort of genes which & g, o/ V& E7 c' v8 ^# }
   define the stem cell state. Historically, ES cell differentiation is ) `& A& U% d/ v( s, x9 s% b
   achieved in culture media containing 10–15% FBS. A direct comparison 0 Y5 e! m) y6 Q+ {; y. W3 p; M6 \
   between ES cell differentiation in serum and a serum-free media was ' t4 m* R1 M# T& [$ _* w
   undertaken. Surprisingly, global gene expression profiles were comparable
   between culture conditions, with the exception of mesoderm derived cardiac
   and haematopoietic transcripts. These results support the use of defined
   serum-free approaches for the directed differentiation of ES cell 1 v" Y, {1 N; ^' k
   commitment. Expression of many KLF family members were enriched in ES 0 ^0 {# b1 `4 O6 [+ i" g% r: ^1 G; f
   cells and rapidly down regulated upon differentiation. KLF2 and KLF4 were
   co-localisation with OCT4 in ES cell nuclei and KLF transcription factor - X! u5 D& C  K) X, `
   binding sites were over-represented within the promoters of the putative
   ES cell gene list (p < 0.01). Taken together, this data strongly suggests
   KLF family members regulate the maintenance of ES cell pluripotency.* z% d% I$ O/ y2 N, Q
   Methods
   ES cell culture, embryoid body formation and immunohistochemistry
   W9.5 ES cell differentiation was performed as described (Bruce et al.,
   Differentiation, in press). For immuno-fluorescence, feeder depleted W9.5 , J( W( [, r7 N6 J! _
   ES cells were seeded onto sterile gelatin-coated glass cover slips in ES ( P, q5 u! |4 D# g/ Y3 v8 A& N
   cell media, fixed in 4% PFA for 10 min then washed in PBS. Cover slips " M# O4 J2 x9 H5 _7 @
   were boiled in 10 mM citric acid (pH 6.4) for 10 min, washed in PBS and 1 R8 R0 \4 @- w; y8 [* @6 D
   permeabilized in 0.18% triton X-100 in PBS for 10 mins. For OCT4/KLF4 1 j- U, N* O( e: F& o. v3 O
   detection, the cover slips were blocked in 1% BSA then incubated with 9 P7 f' m9 S" w7 Q2 @0 C: k& s
   rabbit polyclonal antibody raised against OCT4 (Abcam #ab19857) and goat $ ~6 }- `; p# y' U
   Anti-mouse KLF4 (R&D Systems #AF3158) overnight at 4°C, then incubated in
   goat anti-rabbit Alexa-488 (1:400) (Molecular Probes #A11008) and donkey 0 C' \7 `! B) ~. x
   anti-goat Alexa-647 (1:400) for 1 hour. For co-detection of OCT4/KLF2, the
   Zenon Rabbit IgG labelling kit was used (Molecular Probes #Z-25308). 1 μg
   of KLF2 rabbit polyclonal antibody (Santa Cruz SC-28675) was labeled with $ j: g  a+ i) A5 |" l0 y/ ~
   Alexa647 Fab fragment following the manufacturer's instructions. Cover
   slips already stained for OCT4 (Alexa488) were incubated with labeled KLF2
   for 1 hr then washed and post-fixed in 4% PFA to ensure strong signal . p( ^- J9 d  J; u- z
   detection. DAPI 1:5000 (Molecular Probes #D3571) was used to detect % S* d$ g% m# y8 R4 p, J5 y
   nuclear localization. Cover slips were mounted on SuperFrost® Plus slides
   (Menzel-Glaser) with VectaShield mounting media (Vector Laboratories 8 {1 I0 \0 d4 m; l: T! S
   H-1000). Confocal microscopy was performed on LSM 510 META Carl Zeiss
   microscope system.8 a/ y1 C! T' q& u
   RNA extraction, cDNA synthesis and real-time RT-PCR data analysis
   Total RNA was made using TRIzol (Invitrogen), and cDNA was synthesized
   from 2 μg of DNase1-treated total RNA using Superscript III (Invitrogen)
   and oligo-DT12–15 (Promega) according to the manufacturer's instructions.
   Quantitative RT-PCR (qRT-PCR) was carried out using the Applied Biosystems ; L8 i; M9 E; @8 G- o
   SYBR-green dye system and 7500 Real Time Cycler in 96-well plates. Cycling / {6 u3 F9 N5 z& l! K5 K5 y
   variables were as follows: 50°C for 2 minutes, 95°C for 10 minutes, then
   40 cycles of 15-second denaturation at 95°C and 1-minute at appropriate 3 h0 q/ N% Y+ \" x1 I
   annealing temperatures, optimized for each set of primers based on
   dissociation curves (Additional file 8). Expression levels were normalized 0 k3 c% g8 a1 I4 \/ e% f# }
   to HPRT as determined from the ratio of delta CT values. Mean of relative
   expression ± SD was determined from three biological replicates. * K9 |. b0 l. N+ e4 d0 \3 Q
   Sequencing of gel-purified amplicons was performed to ensure correct 5 }* y: Z: @, J" h8 r
   product amplification., r% N  O9 Y( Y+ G4 M$ u( l2 `
   Additional file 8. Oligonucleotide sequences used for qRT-PCR analysis. ) l5 N7 x: ~6 C: t
   Lists the oligonucleotide sequences and cycling conditions used for the / W7 X+ ]- ?7 i9 ~* F. k
   qRT-PCR analysis.' o2 T/ R' t& R, z, Z
   Format: DOC Size: 48KB Download file2 Q( z- H, x" Z7 I/ |+ W
   This file can be viewed with: Microsoft Word Viewer) z: t. ^  V8 y
   Micro-array hybridization and analysis
   Expression profiling was performed using Sentrix Mouse-6 Expression
   BeadChip arrays from Illumina®. RNA was assessed for integrity using the % ~/ W! o; V3 G* R, ]6 t& \
   Agilent Bioanalyzer 2100 and RNA integrity (RIN) scores above 9.5 were " ^( r: p: w  ]7 B( `2 E
   present in all samples. Amplification was performed with 500 ng of total
   RNA using the Illumina TotalPrep RNA Amplification kit (Ambion) with a 12
   hour in vitro transcription reaction period. The quantity and quality of - ^2 v7 {5 x$ z3 F( p
   biotin-UTP incorporated cRNA was also assessed on the Agilent Bioanalyzer
   2100. Amplified cRNA (1500 ng per array) was hybridized to Mouse-6.v1
   BeadChip arrays according to the manufacturer guidelines and detected with + G0 H2 M* D: A
   Fluorolink Streptavidin-Cy3 (Amersham Biosciences). Arrays were scanned
   using the Illumina BeadStation Scanner. The raw intensity values obtained . _! r) `" i; [* |$ R
   for the scanned array images were compiled using the proprietary ' s* b/ a, t: j4 i# s9 k- V) c; h
   BeadStudio v1.5.1.3 software and imported into GeneSpring GX v7.3.1
   (Agilent). A mouse Illumina probe set was defined in the GeneSpring
   Workgroup using the Illumina targetIDs as the unique identifiers and
   annotated according to array content files supplied by illumina®. Data
   normalization was performed by first setting all measurements less than
   0.01 to 0.01, then applying per chip normalization to the 50th percentile,
   and per gene normalization to the median.* y' V, j. `( c3 u
   From an interpretation which included the three FBS experiments, a
   non-parametric Welch ANOVA (where variances were not assumed equal) was
   performed on all 46,120 probes to find a subset of genes whose expression
   varied significantly throughout the differentiation time course. A
   Benjamini and Hochberg False Discovery Rate multiple testing correction
   was applied to reduce the number of false positives. This yielded a set of
   7,967 probes that showed statistically significant (p < 0.05) differences
   in expression by Welch t-test. This approach simplified running the data ( j9 h' G/ e  J7 K' @
   mining algorithms to find syn-expression patterns by removing genes such
   as housekeeping genes which did not display differential expression, or # F( |: L) L; S. n
   genes which did not reach a threshold of expression at any time points.
   Genome interpretations were generated in GeneSpring GXv7.3.1 (Agilent) in ) U0 I  O9 p1 d
   which the mean relative expression level for each gene was represented at 1 V8 b7 X9 j/ P
   each time point relative to the average of all time points. A number of
   statistical approaches were used to find syn-expression patterns. A + g4 F$ U( P7 w. `" j
   Pearson correlation (between 0.9 and 1.0) was performed to find genes with ) u0 B! [2 g( u+ @
   similar expression profiles to Pouf1/Oct4, nanog, sox2, fgf5, wnt5 and $ D* q; Q0 C( `# ~: F0 Q
   brachyury. Hierarchical clustering was performed using the gene by gene 6 H1 i/ ]! A; U6 h2 |! L
   and group by group 'tree' algorithm within the GeneSpring (Agilent)
   program. This compares each probe with every other in the lists to
   generate similarity statistics and a tree representation of similarity
   based on a Pearson correlation. Also, quality threshold (QT) clustering + e/ I: t3 a+ w
   [92] was used to define distinct subsets of co-expressed genes. The entire 0 \- W$ M  ^0 `5 f7 Z- g
   data set for the experiments is available via GEO as well as via our 7 L3 i! y% i" `% r
   instance of Genespring (contact corresponding author).
   Electro-mobility gel shift assays (EMSA)
   Nuclear extracts were made from undifferentiated ES cells and embryoid ! s- V+ E+ K+ o+ L% r3 h; c
   bodies grown for four days in 10% serum. Electromobility gel shift assays 7 |0 K' _) H1 O. m( l1 R+ |  e
   (EMSA) were performed as previously described [61] using double stranded / t4 O, V/ @. F( m
   oligonucleotide probes corresponding to an extended CACC site in the
   p18INK4c promoter (sense strand 5'-gttgggcggggcgtgggcggggcc-3') (Tallack, ( o5 I( h, y; P; Y) W4 {1 d3 {* w
   et al., submitted). Supershifts were performed with specific antibodies + I! l" A) ?2 K- ~& J: O0 {# c
   raised against SP1 (Santa Cruz SC-059x), SP3 (Santa Cruz SC-644x), KLF2
   (Santa Cruz SC-28675), KLF3 [52] and KLF4 (Santa Cruz SC-12538x).- N$ h. @! I' B* ?
   Bioinformatic searches for CACC sites and other over represented elements 0 [8 z- o* l) T" g& d9 _+ w  l
   in promoters of stem cell genes$ O0 q! \* f' V0 U% B# D) p
   The data-mining tool Biomart (accessed via Ensembl release 36 of the mouse 3 J1 r( J% h+ t
   genome), was used to collate 2 kb of sequences upstream of the
   transcriptional start sites (TSSs) of all genes listed in Table 1. The - E" w+ h; Y* Y1 X
   Clover program, run via the MotifViz web interface [59], was used to
   search for over-representation of position weighted matrices (PWMs) for 7 T+ q$ x' D! U" m0 ~
   octamer [93], Oct4 [60], Oct-Sox [60], KLF4 [54], E-box, GATA-1 [94] and 2 c( [3 z. h# R
   CACC (KLF-A) elements in these promoter sets. The PWMs are listed in , b3 S: x& k) r0 }/ U  c
   Additional file 7. The ECR Browser [95] was used to determine phylogenetic
   conservation of cis elements between mouse, rat and human genomes.
   Abbreviations
   BSA, bovine serum albumin; EB, embryoid body; EMSA, electromobility gel + c8 G- p7 S# [+ u! Y
   shift assay; ES, embryonic stem; FBS, fetal bovine serum; KL,
   Fkruppel-like factor; LIF, leukemia inhibitory factor; MEF, mouse
   embryonic fibroblasts; PFA, paraformaldehyde; PWM, position weighted + }) g$ C7 x# C" J1 c4 a
   matrix; TFBS, transcription factor binding sites.
   Competing interests/ w6 c, Y9 s: V# U
   The author(s) declares that there are no competing interests.
   Authors' contributions
   SB established the EB differentiation assay, collected and purified RNA 9 b" @! J6 [! ~
   samples, designed and carried out the quantitative RT-PCR studies and
   drafted the manuscript. BG carried out the micro array protocol. LB ' {1 x. f/ Q! B& ?6 M* F
   Carried out experimentation of mesoderm specific gene expression. MG
   performed the array statistical analysis and annotation. SG participated ( ]3 U5 j: Z' T
   in the design of the study and performed the statistical analysis. AP 1 N& s$ @0 u( N' `/ B- \9 x
   conceived the study, and participated in its design and coordination and
   helped to draft the manuscript. All authors read and approved the final * ]# z/ {% z" N
   manuscript.

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